Molecular/Cytogenetics Flashcards
Relationship of EGFR and KRAS to colorectal carcinomas?
The EGFR signaling pathway is overexpressed in ~80% of CRCs. When EGF, as well as several other ligands, occupies the EGFR, it activates a signaling pathway cascade through the downstream effectors of the mitogen-activated protein kinases (MAPK) pathway. These effectors (KRAS, BRAF, ERK, and MAPK) influence cellular proliferation, adhesion, angiogenesis, migration, and survival. Blocking EGFR with cetuximab (Erbitux) or panitumumab (Vectibix) blocks all downstream effects of this receptor and is the basis of these therapeutic agents. ~30-40% of CRCs harbor mutations in KRAS that yield a constitutively active protein, where blockage of the EGFR does not affect the downstream signaling cascade of the MAPK pathway. KRAS mutations typically occur in codons 12 or 13 (exon 2) or in codon 61 (exon 3) of the KRAS gene. Only patients whose CRCs carry a wild-type sequence of KRAS have a favorable response to cetuximab or panitumumab; patients with mutations in codons 12 or 13 do not benefit. Hence, the NCCN guidelines with the recommendation that mutation analysis of the KRAS gene on the primary tumor or a site of metastasis should be part of the pretreatment workup for all patients with stage-IV CRC.
TSC1 and TSC2 genes.
Tuberous Sclerosis Complex genes. TSC1 is located on 9q34 and encodes the protein hamartin (130 kDa). TSC2 is located on 16p13.3 and encodes the protein tuberin (198 kDa). Both proteins are highly conserved and ubiquitously expressed. Hamartin and tuberin form a physical and functional heterodimer complex, in which hamartin functions as the regulatory component, stabilizing tuberin and facilitating the tuberin catalytic function as a GTPase-activating protein. Tuberin, which is assumed to be the functional component of this protein complex, is multifunctional, and involved in the regulation of cell size, cell cycles, translation, transcription, apoptosis, and cell differentiation. A major function of the TSC1 (hamartin)-TSC2 (tuberin) complex is its role as a GTPase-activating protein against Rheb (Ras homolog enriched in brain), which in turn regulates mTOR (mammalian target of rapamycin) signaling. The major function of mTOR is to phosphorylate and activate downstream targets p70S6K (p70 ribosomal protein S6 kinase) and 4E-BP1. Deficiency or dysfunction of the encoded proteins, hamartin or tuberin, respectively, result in constitutive activation of mTOR and downstream S6K and 4E-BP1, leading to increased protein translation and, ultimately, to inappropriate cellular proliferation, migration, and invasion.
What is the MC cytogenetic abnormality in ALK positive ALCL?
t(2;5)(p23;q35). The t(2;5) fuses the ALK gene on 2p23 to the nucleophosmin (NPM) gene on 5q35, resulting in the constitutive activation of ALK kinase.
What proteins are associated with integrity and morphology of epithelial units?
E-cadherin and its undercoat proteins, the catenins (alpha, beta, and gamma), which are associated with the cytoplasmic domain of E-cadherin. Beta-catenin, in addition to being a cell adhesion molecule, functions as a downstream transcriptional activator of the Wnt signaling pathway. The level of beta-catenin is regulated by the adenomatous polyposis coli (APC) gene product, by cooperating with glycogen synthase kinase 3 beta (GSK3B) to phosphorylate multiple serine threonine residues coded by exon 3 of the beta-catenin gene. Loss or defective E-cadherin protein or protein degradation contributes to the invasive and metastatic properties of neoplastic cells. In addition, E-cadherin inactivation also occurs by activation of its repressors, such as Snail, Slug, Sip1, Twist, and Ets. Overexpression of Snail and Slug causes down-regulation of E-cadherin in EMT.
Overview of MEN syndromes, prevalence, and gene(s) involved.
MEN1 (eponym Wermer syndrome). MEN2 encompasses MEN 2A (eponym Sipple syndorome), MEN 2B (Gorlin syndrome; multiple other eponyms such as Gorlin-Vickers syndrome, Williams-Pollock syndrome, and Wagenmann-Froboese; also used to be called MEN3), and FMTC (Familial Medullary Thyroid Cancer, no eponym). For prevalences, MEN1 = 1 in 35,000; MEN 2A = 1 in 40,000; MEN 2B = 1 in 40,000. For gene mutations, MEN1 = MEN1 gene; MEN 2A = RET gene; MEN 2B = RET gene; FMTC = RET and NTRK1 genes. MEN1 is due to a variety of germline mutations of the MEN1 gene, which is located on 11q13. MEN2 is due to germline mutations in the RET proto-oncogene, which is located on 10q11.2.
Susceptibility to celiac disease is primarily associated with which human leukocyte antigen alleles?
Susceptibility to celiac disease is primarily associated with the HLA-DQ2 allele (~95% of patients with CD), and also associated with HLA-DQ8 (~5% of patients with CD).
What genetic abnormality do mesoblastic nephroma, infantile fibrosarcoma, and secretory carcinoma of the breast share?
t(12;15).
Alpha-1-antitrypsin deficiency genetics.
AAT deficiency is the most common genetic cause of liver disease in children and a major cause of hereditary liver dysfunction in adults, second only to hereditary hemochromatosis as the leading genetic cause of liver disease in adults. AAT is a glycoprotein produced predominantly in hepatocytes that functions as an inhibitor of serine proteases. AAT deficiency is caused by mutations in the SERPINA1 gene on chromosome 14q31-32.3 and is inherited in an autosomal recessive pattern. The genetic mutation results in a conformational abnormality (misfolding) of the AAT glycoprotein, abnormal retention of a polymerized form in the endoplasmic reticulum of hepatocytes, and low serum levels of AAT (approximately 15% normal). The normal allele is designated PiM and the two most common variant alleles are PiS and PiZ, resulting from single base pair mutations in exons III and V, respectively. The resultant amino acid substitutions in the AAT protein alter the net charge of the protein and allow detection of the normal type (MM), carrier types (MS, MZ) and deficiency variants (SS, SZ, ZZ) by isoelectric focusing (Pi (protease inhibitor) typing). Hepatic cirrhosis is most often associated with PiZZ type.
IDH and gliomas.
IDH1 is a mitochondrial protein that is mutated in most infiltrating gliomas: astrocytomas, oligodendrogliomas, and mixed oligoastrocytomas. In contrast, mutant IDH is rarely detected in other primary CNS tumors: neuronal, circumscribed gliomas, ependymomas, meningiomas, or systemic malignancies, with the exception of a subset of acute myeloid leukemias. Mutations can occur in IDH1 and IDH2; however, IDH1 mutations are more frequently observed. Approximately 70% to 80% of all oligodendrogliomas harbor IDH mutations. The most common IDH1 mutation involves codon 132, where arginine is replaced by histidine (R132H). A monoclonal antibody for the most-common mutant form of IDH1 (R132H) has been developed. This mutant-specific antibody has proven to be very helpful in the differential diagnosis of infiltrating gliomas from other primary CNS tumors and from reactive conditions that can mimic low-grade gliomas. Glial tumors harboring IDH mutations have a more favorable outcome, which may help stratify patient status and to tailor therapy.
Compare and contrast the two main methods of testing for defective mismatch repair function in Lynch syndrome.
IHC and PCR are the two main methods. IHC analysis of mismatch repair protein expression in tumor tissue (MMR-IHC) can reveal the identity of the defective protein for targeted genomic sequencing yet will “miss” a small percentage of cases with expressed but functionally defective MMR proteins. PCR analysis of a panel of microsatellite DNA sequences (MSI-PCR) demonstrates defective mismatch repair function. MSI-PCR, if abnormal, does not elucidate which gene is defective and will “miss” a small percentage of cases with a weak phenotype, as may be seen in MSH6 loss. It should also be noted that loss of MLH1 expression may be associated with non-heritable, epigenetic events, especially in older patients.
What antibody panel is typically used for mismatch repair detection for Lynch syndrome?
Most labs have an MMR-IHC panel consisting of antibodies to four MMR proteins: MLH1, MSH2, MSH6, and PMS2. For efficiency and cost savings, some advocate primary screening with only MSH6 and PMS2. Intact expression of MSH6 and PMS2 generally translates to intact expression of MLH1 and MSH2; aberrant expression of MSH6/PMS2 then requires further IHC characterization. This strategy is based on the endogenous pairing of mismatch repair proteins, and an understanding of pairing is also necessary to interpret staining results. MLH1 and PMS2 form a heterodimer; if MLH1 is lost, PMS2 is also destabilized and expression is undetectable. However, since PMS2 has other binding partners, mutation or loss of PMS2 does not affect expression of MLH1. The MSH2/MSH6 heterodimer functions similarly; an MSH2 defect leads to loss of both MSH2 and MSH6, while an MSH6 mutation shows loss of MSH6 expression and intact MSH2 expression.
For testing for Lynch syndrome, most labs have an MMR-IHC panel consisting of antibodies to four MMR proteins: MLH1, MSH2, MSH6, and PMS2. Why do some advocate primary screening with only MSH6 and PMS2?
For efficiency and cost savings, some advocate primary screening with only MSH6 and PMS2. Intact expression of MSH6 and PMS2 generally translates to intact expression of MLH1 and MSH2; aberrant expression of MSH6/PMS2 then requires further IHC characterization. This strategy is based on the endogenous pairing of mismatch repair proteins, and an understanding of pairing is also necessary to interpret staining results. MLH1 and PMS2 form a heterodimer; if MLH1 is lost, PMS2 is also destabilized and expression is undetectable. However, since PMS2 has other binding partners, mutation or loss of PMS2 does not affect expression of MLH1. The MSH2/MSH6 heterodimer functions similarly; an MSH2 defect leads to loss of both MSH2 and MSH6, while an MSH6 mutation shows loss of MSH6 expression and intact MSH2 expression.
How is the MMR-IHC panel for Lynch syndrome testing interpreted?
Since MMR proteins function in DNA repair, nuclear staining of replicating cells is expected normally. Epithelial cells at the base of normal colonic crypts are convenient internal controls, as are lymphocytes; in the uterus, endometrial stroma and myometrium may serve as internal control. MMR-IHC assays are particularly susceptible to under-fixation, especially MLH1 and PMS2. If internal controls are negative, repeat staining on another tissue block should be attempted; submission of additional tissue sections after longer fixation might also be helpful. MMR proteins may show patchy expression, especially MSH6 in post-chemotherapy specimens. Thus, even a low percentage of cells with nuclear staining should be scored as intact protein expression. In the case of MMR-IHC, positive staining is a normal result, while negative (lack of staining) is abnormal and suggests the need for further testing or genetic counseling. Other considerations in interpretation of IHC results include the fact that loss of expression of MLH1 may be due to a germline mutation in the MLH1 gene (Lynch syndrome), but is perhaps more commonly due to hypermethylation of the MLH1 promoter in older individuals. BRAF point mutations (V600E) are closely associated with this hypermethylation, and many testing algorithms advocate BRAF mutational analysis before embarking on genetic testing in patients whose colon cancers show loss of MLH1 expression.
What is the Vysis UroVysion test?
Vysis UroVysion is a molecular cytology test that detects aneuploidy of chromosomes 3, 7, and 17 and deletion of the 9p21 locus via fluorescence in situ hybridization (FISH) in urine specimens from persons with hematuria suspected of having bladder cancer.
__% of patients with NSCLC test positive for ALK by break-apart FISH probes; an additional __% may test positive by RT PCR due to ALK variants.
3-5% of patients with NSCLC test positive for ALK by break-apart FISH probes; an additional 1-2% may test positive by RT PCR due to ALK variants.
96% of low grade fibromyxoid sarcomas show a characteristic balanced translocation involving the __ gene on chromosome __ and the __ gene on chromosome __.
96% of low grade fibromyxoid sarcomas show a characteristic balanced translocation involving the FUS gene on chromosome 7 and the CREB3L2 gene on chromosome 16.
Almost all cases of mantle cell lymphoma have a balanced chromosomal translocation between (genes and chromosomal locations).
Almost all cases of mantle cell lymphoma have a balanced chromosomal translocation between IgH and CCND1 (cyclin D1), t(11;14)(q13;q32).
What is the classic morphologic appearance, immunophenotype, and cytogenetic abnormality seen in Burkitt lymphoma?
Morphologically, they consist of sheets of medium-sized transformed lymphocytes with minimal pleomorphism, multiple nucleoli, and numerous admixed tingible-body macrophages. Immunophenotypically, they are CD20-positive B-cells that co-express CD10, BCL6, and CD43. They are typically negative for BCL2. Ki-67 is nearly 100%. There are very few admixed T-cells. On cytogenetic studies, >90% of cases show a translocation involving the MYC gene on chromosome 8. Most cases have the MYC gene juxtaposed with the IGH gene on chromosome 14, while a minority involves the kappa (chromosome 2) and lambda (chromosome 22) light chain genes.
What are the PAX2 and PAX8 genes, what is their utility as immunostains?
PAX2 and PAX8 belong to the pair box gene family consisting of 9 members, PAX1 through PAX9, each of which encodes a transcription factor. These transcription factors are expressed in an orderly manner during fetal development. They play a critical role in the formation of tissues and organs during embryonic development and are also crucial for maintaining the normal function of certain cells after birth. Although these 9 transcription factors control the development of a wide range of organs, the roles of PAX2 and PAX8 in ontogenesis are distinctively similar. Both of them are known to control the development of the central nervous system, eye, kidney, thyroid gland, organs deriving from the mesonephric (wolffian) duct, and those related to the müllerian duct. Transcription factors are identified in the nuclei of the cell types that are under their developmental control during organogenesis, but they often disappear in mature tissue. These transcription factors, however, may reexpress in an organ-specific fashion during neoplastic transformation. For example, both PAX2 and PAX8 are abundantly expressed by renal blastemal cells during nephrogenesis, then are noted in only a few renal parenchymal cells in mature kidney, but are identified again in RCC. Tissue expression of transcription factor therefore has been used as a specific marker for tumor diagnosis.
Spinal muscular atrophy is a recessive disorder caused by loss of what genes?
Spinal muscular atrophy is a recessive disorder caused by loss of the survival motor neuron (SMN1 and SMN2) genes. Spinal muscular atrophy, types I, II, III, and IV, reflect increasing age of onset and decreasing disease severity.
Amyotrophic lateral sclerosis is a disease of motor neurons, linked to the accumulation of pathogenic proteins in the central nervous system, including __ and __. About __% of individuals with ALS have at least one other affected family member (familial ALS). Superoxide dismutase gene mutations occur in __% of patients with familial ALS and __% of sporadic cases.
Amyotrophic lateral sclerosis is a disease of motor neurons, linked to the accumulation of pathogenic proteins in the central nervous system, including TDP-43 and ubiquitin. About 10% of individuals with ALS have at least one other affected family member (familial ALS). Superoxide dismutase gene mutations occur in 20% of patients with familial ALS and 3% of sporadic cases.
Pulmonary hamartoma.
While a hamartoma is defined as disordered growth of tissues normally present within the organ, pulmonary hamartomas are clonal proliferations and therefore neoplastic. They contain chromosomal rearrangements of 12p15 and 6p21. Among the benign neoplasms of lung, which are overall rare, hamartomas are the exception. They can occur in the periphery or endobronchially and can vary in size, although they are usually less than 4 cm. Their distinct radiologic appearance (lobulated, ‘‘popcorn’’ calcification) can be diagnostic, therefore avoiding the need for resection. Pulmonary hamartomas contain at least 2 benign/mature mesenchymal tissues, one of which is often cartilage. Adipose tissue is also frequently present, especially in central lesions. The epithelium is thought to be entrapped reactive epithelium. In tumors with only 1 mesenchymal element, a corresponding benign mesenchymal neoplasm, such as a lipoma or chondroma, should be the main diagnostic consideration.
What two syndromes have Lynch syndrome-like findings (can be considered Lynch syndrome variants) and may also have germline mismatch repair gene mutations?
Muir-Torre syndrome (sebaceous neoplasms of the skin and internal malignancies typical of Lynch syndrome) and Turcot syndrome (central nervous system tumors and colorectal adenomas or cancer).
In what syndrome is the STK11 gene mutated?
Peutz-Jeghers syndrome. The STK11 gene is also called STK11/LKB1. STK11 = Serine-Threonine Kinase 11. LKB1 = Liver Kinase B1. It encodes a member of the serine/threonin kinase and regulates cell polarity and functions as a tumor suppressor.
STK11/LKB1 gene.
The STK11/LKB1 gene, also called just the STK11 gene, encodes a member of the serine/threonine kinase, and regulates cell polarity and functions as a tumor suppressor. It is located on chromosome 19p13.3. The protein kinase encoded by the gene is Serine/threonine kinase 11 (STK11), AKA liver kinase B1 (LKB1), AKA renal carcinoma antigen (NY-REN-19). Germline mutations in this gene have been associated with Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by the growth of polyps in the GI tract, pigmented macules on the skin and mouth, and other neoplasms. Recent studies have uncovered a large number of somatic mutations of the LKB1 gene that are present in lung, cervical, breast, intestinal, testicular, pancreatic and skin cancer.
What gene mutation is associated with fibrous dysplasia and McCune Albright syndrome?
Molecular findings in FD, and especially those for MAS, suggest it is caused by a somatic mutation early in embryonic life that causes a gene mosaicism. The earlier the mutation occurs, the more widespread the effects will be. The gene is located on 20q13, an area that codes for the alpha subunit on G-protein receptors. This mutation is also present in various endocrine tumors as well as FD. The G-proteins begin a cascade that ultimately leads to activation of the enzyme adenylyl cyclase that produces cAMP. In MAS, there is a missense mutation that causes the substitution of arginine in position 201 of the Gs-alpha gene. Normally, there is an almost immediate deactivation of adenylyl cyclase and a breakdown of the cAMP. In MAS, that does not occur. Overproduction of cAMP leads to increased amounts of activity that affect each tissue differently based on its designated function.
Whereas autosomal-dominant hyperimmunoglobulin E syndrome is caused by mutations in (gene), (gene) and (gene) mutations are implicated in the comparatively rare autosomal-recessive forms of the disease.
Whereas autosomal-dominant hyperimmunoglobulin E syndrome is caused by mutations in STAT3, DOCK8 and TYK2 mutations are implicated in the comparatively rare autosomal-recessive forms of the disease.
What is pyrosequencing?
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides. The technique was developed in Stockholm in 1996. The desired DNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide by the fact that only one out of four of the possible A/T/C/G nucleotides are added and available at a time so that only one letter can be incorporated on the single stranded template (which is the sequence to be determined). The intensity of the light determines if there are more than one of these “letters” in a row. The previous nucleotide letter (one out of four possible dNTP) is degraded before the next nucleotide letter is added for synthesis: allowing for the possible revealing of the next nucleotide(s) via the resulting intensity of light (if the nucleotide added was the next complementary letter in the sequence). This process is repeated with each of the four letters until the DNA sequence of the single stranded template is determined.
What are pros and cons of pyrosequencing compared to Sanger sequencing?
First, compared to Sanger sequencing, pyrosequencing has has higher sensitivity. Sanger sequencing needs greater than 20 % of tumor load in a specimen to render a reliable result, while pyrosequencing can render a reliable result with 5 % tumor load. Second, pyrosequencing is faster than Sanger sequencing. Third, pyrosequencing is more cost effective. One of the disadvantages of pyrosequencing is that it can only sequence a short length of nucleotide sequence. Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain termination methods (e.g. Sanger sequencing). This can make the process of genome assembly more difficult, particularly for sequences containing a large amount of repetitive DNA. As of 2007, pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available. The other disadvantage is that pyrosequencing data analysis sometimes can be complex and challenging, and some date analysis is done by a manual process.
Fabry disease. Other names.
Fabry disease (also known as Fabry’s disease, Anderson-Fabry disease, angiokeratoma corporis diffusum, and alpha-galactosidase A deficiency) is a rare X-linked lysosomal, which can cause a wide range of systemic symptoms. It is a form of sphingolipidosis, as it involves dysfunctional metabolism of sphingolipids. The disease is named after one of its discoverers, Johannes Fabry (June 1, 1860–June 29, 1930).
Lymphangioleiomyomatosis. Locations? Association with a syndrome?
Lymphangioleiomyomatosis (or lymphangiomyomatosis) (LAM) occurs almost exclusively in reproductive age women and consists of a proliferation of immature myoid cells which are currently thought to derive from perivascular epitheliod cells (PEC). LAM may involve the lungs and axial thoraco-abdominal lymphatic system including both lymph nodes and the thoracic duct. Supraclavicular and inguinal nodes may rarely be involved. In most cases, the pulmonary manifestations dominate but LAM may occasionally present exclusively in the abdomen. Abdominal LAM may mimic ovarian carcinoma radiographically and patients may present with pain, often due to hemorrhage. LAM may occur in association with tuberous sclerosis (TS) or in isolation (sporadic LAM), although in either form the LAM cells may be associated with mutations of the TS genes, TSC-1 or TSC-2. Whether LAM is sporadic or associated with TS, the histologic appearance is the same but lymph node involvement is more frequent in patients without TS.
Synovial sarcoma is a spindle cell tumor arising in soft tissue which displays variable epithelial differentiation including glandular formation. It also has a specific genetic alteration, a chromosomal translocation ___, that results in a fusion between ___ and ___.
Synovial sarcoma is a spindle cell tumor arising in soft tissue which displays variable epithelial differentiation including glandular formation. It also has a specific genetic alteration, a chromosomal translocation t(X;18), that results in a fusion between the SS18 (SYT) gene on chromosome 18 and one of the SSX genes on the X chromosome, creating SS18-SSX1, SS18-SSX2, or SS18-SSX4 chimeric genes.
Synovial sarcoma and TLE-1.
TLE1 is one of four transducin-like enhancer of split (TLE) genes that encode human transcriptional repressors. It is an important protein in the Wnt/Beta-catenin pathway, a signaling pathway that is strongly associated with SS. Strong nuclear TLE1 expression is a robust IHC biomarker for SS, particularly in those cases that do not exhibit biphasic histology, and helps distinguish this tumor from other spindle cell tumors. In an appropriate clinical background and morphology, strong nuclear TLE1 expression in a CD34-negative spindle cell tumor containing scattered CK-positive cells can be regarded as diagnostic of SS.
Clear cell sarcoma overview.
Clear cell sarcoma of soft tissue is a rare tumor that affects the extremities in young adults and shows melanocytic differentiation. This tumor is usually associated with tendons or aponeuroses and presents as a slow growing mass. Microscopically, the tumor grows in a uniform, nested to fascicular pattern with thin fibrous septa. Tumor cells are polygonal to spindle-shaped with abundant clear or eosinophilic cytoplasm and prominent nucleoli. Multinucleated giant cells are present in half of cases, and intracellular melanin can occasionally be seen. Clear cell sarcoma is positive for S100 protein, HMB45, and other melanocytic markers and shows a reciprocal translocation, t(12;22)(q13;q12), that results in EWS/ATF1 gene fusion.
Ki-67 protein and antibodies against it.
Antigen KI-67, also known as Ki-67 or MKI67 is a protein that in humans is encoded by the MKI67 gene. It is a nuclear protein that is associated with and may be necessary for cellular proliferation. Furthermore it is associated with ribosomal RNA transcription. Inactivation of antigen KI-67 leads to inhibition of ribosomal RNA synthesis. The Ki-67 protein (also known as MKI67) is a cellular marker for proliferation. It is strictly associated with cell proliferation. During interphase, the Ki-67 antigen can be exclusively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0). Ki-67 is an excellent marker to determine the growth fraction of a given cell population. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of cancer. Ki67 and MIB-1 monoclonal antibodies are directed against different epitopes of the same proliferation-related antigen. The Ki-67 protein was originally defined by the prototype monoclonal antibody Ki-67; The name is derived from the city of origin (Kiel, Germany) and the number of the original clone in the 96-well plate.Whereas Ki67 works only on frozen sections, MIB1 may be used also on fixed sections. MIB-1 is used in clinical applications to determine the Ki-67 labeling index. One of its primary advantages over the original Ki-67 antibody (and the reason why it has essentially supplanted the original antibody for clinical use) is that it can be used on formalin-fixed paraffin-embedded sections, after heat-mediated antigen retrieval.
How are podoplanin and D2-40 different?
Basically, podoplanin is the protein, and D2-40 is the antibody against that protein. Podoplanin is a mucin-type transmembrane glycoprotein that is expressed in lymphatic endothelial cells (and several other cell types). The mouse monoclonal antibody D2-40 was originally raised against an oncofetal antigen, M2A antigen, which is an O-linked sialoglycoprotein with a simple mucin-type carbohydrate epitope associated with germ cell neoplasms, but then this antibody clone was found to be reactive with other antigens/cell types as well. D2-40: = anti-podoplanin.
What CYP2C9 allele combinations are seen in normal/extensitve metabolizers, intermediate metabolizers, poor/hypo-metabolizers, and ultra/hypermetabolizers of clopidogrel (Plavix)?
Normal/extensitve metabolizers: 11. Intermediate metabolizers: 12. Poor/hypo-metabolizers: 22. Ultra/hypermetabolizers: 117 or 1717. Also, 217 may be considered indeterminate.
Clopidogrel (Plavix), prasugrel (Effient), ticagrelor (Brilinta). Which are prodrugs that are converted to active form in the liver?
Clopidogrel is converted by CYP2C9 to active form in the liver, and prasugrel is converted by CYP3A4 and CYP2B6 to active form in the liver. Ticagrelor is already in active form.
Solid-pseudopapillary neoplasm/solid-pseudopapillary tumor of pancreas. Gender prevalence and mean age. Common mutation seen in this neoplasm. Cell of origin. Current grading and staging systems.
SPNs occur mostly in females (~90%) with a mean age of presentation of 28-35. In males, the mean age at presentation tends to be 5-10 years older than that of females. This neoplasm has been shown to consistently harbor mutations in exon 3 of the beta-catenin gene, which leads to upregulation of known oncogenes such as cyclin D1. The cell of origin for SPN is unknown. Currently, no grading or staging schemes for SPN exist; if malignant features, such as invasion into adjacent structures, metastases, or malignant histology are present, the staging of these “solid pseudopapillary carcinomas” follows that of other exocrine pancreatic carcinomas.
Stains for solid-pseudopapillary neoplasm of the pancreas.
SPN stains positive for alpha-1 antitrypsin, PR, vimentin, alpha-1 antichymotrypsin, CD10, CD56, NSE, beta-catenin and cyclin D1. Alpha-1 antitrypsin will generally also stain the intracytoplasmic hyaline globules that are also typically PAS-positive. Vimentin and NSE stain in a diffuse manner. PR staining is present in both male and female patients. Nuclear staining of beta-catenin is seen due to mutations in the beta-catenin gene which leads to unusual nuclear accumulation of beta-catenin, and since beta-catenin mutations lead to cyclin D1 upregulation, cyclin D1 is usually positive in SPN. SPNs stain variably for cytokeratins and synaptophysin. Only in rare instances are positive staining results seen for S100, pancreatic enzymes (trypsin, amylase, chymotrypsin, etc.), or pancreatic hormones (insulin, glucagon, and somatostatin). Most report chromogranin to be negative in SPN. SPNs characteristically lose E-cadherin expression. CD99 has a characteristic paranuclear dotlike pattern.
The principal differential diagnosis of mesenchymal dysplasia, both clinically and pathologically, is partial hydatidiform mole due to the similar cystic changes and the admixture of normal and abnormal villi. How can they be distinguished?
Unlike partial hydatidiform mole, mesenchymal dysplasia shows no trophoblast hyperplasia, stromal trophoblastic inclusions, or scalloping of the villous surface. The fetus associated with partial molar pregnancy is often small and malformed, unlike the more normal fetus associated with mesenchymal dysplasia. DNA ploidy may also help in distinguishing these entities, as partial hydatidiform molar pregnancy is typically triploid, whereas mesenchymal dysplasia is typically diploid.
Overview of myxoid liposarcoma with a round cell component (myxoid/round cell liposarcoma).
Myxoid liposarcoma and round cell liposarcoma were historically considered separate subtypes of liposarcoma. The frequent transition areas between myxoid liposarcoma and round cell areas in the same tumor, along with the shared t(12;16)(q13;p11) chromosomal translocation, support a single classification as myxoid/round cell liposarcoma. The translocation results in fusion of the FUS (TLS) and CHOP (DDIT3) genes on chromosomes 16 and 12, respectively. Myxoid/round cell liposarcoma accounts for approximately 30–35% of liposarcomas. These tumors occur almost exclusively in adults, usually in the third to eighth decade with a male preponderance (male: female ratio of 3:1). Myxoid/round cell liposarcoma is somewhat unusual among soft tissue tumors in that it often metastasizes to unusual sites such as the soft tissue of the trunk or axilla before metastasizing to the lung.
Ancillary studies for myxoid/round cell liposarcoma.
In small biopsy specimens or cases with few, if any, lipoblasts, the detection of the t(12;16) translocation may be extremely helpful in the differential diagnosis. Fresh tissue placed in RPMI may be used for complete karyotyping. Fluorescence in situ hybridization (FISH) for chromosome 12 (CHOP (DDIT3), 12q13) and reverse transcriptase polymerase chain reaction (RT-PCR) for the FUS-CHOP (TLS/DDIT3) fusion transcripts may be performed on formalin-fixed paraffin-embedded tissue. Immunohistochemistry is of limited utility.
Lynch syndrome is the most common hereditary CRC syndrome. What are the 4 genes/proteins involved and what chromosomes are they on?
The four genes that are involved in encoding proteins that participate in DNA mismatch repair are: MLH1 (3p21), MSH2 (2p22-p21), MSH6 (2p16), and PMS2 (7p22). Tumors in patients with Lynch syndrome have a germline mutation of one allele of a DNA MMR gene and a somatic mutation in the other allele. The presence of two inactivating mutations results in defective DNA mismatch repair. If the MSI or DNA MMR IHC results show evidence of defective DNA MMR, then the patient may be evaluated further for Lynch syndrome by assessing a peripheral blood sample for germline mutations for one of the four DNA MMR genes.
What is the significance of the five common staining combinations seen in DNA mismatch repair protein IHC (MLH1, PMS2, MSH2, MSH6) in CRC? Give the MSI phenotype, clinical interpretation, and etiology.
Pattern #1: MLH1+, PMS2+, MSH2+, MSH6+; MSI phenotype is MSS or MSI-L; clinical interpretation is most likely sporadic CRC (Lynch syndrome very unlikely). Pattern #2: MLH1-, PMS2-, MSH2+, MSH6+; MSI phenotype is MSI-H; clinical interpretation is LS or sporadic CRC; etiology is germline hMLH1 mutation or hMLH1 promoter hypermethylation. Pattern #3: MLH1+, PMS2+, MSH2-, MSH6-; MSI phenotype is MSI-H; clinical interpretation is LS; etiology is germline hMSH2 mutation. Pattern #4: MLH1+, PMS2+, MSH2+, MSH6-; MSI phenotype is MSI-H; clinical interpretation is LS; etiology is germline hMSH6 mutation. Pattern #5: MLH1+, PMS2-, MSH2+, MSH6+; MSI phenotype is MSI-H; clinical interpretation is LS; etiology is germline PMS2 mutation.
Somatic BRAF mutations are (often seen/generally not seen) in tumors from those with Lynch syndrome.
Somatic BRAF mutations are generally not seen in tumors from those with Lynch syndrome. In patients with a BRAF mutation, LS can be ruled out in most cases.
What FISH and IHC studies are useful in mantle cell lymphoma?
FISH studies for cyclin D1 are very important in making a definitive diagnosis of MCL. Translocation t(11;14)(q13;q32) is the chromosomal rearrangement juxtaposing the cyclin D1 gene locus (CCND1) on chromosome 11q13 with the immunoglobulin heavy chain locus (IGH) located on chromosome 14q32, placing CCND1 under control of IGH enhancer sequences, and leading to over-expression of cyclin D1 protein. In addition to cyclin D1 immunoreactivity, MCL tumor cells are positive for pan-B-cell markers, CD19 and CD20. Surface immunoglobulin and IgM are also positive, with or without associated IgD positivity. Tumor cells also show CD5 immunoreactivity, and are negative or only weakly positive for CD23, a marker helpful in distinguishing between chronic lymphocytic leukemia/small lymphocytic lymphoma, which is typically positive for CD23.
Cytogenetic and IHC characteristics of hibernoma?
Cytogenetically, hibernomas are often associated with rearrangements of chromosomal bands 11q13-21; however, other benign lipomatous tumors may also display such abnormalities. By IHC, hibernomas are variably positive for S100 protein and spindle cell components are positive for CD34.
What are the 4 major molecular subtypes of breast cancer?
Luminal A, luminal B, triple negative/basal-like, and HER2 type. Other less common molecular subtypes have also been described, including normal breast-like, apocrine molecular subtype, luminal ER-/AR+, and claudin-low type. Luminal A subtype (40-60% prevalence) is ER+ and/or PR+, HER2-, low Ki67. Luminal B subtype (10-20% prevalence) is ER+ and/or PR+, HER2+ (or HER2- with high Ki67). Triple negative/basal-like subtype (10-20% prevalence) is ER-, PR-, HER2-, CK5/6+ and/or HER1+. HER2 type (10-15% prevalence) is ER-, PR-, HER2+.
What is the most common chromosomal anomaly in CLL/SLL?
Deletion of 13q14 (>50% of cases). Other frequent findings are trisomy 12 (15-20%), del(11q), del(14q), and del(17p).
A subset of mantle cell lymphoma (MCL) with a tendency for peripheral blood involvement tend to have cytogenetic aberrations involving __, __, and __ (in addition to the requisite t(11;14)).
A subset of mantle cell lymphoma (MCL) with a tendency for peripheral blood involvement tend to have cytogenetic aberrations involving 8, 17, and 21 (in addition to the requisite t(11;14)).
What are the myc genes?
They are a family of retrovirus-associated DNA sequences (myc) originally isolated from an avian myelocytomatosis virus (myc for MYeloCytomatosis). The proto-oncogene myc (c-myc) codes for a nuclear protein which is involved in nucleic acid metabolism and in mediating the cellular response to growth factors. Truncation of the first exon, which appears to regulate c-myc expression, is crucial for tumorigenicity. The human c-myc gene is located at 8q24.
Amplification of MDM2 region in 12q15 and CHOP/DDIT3 region in 12q13 is associated with what sarcoma?
Liposarcoma. Mouse Double Minute 2 homolog (MDM2) is a protein that is a negative regulator of p53 encoded by the MDM2 gene. DNA Damage Inducible Transcript 3, AKA C/EBP homologous protein (CHOP), is a transcription factor encoded by the DDIT3 gene.
Approximately 90% of Wilms tumors are sporadic, while a minority are associated with syndromes and/or mutations in the tumor suppressor genes ___ or ___.
Approximately 90% of Wilms tumors are sporadic, while a minority are associated with syndromes and/or mutations in the tumor suppressor genes WT1 (chromosome 11p13) or WT2 (chromosome 11p15).
What translocation is seen in myxoid/round cell liposarcoma?
A t(12;16)(q13;p11) chromosomal translocation, resulting in fusion of the FUS (TLS) and CHOP (DDIT3) genes on chromosomes 16 and 12, respectively.
Myxoid/round cell liposarcoma overview.
Myxoid liposarcoma and round cell liposarcoma were historically considered separate subtypes of liposarcoma. The frequent transition areas between myxoid liposarcoma and round cell areas in the same tumor, along with the shared t(12;16)(q13;p11) chromosomal translocation, support a single classification as myxoid/round cell liposarcoma. The translocation results in fusion of the FUS (TLS) and CHOP (DDIT3) genes on chromosomes 16 and 12, respectively. Myxoid/round cell liposarcoma is the most common subtype of liposarcoma, accounting for approximately 30–35% of liposarcomas. These tumors occur almost exclusively in adults, usually in the third to eighth decade with a male preponderance (male: female ratio of 3:1). Myxoid/round cell liposarcoma is somewhat unusual among soft tissue tumors in that it often metastasizes to unusual sites such as the soft tissue of the trunk or axilla before metastasizing to the lung. Lymph node metastases are also more common in myxoid liposarcoma than many other soft tissue malignancies.
What are some nonmolecular methods and molecular methods for detecting M. tuberculosis complex (with or without the simultaneous detection of genes conferring drug resistance)?
Nonmolecular methods: Microscopic observation direct susceptibility assay (MODS assay). Light-emitting diode microscopy. MDR-XDRTB Colour Test. Colorimetric assays. Phage amplification assays. Molecular methods: Line probe assays. Automated nucleic acid amplification tests. Loop-mediated isothermal amplification. Oligonucleotide microarray.
___ deletions are detected in ~70% of atypical teratoid/rhabdoid tumors; however, loss of the corresponding ___ protein is even more common than the genetic alteration. Loss of ___ nuclear immunoreactivity in tumor cells is used as a surrogate for genetic testing to demonstrate biallelic inactivation of the gene.
INI1/BAF47 deletions are detected in ~70% of atypical teratoid/rhabdoid tumors; however, loss of the corresponding INI1 protein is even more common than the genetic alteration. Loss of INI1 nuclear immunoreactivity in tumor cells is used as a surrogate for genetic testing to demonstrate biallelic inactivation of the gene.
Isocitrate dehydrogenase (IDH1/IDH2) mutations have been detected in the majority of diffuse gliomas, with the notable exception of ___, and appear to play a fundamental and early role in oncogenesis. The most common mutation is in the IDH1 gene (R132H), and is recognized by monoclonal antibody IDH-1. Diagnostically, the IHC stain shows greatest promise for its potential to distinguish low-grade diffuse glioma from gliosis. Prognostically, the presence of this mutation is favorable, as such tumors appear to show greater response to therapy.
Isocitrate dehydrogenase (IDH1/IDH2) mutations have been detected in the majority of diffuse gliomas, with the notable exception of primary (de novo) GBM, and appear to play a fundamental and early role in oncogenesis. The most common mutation is in the IDH1 gene (R132H), and is recognized by monoclonal antibody IDH-1. Diagnostically, the IHC stain shows greatest promise for its potential to distinguish low-grade diffuse glioma from gliosis. Prognostically, the presence of this mutation is favorable, as such tumors appear to show greater response to therapy.
Chronic myelogenous leukemia. What ancillary studies are done for blood and marrow.
FOR BLOOD: LAP (NAP) score low. B12 increased. FOR MARROW: Cytogenetic analysis: t(9;22) or variant (more than 95% of cases). Molecular: BCR-ABL1+ by FISH or PCR (100% of cases). Most cases: major BCR-ABL1, (e13 or14/a2 or 3), p210 protein. Rare cases: minor BCR-ABL1, (e1/a2 or 3), p190 protein. Rare cases: mu BCR-ABL1, (e19/a2 or 3), p230 protein.
What is cherubism?
Cherubism is an inherited disease characterized by progressive, painless, symmetrical expansion of the jaws, resulting in a cherubic facial appearance. M:F = 2:1, with 100% penetrance in males and 50-70% penetrance in females. Mutations in the SH3BP2 gene have been identified in about 80 percent of people with cherubism. In most of the remaining cases, the genetic cause of the condition is unknown. There are germline point mutations in the SH3BP2 gene on chromosome 4p16.3.
What is the genetic mutation in fibrous dysplasia?
Postzygotic, somatic, activating missense mutations in GNAS1 gene coding for alpha subunit of stimulatory G protein are a consistent finding (~3/4 of cases). The GNAS1 gene is on chromosome 20q13.2-13.3. The abnormal G1 protein stimulates cAMP, and the osteoblastic cells expressing this mutation have a higher rate of DNA synthesis than normal cells. This abnormal growth leads to the formation of a disorganized fibrotic bone matrix with primitive bone formation, and lack of maturation to lamellar bone. Mineralization is also abnormal. There is a failure of the bone to align in response to mechanical stress. The extent of disease is related to the stage at which the postzygotic mutation in Gsα has occurred, whether during embryonic development or postnatally.
Postzygotic, somatic, activating missense mutations in GNAS1 gene coding for alpha subunit of stimulatory G protein are a consistent finding (~3/4 of cases) in what condition?
Fibrous dysplasia.
Molecular genetics of ameloblastoma.
ING family of tumor suppressor gene has high frequency loss of heterozygosity (ING5 locus LOH correlated to solid tumor type). Notch signaling molecules may be associated with specific tumor phenotypes. Dysregulation of a number of genes involved in normal tooth development may play a role: FOS oncogene most overexpressed gene; underexpressed genes include SHH, TRAF3, DCC, CDH12, TDGF1, TGFB1.
The genes PRSS-1, PST1, and CFTR have all been implicated as causes of recurrent ___.
The genes PRSS-1 (cationic trypsinogen), PSTI (pancreatic secretory trypsin inhibitor), and CFTR (cystic fibrosis transmembrane conductance regulator) have all been implicated as causes of recurrent pancreatitis.
Keratocystic odontogenic tumor. Microscopic appearance. DDx.
AKA odontogenic keratocyst AKA odontogenic keratocystoma AKA primordial cyst. Micro: Epithelial lining (6-8 cells thick; lacks rete ridges and epithelium is often detached from fibrous wall, creating cleft; basal layer shows palisading and hyperchromicity; parakeratotic epithelial cells; wavy or corrugated surface keratinization; keratineceous debris in lumen) and fibrous connective tissue (may be detached from overlying epithelium). When inflamed, epithelium is altered and rete ridges are noted. Epithelial hyaline bodies (Rushton bodies) may be seen. By IHC, high Ki-67 confirms proliferation, and TP53 is overexpressed. By molecular genetics, there are mutations in the PTCH gene (chromosome 9q22.3-q31), a tumor suppressor gene, with loss of function. The mutation results in overexpression of BCL-1 and TP53. DDx: Orthokeratinized odontogenic cyst. Dentigerous cyst. Periapical cyst.
Multiple odontogenic keratocysts are associated with ___ syndrome.
Multiple odontogenic keratocysts are associated with Gorlin syndrome (nevoid basal cell carcinoma syndrome). Mutations in PTCH gene (chromosome 9q22.3-q31), a tumor suppressor gene, with loss of function.
Cytogenetics of pleomorphic adenoma.
4 major cytogenetic abnormalities, with rearrangements involving: 8q12 (39%), 12q13-15 (8%), sporadic clonal rearrangements of other genes (23%), normal karyotype (30%). Currently identified 5 PLAG1 (8q12) and HMGA2 (12q13-15) -containing fusion genes are tumor specific.
Warthin tumor. Micro. IHC. Cytogenetics. DDx.
Micro: Papillary and cystic lesion composed of epithelial and lymphoid components. Epithelial component lining the papillary projections composed of double layer of granular eosinophilic cells (referred to as oncocytic epithelia). Inner or luminal cells: Nonciliated, tall columnar cells with nuclei aligned toward luminal aspect. Outer or basal cells: Round, cuboidal, or polygonal cells with vesicular nuclei. Lymphoid component predominantly composed of mature lymphocytes containing lymphoid follicles with germinal centers. Epithelioid and lymphoid components are sharply demarcated from one another. Other inflammatory cells may be seen, including plasma cells, histiocytes, mast cells, and occasional multinucleated (Langhans-type) giant cells. Lumens of cysts may contain thick secretions, cholesterol crystals, cellular debris, or corpora amylacea-like laminated bodies. Squamous metaplasia and focal necrosis may be seen in association with secondary inflammation. Due to presence of oncocytic cells, WT is subject to degenerative alterations occuring spontaneously or following manipulation. Metaplastic or infarcted variant of WT accounts for
Warthin tumor and many mucoepidermoid carcinomas have a similar translocation and fusion transcript, suggesting evidence for a common genetic association. What is it?
t(11;19) translocation and CRTC1 (AKA MECT1, TORC1, or WAMTP1)/MAML2 fusion transcript.
Mucoepidermoid carcinoma of salivary gland. Micro. IHC. Cytogenetics.
Cystic spaces: often filled with mucin, occ papillary projections present. Epidermoid cells: nests or scattered; polygonal cells. Intermediate cells: large polygonal epidermoid cells; small basal cells; often in nests or sheets. Mucous cells: intracytoplasmic mucin; large or ovoid; vacuolated or clear cytoplasm; in groups or individual cells. Clear cells: usually
Adenoid cystic carcinoma in salivary gland. Micro. HC and IHC. DDx. Cytogenetics.
Micro: Infiltrative (PNI commonly seen; infiltrative edges into fat, skeletal muscle, soft tissue). Combinations of patterns usually present, although one predominates. Patterns: cribriform, tubular, and solid. Cytologic features: small to medium cells with eosinophilic to clear cytoplasm; nuclei are oval to sharply angulated with coarse, basophilic chromatin and occasional small nucleoli; mitotic figures are rare except for in the solid pattern. HC: Alcian blue and PAS highlight basement membrane material of pseudolumina. IHC: May have limited practical use, as tumors in DDx often react similarly. DDx: PLGA, PA, basal cell adenocarcinoma, basal cell adenoma, epithelial-myoepithelial carcinoma, CExPA, basaloid SCC, cylindroma, sialoblastoma, NEC. Cytogenetics: No particular gene profile identified. 30% have translocations involving 6q and 9p (MYB-NFIB translocation). ~50% have loss of chromosome 12q12. LOH at 6q23-25 is associated with poorer prognosis. Alteration of p53 is associated wtih tumor recurrence and progression to solid type.
Acinic cell carcinoma in salivary gland. Micro. HC. IHC. Cytogenetics.
Micro: Tumor extension into normal tissue is common, although “apparent” encapsulation is present. Although one pattern and cell type dominate, combination and spectrum is common. Patterns: solid/lobular > microcystic > papillary-cystic > follicular. Several cell types are present: serous acinar cells > intercalated duct type cells > vacuolated cells > nonspecific glandular cells > clear cells. Lymphoid infiltrate, sometimes prominent with germinal center formation, can be seen. Stromal fibrosis or desmoplasia is uncommon. HG transformation (dedifferentiation) into HG carcinoma (including small cell carcinoma) is rare and heralds poor prognosis. HC: PAS+, diastase-resistant zymogen granules (reaction can be patchy and limited). Neg or only focally pos granules with mucicarmine. IHC: Immunoprofile is nonspecific and unpredictable, so seldom of diagnostic value. Cytogenetics: No consistent or specific structural chromosomal alterations. DDx: normal salivary gland, papillary cystadenocarcinoma, mucoepidermoid carcinoma, metastatic thyroid carcinoma, clear cell tumors (epithelial-myoepithelial CA, clear cell adenoCA, clear cell oncocytoma, met RCC), PLGA.
Polymorphous low-grade adenocarcinoma. Micro. IHC. Cytogenetics.
Micro: Infiltrative growth, architectural diversity, and cytologic uniformity, set in characteristic matrix. Infiltrative growth: Unencapsulated, but usually well circumscribed. Encased, entombed, incarcerated, or completely surrounded minor salivary glands. Invades into soft tissues, especially fat. Bone invasion can be seen. Significant PNI. Patterns of growth: Striking variety of growth patterns. Low power gives “eye of the storm,” “streaming” or “whorled” appearance. Characteristic concentric layering of cells around central nidus, creating targetoid tableau. Periphery often shows linear, single-file cell infiltration. Arranged in lobules, theques, glandular profiles, tubules, trabeculae, and cribriform nests. Papillae, if identified, are focal and not dominant pattern. Cellular features: Uniformly bland round to polygonal or fusiform tumor cells. Small to medium, with indistinct cellular borders. Ample pale to eosinophilic cytoplasm. Round to oval nuclei with open, vesicular nuclear chromatin. Inconspicuous to small nucleoli. Matrix material: Slate gray-blue stroma usually only focal. Hyalinized, slightly eosinophilic stroma separates cells. Inconspicuous mitoses. Tyrosine-like crystals are identified in <5%. Rare metaplastic changes: squamous, sebaceous, mucous, clear, oncocytic. IHC: Variable expression of epithelial and myoepithelial markers. Cytogenetics: Chromosome 12 abnormalities are most common: p or q arms (12q22 and 12p12.3).
Salivary duct carcinoma. Micro. HC. IHC. Cytogenetics.
Micro: Variably sized, rounded, solid or cystic nodules of tumor cells that resemble intraductal or infiltrating ductal carcinoma of the breast. Small nodules are filled with neoplastic cells. Larger cystic nodules with irregular shape. Comedonecrosis is conspicuous. PNI and LVI frequent. Marked, dense, desmoplastic (hyalinized) fibrosis is conspicuous. Lymphoplasmacytic inflammator cell infiltrate is frequently present. Cells are arranged in cribriform, band-like solid, and papillary patterns. Small tumor nests infiltrate between larger nodules. Epithelial cells have moderate to marked pleomorphism, are cuboidal to polygonal, with ample eosinophilic granular, oncocytic cytoplasm. Nuclei are round, centrally located, with large, prominent nucleoli and hyperchromatic chromatin. Mitotic figures, including atypical forms, are usually easily identified. Uncommonly, psammoma bodies and areas of squamous differentiation seen. Variants: sarcomatoid, micropapillary, mucin-rich, osteoclast-type giant cell, low-grade SDC (controversial entity). HC: Nonreactive with mucicarmine and Alcian blue. IHC: Pos for epithelial markers, androgen receptor, HER-2/neu. Cytogenetics: Frequent LOH involving 9p21, 6q, 16q, 17p, and 17q regions.
What is IMP3 gene and protein?
IMP3 protein is a member of the insulin-like growth factor II mRNA-binding proteins that consist of IMP1, IMP2, and IMP3. The IMP3 gene is located on band 7p11.2 and is identical to the KH domain–containing protein overexpressed in cancer (KOC) protein. IMP3 is an oncofetal protein involved in embryogenesis. Recent studies have shown that IMP3 is an important cancer-specific gene that is associated with many aggressive and advanced cancers and is specifically expressed in malignant tumors but is not found in benign tissues. Moreover, IMP3 promotes tumor cell proliferation, adhesion, invasion, and metastasis. IMP3 is an important prognostic biomarker for localized RCC (predicts metastasis and prognosis) and superficial urothelial carcinoma (predicts aggressive superficial UCs).
Xp11.2 translocation renal cell carcinoma is a subtype of renal cancer defined by a breakpoint at Xp11.2 with gene fusions between the transcription factor E3 (TFE3) gene and 6 other genes. The most common fusion partners are ___.
Xp11.2 translocation renal cell carcinoma is a subtype of renal cancer defined by a breakpoint at Xp11.2 with gene fusions between the transcription factor E3 (TFE3) gene and 6 other genes. The most common fusion partners are the papillary renal cell carcinoma (PRCC, 1q21) gene and alveolar soft part sarcoma chromosome region, candidate 1 (ASPSCR1, 17q25) gene. The same ASPSCR1-TFE3 gene fusion is seen in alveolar soft part sarcoma, a rare pediatric tumor. Another renal translocation carcinoma exhibits the t(6;11)(p21;q12) translocation with a gene fusion between the alpha gene and the transcription factor EB (TFEB) gene. Both TFEB and TFE3 are members of the MiTF/TFE family of transcription factors.
What are the FDA-approved criteria for a positive UroVysion FISH test result?
UroVysion is a multitarget multicolor FISH assay that examines 4 chromosomal abnormalities that commonly occur in UC. The assay is done on exfoliated urothelial cells using centromeric fluorescent denatured chromosome enumeration probes for chromosomes 3, 7, and 17, as well as a locus-specific identifier probe for 9p21. Normal cells are diploid or possess 2 copies of each chromosome. The types of genetic abnormalities observed by FISH include gains (3 or more copies) of one or more chromosomes, monosomy (1 copy), or deletions (no copies). The abnormalities that have been found to be associated with UC are polysomy (including tetrasomy), trisomy, and 9p21 deletion. The FDA-approved criteria for a positive FISH result are (1) 4 or more cells with polysomy (3 or more copies of 2 or more chromosomes), or (2) 9p21 deletion in 12 or more cells. A minimum of 25 morphologically abnormal cells need to be scanned.
Direct detection of the mutations of thyroid cancer is feasible because only about 7 genes are mutated in the vast majority of thyroid cancers. The mutations in these genes are almost always mutually exclusive. The various mutations correlate closely with tumor type: Mutations in ___, ___ and ___ are restricted to papillary thyroid carcinomas, ___ is strongly associated with the follicular variant of papillary thyroid carcinoma, ___ is largely restricted to follicular neoplasms, and ___ and ___ (less common) are more likely in follicular neoplasms than papillary thyroid carcinomas.
Direct detection of the mutations of thyroid cancer is feasible because only about 7 genes are mutated in the vast majority of thyroid cancers. The mutations in these genes are almost always mutually exclusive. The various mutations correlate closely with tumor type: Mutations in B-RAF, RET and TRK are restricted to papillary thyroid carcinomas, N-RAS is strongly associated with the follicular variant of papillary thyroid carcinoma, PAX8/PPARgamma is largely restricted to follicular neoplasms, and H-RAS and K-RAS (less common) are more likely in follicular neoplasms than papillary thyroid carcinomas.
What are issues and problems with mutation testing of thyroid cancers?
Though only 7 genes appear involved in initiation of the vast majority of thyroid cancers of follicular origin, testing is complicated by the presence of multiple potential point mutations in RAS and B-RAF genes, and many different translocations of RET and TRK (at least 11 and 5, respectively). Testing for point mutations (eg, B-RAF, N-RAS, H-RAS, and K-RAS) requires only a small amplicon size, an assay that works on the relatively poor substrates of FFPE material. Detecting translocations is much more challenging. The translocations of RET and TRK are mostly intrachromosomal, making them relatively difficult to detect by FISH. Studies using FISH to detect RET translocations have found alternating positive and negative areas within morphologically malignant cells of papillary thyroid carcinoma—a finding that may reflect insensitivity of the FISH technique rather than the implausible alternative of multiple independent RET translocations. Polymerase chain reaction–based detection of the translocations is also difficult because the translocations can take place over a large distance. Use of messenger RNA as the basic substrate makes the translocations easier to detect, because relatively long introns are spliced out of the messenger RNAs, though working with RNA requires special attention to the method of preservation.
As of 2013, molecular genetic, biochemical, and cytogenetic tests for approximately ___ inherited genetic diseases are offered in clinical laboratories for diagnosing disease, screening carriers, predicting clinical disease susceptibility, assessing risk, and prognosticating on the course of disease.
As of 2013, molecular genetic, biochemical, and cytogenetic tests for approximately 2300 inherited genetic diseases are offered in clinical laboratories for diagnosing disease, screening carriers, predicting clinical disease susceptibility, assessing risk, and prognosticating on the course of disease.
List in vitro assays that have been developed for identification of cancer stem cells.
Animal studies are relatively expensive and require considerable time and effort. Therefore, numerous in vitro assays have been developed for CSC identification, which include: The colony forming cell assay/colony assay (assesses the ability of progenitor cells, in a semisolid media or methylcellulose-based culture media, to proliferate and differentiate into colonies in response to cytokine stimulation). The microsphere assay/sphere assay (assesses the ability of neural stem cells from mammalian adult brain subventricular neural cells to grow in serum-free medium in nonadherent conditions, forming sphere-shaped cell aggregates). The “side population” (SP) assay by Hoechst labeling (measures upregulation of P-glycoprotein by CSCs, which leads to active transport of some dyes, such as Hoechst or rhodamine, out of the cells). Staining for CSC surface antigens (such as ABCG2 and other markers). The aldehyde dehydrogenase (ALDH) activity assay (high levels of ALDH activity have recently been identified as a characteristic of CSCs, and the Aldefluor flow cytometric assay has been widely used for isolation and study of CSCs). Label-retaining cell assay using PKH (Paul Karl Horan) dyes (identifies CSCs by demonstrating their asymmetric division).
Brief overview of low-grade myofibroblastic proliferations of the urinary bladder.
The low-grade myofibroblastic proliferations of the urinary bladder are rare lesions affecting males more often than they do females. The most-common signs and symptoms are hematuria and dysuria. Histopathologically, they are spindle cell proliferations in a loose myxoid stroma, even though compact proliferations or hypocellular fibrous patterns can be found. Typically, there are varying amounts of acute, chronic, or mixed inflammatory infiltrates. Necrosis is rare to absent. Muscularis propria infiltration is common, while perivesical soft tissue invasion is uncommon. IHC is nonspecific, except for ALK-1 positivity (20%–89%). FISH has demonstrated clonal genetic aberrations involving the ALK gene in 50% to 60% of cases. After surgery, only 6% of patients experience local recurrence, without metastases or deaths from the disease. Malignant transformation has been reported exceptionally. These myofibroblastic proliferations are probably part of a continuum with, at one end, benign pseudosarcomatous proliferations and, at the opposite end, more-aggressive lesions.
Molecular studies for primary effusion lymphoma.
Epstein-Barr virus infection can be demonstrated by in situ hybridization for EBV-encoded small RNA (EBER); immunohistochemical studies for EBV latent membrane protein 1 are negative. Molecular studies demonstrate clonal immunoglobulin gene rearrangements and somatic hypermutation, indicating that the cell of origin is a postgerminal center B cell. PCR can demonstrate presence of the viral genome. Cytogenetic studies have not demonstrated any recurrent chromosomal abnormalities.
Currently, nucleic acid amplification tests are used most often for diagnosis of viral infections of the central nervous system. Other diagnostic methods are ___ (primarily for detection of enteroviruses, although PCR is preferred) and ___ for viruses that cause encephalitis (western equine, eastern equine, Venezuelan equine, St Louis, Japanese, and La Crosse and West Nile).
Currently, nucleic acid amplification tests are used most often for diagnosis of viral infections of the central nervous system. Other diagnostic methods are conventional cell culture (primarily for detection of enteroviruses, although PCR is preferred) and serologic tests for viruses that cause encephalitis (western equine, eastern equine, Venezuelan equine, St Louis, Japanese, and La Crosse and West Nile).
Overview of chondromyxoid fibroma.
A rare benign cartilaginous bone tumor arising in young adults aged 15-25 years that often occurs in the metaphysis of long tubular bones or in the small bones of the feet. Grossly: well circumscribed, solid and glistening. It is often lobulated with zonation. Older tumors are more hyalinized. Microscopic: well circumscribed, hypocellular lobules of poorly formed hyaline cartilage composed of chondroblasts with abundant pink cytoplasm, and myxoid tissue with fibrous septae containing spindle cells and osteoclasts. Atypia is common, including large, hyperchromatic nuclei. Scattered calcification and osteoclast-like giant cells are present. At the periphery, the tumor is more cellular and vascular. No/rare mitotic activity. Tumors often have 6q13 rearrangements, with recurrent 6p25 and 6q25 anomalies.
HIV testing is accomplished by ELISA, and repeat-reactive tests are confirmed by western blot testing for the presence of antibodies directed against the HIV proteins of the ___, ___, and ___ genes.
HIV testing is accomplished by ELISA, and repeat-reactive tests are confirmed by western blot testing for the presence of antibodies directed against the HIV proteins of the gag, pol, and env genes.
What HLA loci are present on platelets?
Platelets only have HLA-A, HLA-B, and HLA-C antigens, which are all part of the HLA class I structure. In routine clinical practice, only HLA-A and HLA-B are generally considered when the transfusion service is matching platelets in immune refractory patients.
HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR. Which of these are HLA class I antigens, and which are HLA class II antigens?
HLA-A, HLA-B, and HLA-C antigens are part of the HLA class I structure. HLA-DP, HLA-DQ, HLA-DR antigens are part of the HLA class II structure.
What methods are used in HLA typing for compatible platelets?
A number of methods are utilized in HLA typing for compatible platelets (primarily patient lymphocytes are used in testing because of the increased density of the class I HLA antigens), including: Anti-Human Globulin augmented Complement Dependent lymphoCytotoxicity Assay (AHG-CDC) – Antibodies against the HLA antigen will fix complement and cause lysis, visualized with a supravital stain. ELISA – Purified HLA gene products are adsorbed to the plate wells. Flow cytometry – Fluorescent markers identify cell surface antigens.
A distinguishing features of lobular carcinoma is the loss of expression of E-cadherin, a protein involved in calcium-dependent cellular adhesion. What are 3 mechanisms of loss of the E-cadherin protein that are seen?
Mechanisms of loss of E-cadherin protein include mutations in the gene, which is located on chromosome 16q22.1, loss of heterozygosity of chromosome 16, and silencing of gene expression by promoter methylation.
Familial neutrophilia, an autosomal dominant disorder of prominent leukocytosis (>20,000/mL), splenomegaly, and widened diploe of the skull, is caused by a mutation in the ___ gene.
Familial neutrophilia, an autosomal dominant disorder of prominent leukocytosis (>20,000/mL), splenomegaly, and widened diploe of the skull, is caused by a mutation in the G-CSF receptor gene (CSF3R). Neutrophils in this disorder are functionally normal and the leukocytosis has no clinical consequences.
Pelger-Huet anomaly (PHA) is characterized by mature neutrophils with bilobed nuclei, rather than the characteristic multilobed nuclear morphology. PHA is caused by a mutation in the ___ gene.
Pelger-Huet anomaly (PHA) is characterized by mature neutrophils with bilobed nuclei, rather than the characteristic multilobed nuclear morphology. PHA is caused by a mutation in the lamin B receptor gene. Neutrophil function in PHA is normal, but automated cell counters may indicate a left-shifted WBC because they mistakenly classify the cells as immature granulocytes.
Transient myeloproliferative disorder (TMD) is seen in up to __% of patients with Down syndrome (trisomy 21). TMD may also be seen in patients with trisomy 21 mosaicism who are phenotypically normal. TMD is characterized by peripheral blood leukocytosis in early infancy, and may include circulating myeloblasts in association with an accumulation of megakaryoblasts in the blood, liver, and marrow. TMD typically persists for several weeks and resolves spontaneously in most patients, but up to __% of affected patients later develop acute megakaryoblastic leukemia.
Transient myeloproliferative disorder (TMD) is seen in up to 10% of patients with Down syndrome (trisomy 21). TMD may also be seen in
patients with trisomy 21 mosaicism who are phenotypically normal. TMD is characterized by peripheral blood leukocytosis in early infancy, and may include circulating myeloblasts in association with an accumulation of megakaryoblasts in the blood, liver, and marrow. TMD typically persists for several weeks and resolves spontaneously in most patients, but up to 30% of affected patients later develop acute megakaryoblastic leukemia.
List DNA methylation screening techniques.
Blotting, genomic sequencing, bisulfite sequencing, methylation-specific PCR, methylated DNA immunoprecipitation, microarray analysis, matrix-assisted laser desorption time-of-flight mass spectroscopy (MALDI-TOF MS), a nanowire transistor detection procedure, a quantum-dot (QD) nanoassay, single-molecule real-time (SMRT) detection, a fluorimetric assay, electrochemical detection, and atomic force spectroscopy.
What is DNA methylation? What pathogenic and normal processes is it seen in?
DNA methylation, an epigenetic alteration, plays a key role in transcriptional control; it occurs when a methyl group is added to the fifth carbon of cytosine or at the sixth carbon of adenine and is catalyzed by specific DNA methyl transferases. Aberrant cytosine methylation is associated with silencing of tumor suppressor genes and plays a decisive role in the development of many cancers, such as colorectal, lung, liver, and breast cancers. Apart from carcinogenesis, DNA methylation is crucial for a variety of processes, such as genomic imprinting, X-chromosome inactivation, and suppression of repetitive elements.
How do hypomethylation and hypermethylation affect DNA expression? What is the relationship of DNA hypomethylation and hypermethylation with cancer?
Genome-wide hypomethylation results in chromosomal instability and increased mutation rates, whereas promoter hypermethylation suppresses gene transcription by either preventing transcription factors from binding to the gene or altering chromatin structure. DNA hypermethylation contributes to oncogenesis by point mutation and inactivation of tumor suppressor gene, whereas hypomethylation may lead to chromosomal instability and activation of proto-oncogene. Tumor growth is characterized by genome-wide hypomethylation, accompanied by hypermethylation of tumor suppressor gene promoters caused by increased expression of DNA methyltransferases. Changes in DNA methylation in cancer include both global hypomethylation and gene-specific hypermethylation. Whether global hypomethylation and gene-specific hypermethylation are mutually exclusive or not remains ambiguous.
As of 2012, what is the only FDA-approved device for circulating tumor cell detection?
CellSearch (Veridex LLC, Raritan, NJ). The CellSearch system, which is the current industry standard, uses EpCAM-coated immunomagnetic particles to capture and align CTCs for subsequent microscopic analysis.
Microfluidic isolation can be based on what differential physical properties of circulating tumor cells?
Size filtration, size streamline sort, size and particle polarizability, binding cell surface markers, binding cell surface markers and electrokinetic manipulation, size and binding cell surface markers.
Pathogenic variants for hypertrophic cardiomyopathy have been described in eight genes encoding sarcomere proteins, with most (∼80%) present in the ___ and ___ genes. As is typical for structural proteins, most sarcomere variants are believed to act in a dominant negative manner (ie, by adversely affecting the normal gene product). Loss-of-function variants leading to haploinsufficiency occur less frequently but are prevalent in the ___ gene. Collectively, sarcomere variants are identified in up to 60% of patients with HCM who also have a family history and in ∼40% of patients with sporadic HCM.
Pathogenic variants for HCM have been described in eight genes encoding sarcomere proteins, with most (∼80%) present in the MYH7 and MYBPC3 genes. As is typical for structural proteins, most sarcomere variants are believed to act in a dominant negative manner (ie, by adversely affecting the normal gene product). Loss-of-function variants leading to haploinsufficiency occur less frequently but are prevalent in the MYBPC3 gene. Collectively, sarcomere variants are identified in up to 60% of patients with HCM who also have a family history and in ∼40% of patients with sporadic HCM.
Pathogenic variants for hypertrophic cardiomyopathy have been described in eight genes encoding sarcomere proteins, with most (∼80%) present in the ___ and ___ genes. Storage cardiomyopathies masquerading as HCM are caused by mutations in ___ (Danon disease), ___ (Wolff-Parkinson-White syndrome), and ___ (Fabry disase).
Pathogenic variants for HCM have been described in eight genes encoding sarcomere proteins, with most (∼80%) present in the MYH7 and MYBPC3 genes. Storage cardiomyopathies masquerading as HCM are caused by mutations in LAMP2 (Danon disease), PRKAG2 (Wolff-Parkinson-White syndrome), and GLA (Fabry disase).
2011 clinical guidelines for hypertrophic cardiomyopathy recommend comprehensive testing for what five HCM genes?
2011 clinical guidelines for hypertrophic cardiomyopathy recommend comprehensive testing for five HCM genes (MYBPC3, MYH7, TNNI3, TNNT2, and TPM1).
Compared with hypertrophic cardiomyopathy, which is mainly a disease of the sarcomere, dilated cardiomyopathy shows a considerably higher degree of locus heterogeneity with a steadily growing number of genes implicated (currently ∼40, with HCM
Compared with HCM, which is mainly a disease of the sarcomere, DCM shows a considerably higher degree of locus heterogeneity with a steadily growing number of genes implicated (currently ∼40, with HCM
Most pathogenic arrhythmogenic right ventricular cardiomyopathy variants are present in what five genes?
Most pathogenic ARVC variants are present in five genes encoding desmosomal proteins [plakoglobin (JUP), desmoplakin (DSP), desmocollin-2 (DSC2), desmoglein-2 (DSG2), and plakophilin-2 (PKP2)].
Overview of left ventricular noncompaction.
Isolated LVNC is characterized by a heavily trabeculated or spongy appearance of the LV myocardium. An arrest of myocardial compaction during the first trimester of embryonic development is widely believed to be a cause. The left ventricle is typically affected, but 50% of patients with LVNC also have right ventricular involvement. There is a suggestion that LVNC is frequently associated with mitochondrial disorders, followed by Barth syndrome. Because LVNC is rare, its genetic etiology is not well understood. Variants have been described in known DCM and HCM genes encoding components of the sarcomere (ACTC1, MYH7, MYBPC3, and TNNT2), the Z-disk (LDB3), the nuclear lamina (LMNA), the dystrophin-associated glycoprotein complex (DTNA), as well as the Barth syndrome gene tafazzin (TAZ), a nuclear-encoded mitochondrial protein.
Activating somatic point mutations of the ___ gene are associated with mastocytosis and are encountered in most (>80%) of sporadic cases.
Activating somatic point mutations of the KIT gene are associated with mastocytosis and are encountered in most (>80%) of sporadic cases.
Enteropathy-associated T-cell lymphoma is subclassified into classic type and type II, based on the assessment of histomorphology and immunophenotype. How do classic EATL and type II EATL differ in their epidemiology?
Classic EATL: Represents 80-90% of all EATL. Is a complication of celiac disease, with >90% having celiac disease-associated HLA-DQ2/-DQB. Patients with refractory celiac disease are at highest risk. Most common in Northern Europe (population has a high prevalence of celiac disease). Type II EATL: Represents 10-20% of all EATL. May be associated with celiac disease, but in most cases occurs sporadically in individuals with celiac-associated HLA types similar to those in the general population. Most common in Asia (celiac disease is uncommon in the population).
Enteropathy-associated T-cell lymphoma is subclassified into classic type and type II, based on the assessment of histomorphology and immunophenotype. How do classic EATL and type II EATL differ in their immunophenotype?
Classic EATL: CD3+, CD5-, CD7+, 80% CD8-, >90% CD56-. Type II EATL: CD3+, CD5-, CD7+, 80% CD8+, >90% CD56+.
Enteropathy-associated T-cell lymphoma is subclassified into classic type and type II, based on the assessment of histomorphology and immunophenotype. How do classic EATL and type II EATL differ in their genetic aberrations?
Classic EATL: 86% +9q31.3 or -16q12.1. 73% +1q32.2-q41. 80% +5q34-q35.2 27% +8q24 (MYC). Type II EATL: 83% +9q31.3 or -16q12.1. 27% +1q32.2-q41. 20% +5q34-q35.2 73% +8q24 (MYC).
Pharmacogenetic testing for routine clinical practice is becoming increasingly important for select medications used today. The FDA’s Center for Drug Evaluation and Research (CDER) has stimulated pharmacogenomic testing by requiring manufacturers to provide pharmacogenetic information on their product labels and stating that pharmacogenomic testing is recommended before initial dosing of the drug. What are the 5 genes for which the CAP has proficiency testing program available?
Variants in the gene for UPD-glucuronosyltransferase (UGT1A1) were included by the CAP Working Committee for PT because patients with colon cancer who have the 7 TA repeats (28) are at increased risk for hematopoietic toxicity while taking irinotecan. Patients with variant alleles for cytochrome P450 (CYP) 2C9 (2, 3) and vitamin K epoxide reductase complex subunit 1 (VKORC1) (A alleles at −1639 promoter region) have increased sensitivity toward warfarin. As the CYP2C19 enzyme is important for the metabolism of clopidogrel to an active metabolite, the presence of loss-of-function variant alleles (2, *3) results in a higher rate of adverse cardiac events in patients undergoing percutaneous coronary intervention. The FDA has included pharmacogenetic information regarding CYP2D6 testing for dozens of drugs including codeine, tamoxifen, atomoxetine, and the tricyclic antidepressants.
What are the 2 most commonly used methods for DNA sequencing today?
Sanger (AKA terminator sequencing) and pyrosequencing (AKA sequence-by-synthesis). Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing.
How does Sanger sequencing work?
The Sanger method is also known as terminator sequencing because DNA fragments of varying lengths are synthesized by incorporating both nucleotides and dideoxy terminators (deoxyribonucleotide triphosphates [dNTPs] and dideoxynucleotide triphosphates [ddNTPs], respectively). Random incorporation of the ddNTPs causes chain termination that produces DNA fragments of every possible length. In a more recent adaptation, each ddNTP (A, C, T, or G) carries a unique, fluorescent molecule, such that the extension products are both terminated and labeled with the appropriate fluorophore. Terminated products must be purified from unincorporated ddNTPs, and the fragments are subsequently separated by size using capillary electrophoresis, in which the terminal nucleotide of each fragment is detected by fluorescence at wavelengths unique to each of the terminators. PCR amplification and DNA sequencing reactions are most commonly run separately, although they can be combined into a single reaction. Read lengths have increased for Sanger sequencing, and 800 base reads can now be achieved routinely.
How does pyrosequencing work?
Pyrosequencing is designated as a sequence-by-synthesis technique because DNA synthesis is monitored in real time. After an oligonucleotide is annealed to the template strand of DNA to be sequenced, a DNA polymerase synthesizes DNA by extending the 3′ end of the nascent strand using the information encoded in the template strand. During pyrosequencing, dNTPs are sequentially dispensed into the chamber containing the template with the primer and DNA polymerase bound. When the correct complementary dNTP is injected and added by the polymerase, inorganic pyrophosphate (PPi) is released during a condensation reaction. Through a sequence of another 2 reactions, the released pyrophosphate is converted into ATP, a cofactor for the enzyme luciferase, oxidizing luciferin to oxyluciferin and light. Thus, light emission is proportional to the amount of pyrophosphate produced, which is directly proportional to the number of nucleotides added. Apyrase catalyzes the degradation of excess dNTPs via the following reaction and before the next dNTP is dispensed.
What are strengths, weaknesses, and common applications of these 3 sequencing methods: Sanger sequencing. Pyrosequencing. Next-generation sequencing.
Sanger sequencing: Strengths are longer read lengths (800 bases). Weaknesses are worse limit of detection (~20%). Common applications are somatic and germline mutation detection. Pyrosequencing: Strengths are better limit of detection (~5%). Weaknesses are shorter read lengths (100-400 bases); homopolymers. Common applications are oncogene detection, methylation analyses, and resolving complex Sanger results. Next-generation sequencing: Strengths are massively parallel sequencing or extremely high depth of coverage; cost reduction for gene panels. Weaknesses are costs. Common applications are whole-genome sequencing, whole-exome sequencing, RNASeq, amplicon sequencing, rapid microbial identification, gene panels, early detection of cancer.
In pyrosequencing, why does the natural deoxyadenosine triphosphate (dATP) result in false signals?
Because, like ribose adenosine 5’-triphosphate (rATP), it is a substrate for luciferase. Accordingly, the dATP analog, deoxyadenosine alpha-thio triphosphate (dATP-alpha-S), is used in lieu of dATP, but that produces a higher peak than the other dNTPs, which needs to be considered when comparing homopolymers of A that are equal in length to the other nucleotides.
PAX-5 immunostain. What is it? What is it used for?
Also called BSAP, a B cell-lineage specific activator protein at 9q13. Member of paired box (PAX) family of transcription factors, which have a novel, highly conserved DNA-binding motif, known as the paired box; encodes BSAP expressed at early stages of B-cell differentiation, also in developing CNS and testis. Detected in B cells from pro-B cell stage to plasma cell stage where it is downregulated. Required for progression of B cell development beyond the early pro-B cell stage. Uses: Detection of pre-B cells (PAX5+, more sensitive and specific than CD20). Diagnosis of Reed-Sternberg cells in classic Hodgkin lymphoma (PAX5+) versus T / null cell anaplastic large cell lymphoma (PAX5-), although rarely positive in T cell lymphomas. Diagnosis of lymphoplasmacytic lymphoma / plasmacytoid differentiation in marginal zone lymphoma (PAX5+) versus plasmacytoma (PAX5-).
What is the PTEN gene? In what tumors is it often mutated?
Phosphatase and tensin homolog (PTEN) gene is on chromosome band 10q23.31. In addition to its role as a tumor suppressor, it has important roles in embryogenesis and maintenance of physiologic functions in many organ systems and is constitutively expressed in normal tissues. It is one of the most frequently inactivated genes in sporadic cancer. Sporadic mutations of PTEN occur frequently in many tumors such as glioblastoma, breast carcinoma, endometrial carcinoma, thyroid neoplasms, skin neoplasms, and advanced prostate cancer.
What is the mechanism by which PTEN acts as a tumor suppressor?
The role of PTEN as a tumor suppressor is due to its lipid phosphatase activity, including dephosphorylating phosphatidylinositol-3,4,5 triphosphate (PIP3), the product of phosphatidylinositol 3-kinase (PI3K) function, to form phosphatidylinositol-3,4 bisphosphate. The dephosphorylation of PIP3 antagonizes the PI3K function, thereby abolishing the PIP3-mediated activation of survival kinases, such as phosphoinositide-dependant kinase 1, and the AKT/mammalian target of rapamycin (mTOR) pathway. PTEN is the only known lipid phosphatase abrogating the PI3K pathway, and therefore, loss of PTEN has a significant impact on multiple aspects of tumorigenesis.
Clear cell sarcoma (CCS), which is also known as melanoma of soft parts, is a rare aggressive tumor that preferentially affects young adults and typically arises in the deep soft tissues of the lower extremities. CCS share many features with malignant melanoma, including expression of melanoma markers, but how are they different in regard to molecular mutations?
CCS share many features with malignant melanoma, including expression of melanoma markers, but, in contrast to most melanomas, they lack BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. In addition, most CCS show rearrangement of the EWSR1 (Ewing sarcoma breakpoint region 1) locus, which in most cases leads to fusion of the EWSR1 gene with the activating transcription factor-1 gene (ATF1) in a recurrent translocation, t(12;22)(q13;q12). This translocation is not present in melanoma. On rare occasions, CCS of soft tissues can also show EWSR1-CREB1 (cAMP responsive element–binding protein 1) gene fusion.
What is the E-cadherin complex?
The E-cadherin complex, essential for the formation of intercellular tight junctions, is composed of the transmembrane E-cadherin protein and α, β, γ, and p120 catenins, which anchor the E-cadherin protein to the cytoplasmic actin filaments. The α and β catenins are complexed with the carboxy-terminal cytoplasmic tail of E-cadherin, whereas the p120 catenin is anchored to the E-cadherin in a juxtamembranous site. p120 is actively involved in cell motility, E-cadherin trafficking and turnover, promotion of cell junction formation, and regulation of the actin cytoskeleton. β-Catenin is a cofactor implicated in the Wnt-signaling pathway, a powerful regulator of cell proliferation.
Pleomorphic lobular carcinoma has morphologic and immunophenotypic characteristics that overlap between classical invasive lobular carcinoma and invasive ductal carcinoma. What genetic mutations do PLC have in common with classical ILC and with IDC?
Molecular studies have demonstrated that PLC and ILC share several molecular features such as alterations in the gene CDH1 on chromosome band 16q22 that results in changes in E-cadherin protein function, as well as recurrent genomic changes affecting 1q+, 11q−, 16p+, and 16q− and genomic amplifications in the region of 8q24, 11q13, 12q13, 17q12, and 20q13. The aggressive biology of pleomorphic lobular carcinoma relates to the acquisition of genetic alterations typical of high-grade ductal carcinoma, such as overexpression of HER2/neu and c-myc, p53 positivity, and amplification of 8q24, 12q13, and 20q13.
In what brain tumors can IDH1 and/or IDH2 gene mutations be seen?
Somatic mutations in the IDH1 gene (on 2q33.3, exon 4 (codon R132), mutations R132H, C, L, S, or G) encoding cytosolic NADP+ - dependent isocitrate dehydrogenase have been shown in the majority of astrocytomas, oligodendrogliomas, and oligoastrocytomas of WHO grades II and III. IDH2 (on 15q26.1, exon 4 (codon R172), mutations R172G, M, or K) encoding mitochondrial NADP+ - dependent isocitrate dehydrogenase can also be mutated in gliomas, although in much lower frequencies. Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and mutations inactivate enzyme activities.
By what mechanisms do gliomas with inactivation of the MGMT gene have increased sensitivity to alkylating chemotherapy?
The DNA-repair enzyme MGMT (O^6-methylguanine-DNA methyltransferase) rescues the cell from damage induced by TMZ (temozolomide) and leads to resistance to therapy. Gliomas with inactivation of the MGMT gene are less capable of repairing DNA, which leads to increased sensitivity to alkylating chemotherapy.
What product is formed by t(9;22)(q34;q11.2) AKA Philadelphia chromosome?
It produces a BCR-ABL1 fusion with an abnormal tyrosine kinase activity that promotes the characteristic proliferation of progenitor cells in CML and ALL. BCR-ABL1 kinase signaling constitutively activates downstream proteins that promote growth factor-independent proliferation, altered adhesion, resistance to DNA repair, and inhibition of apoptosis that culminates in the malignant transformation of hematopoietic stem cells. The BCR-ABL1 fusion transcript and its resulting kinase have become a key target and biomarker in the treatment and monitoring of CML.
Dx of polycythemia vera can be made when JAK2 V617F or exon 12 mutation is detected, along with increased hemoglobin and low or normal levels of erythropoietin. Clinically, what differences are seen in those with V617F mutation and in those with exon 12 mutation?
Clinically, patients with exon 12 mutations typically present with isolated erythrocytosis and suppressed erythropoietin, in contrast to the trilineage hyperplasia characteristic of patients with V617F mutation. Bone marrow from patients with exon 12 mutation often exhibits nonspecific morphology, with isolated erythroid proliferation and absence of prominent megakaryocyte atypia and clustering. Demonstration of exon 12 mutation in these patients is particularly helpful for ruling out reactive erythrocytosis.
What 2 mutually exclusive mutations are seen in virtually all cases of polycythemia vera?
JAK2 V617F (~96%) or JAK2 exon 12 mutations (~3%).
True or false. JAK2 and MPL mutations are completely specific for MPN.
False. Other hematological neoplasms (eg, myelodysplastic syndrome, chronic myelomonocytic leukemia, acute myeloid leukemia, and acute lymphoblastic leukemia) may harbor JAK2 and MPL mutations in low frequencies. Because JAK2 and MPL mutations are not completely specific for MPN, finding of these mutations in isolation does not warrant a diagnosis of MPN.
JAK2 gene. Chromosome? What does it encode? What happens with mutation?
The JAK2 gene maps to chromosome band 9p24 and encodes a tyrosine kinase protein composed of 1132 amino acids. It contains three critical domains: JH1, JH2, and four-point-one, ezrin, radixin, moesin (FERM) homolog domains. JAK2 protein kinase activity is activated by phosphorylation of its kinase domain. Activation of JAK2 induces signal transduction from both type 1 and type 2 cytokine receptors. Constitutive activation of JAK2 by either point mutation or fusion protein causes activation of the JAK/STAT pathway. The activated JAK2 causes phosphorylation of STATs, which then dimerize and translocate to the nucleus, where they regulate gene transcription. The constitutive tyrosine phosphorylation activity promotes cytokine hypersensitivity.
MPL (myeloproliferative leukemia virus oncogene) gene. Chromosome? What does it encode? What happens with mutation?
The MPL gene maps to chromosome band 1p34 and encodes the thrombopoietin receptor, which binds to thrombopoietin, the primary cytokine that regulates megakaryocyte development and platelet production, as well as hematopoietic stem cell homeostasis. Binding to thrombopoietin to MPL leads to activation of JAK2, which phosphorylates MPL and initiates a cascade of downstream signaling events that regulate cell survival, proliferation, and differentiation. The mutation W515L results in impaired function of the autoinhibitory region and subsequent ligand-independent thrombopoietin receptor activation. This then leads to subsequent activation of downstream tyrosine kinases and activation of transcription factors STAT3 and STAT5, which in turn leads to transformation of hematopoietic cells into cytokine-independent clones, resulting in megakaryocytic hyperplasia and marrow fibrosis. The mutation Y252H, which is located in the extracellular domain of MPL, confers hypersensitivity to thrombopoietin and increases the generation of megakaryocyte colonies in vitro and leads to increased thrombopoietin signaling and cell growth and survival.
EWS/EWSR1 translocations in soft tissue tumors. Which tumors? What is the mechanism?
The Ewing sarcoma breakpoint region 1 (EWSR1; also known as EWS) represents one of the most commonly involved genes in sarcoma translocations. In fact, it is involved in a broad variety of mesenchymal lesions which includes Ewing’s sarcoma/peripheral neuroectodermal tumor, desmoplastic small round cell tumor, clear cell sarcoma, angiomatoid fibrous histiocytoma, extraskeletal myxoid chondrosarcoma, and a subset of myxoid liposarcoma. The fusion products between EWSR1 and partners usually results in fusion of the N-terminal transcription-activating domain of EWSR1 and the C-terminal DNA-binding domain of the fusion partner, eventually generating novel transcription factors.
EWS translocations occur in what soft tissue tumors?
Ewing’s sarcoma/peripheral neuroectodermal tumor, desmoplastic small round cell tumor, clear cell sarcoma, angiomatoid fibrous histiocytoma, extraskeletal myxoid chondrosarcoma, and a subset of myxoid liposarcoma.
The Ewing sarcoma/PNET family of tumors is characterized by chromosomal translocations involving the EWSR1 gene on chromosome 22 with one of several members of the ETS family of transcription factors on various different chromosomes including 2, 7, 11, 17 and 21. The most common translocation (over 70% of cases in bone/soft tissue) is t(11;22)(__;__) which leads to the formation of the EWS-__ fusion protein.
The Ewing sarcoma/PNET family of tumors is characterized by chromosomal translocations involving the EWSR1 gene on chromosome 22 with one of several members of the ETS family of transcription factors on various different chromosomes including 2, 7, 11, 17 and 21. The most common translocation (over 70% of cases in bone/soft tissue) is t(11;22)(q24;q12) which leads to the formation of the EWS-FLI1 fusion protein.
Typically in bone and soft tissues, PNET tumors with variant gene fusions (non-FLI1) are associated with a (better/worse) outcome.
Typically in bone and soft tissues, PNET tumors with variant gene fusions (non-FLI1) are associated with a worse outcome.
MSI testing is performed by extracting DNA from paraffin-embedded neoplastic and nonneoplastic tissue. PCR is used to amplify 5 specific microsatellite regions: ___, ___, ___, ___, ___. These regions from the tumor are compared to those from nonneoplastic tissue to assess for differences in length, indicative of MSI.
MSI testing is performed by extracting DNA from paraffin-embedded neoplastic and nonneoplastic tissue. PCR is used to amplify 5 specific microsatellite regions: BAT25, BAT26 (both of which are regions of mononucleotide repeats), D2S123, D5S346, and D17S250 (regions of dinucleotide repeats). These regions from the tumor are compared to those from nonneoplastic tissue to assess for differences in length, indicative of MSI.
RCCs in vHL tend to present in young patients (mean 37 to 44 yo) and tend to be multifocal and bilateral. Are they more or less aggressive than the sporadic tumors?
They are somewhat more indolent than sporadic tumors (metastasize only when quite large).
What tumors are seen in patients with vHL disease?
Hemangioblastomas (CNS and retinal), pheochromocytoma, clear cell RCC, pancreatic cysts, islet cell tumors, epididymal and ovarian cystadenomas, endolymphatic sac tumors.
~__% of cases of vHL disease are caused by de novo mutations, with the remaining caused by inherited vHL gene mutations.
~20% of cases of vHL disease are caused by de novo mutations, with the remaining caused by inherited vHL gene mutations.
vHL disease can be subtyped according to the clinical manifestations (although these groups often correlate with certain types of mutations present in the VHL gene). What are the subtypes?
Type 1 often has deletion or nonsense mutations. This group manifests mostly as hemangioblastomas whereas clear cell RCC and pheos are rare. Type 2 is subdivided into types 2A, 2B, and 2C, and are characterized mostly by missense mutations. Type 2A is at risk of hemangioblastomas and pheos, but not clear cell RCC. Type 2B is at risk of all 3 tumors, with a higher risk of clear cell RCC. Type 2C is at risk for only pheos. Type 3 has a risk of Chuvash polycythemia.
VHL disease is an AD disorder caused by a germline mutation in the VHL gene, a tumor suppressor gene on (chromosome).
VHL disease is an AD disorder caused by a germline mutation in the VHL gene, a tumor suppressor gene on 3p25-26. The VHL protein (pVHL) is involved in the regulation of the protein hypoxia inducible factor 1-alpha.
What tumors are seen commonly in tuberous sclerosis (the “major features” as set forth in a consensus statement from the Diagnostic Criteria Committee of the National Tuberous Sclerosis Association)?
Facial angiofibromas (adenoma sebaceum) or forehead plaque. Nontraumatic ungual or periungual fibroma. >3 hypomelanotic macules. Shagreen patch (connective tissue nevus). Multiple retinal nodular hamartomas. Cortical tuber. Subependymal nodule. SEGA. Cardiac rhabdomyoma, single or multiple. LAM. Renal AML. Definite TSC is either 2 major features or one major plus 2 minor features.
__% of all women with breast cancer and __% of Ashkenazi Jewish women with breast cancer have a BRCA mutation. The lifetime risk of breast cancer in women is __% and the lifetime risk in BRCA1 or BRCA2 is __%.
5% of all women with breast cancer and 25% of Ashkenazi Jewish women with breast cancer have a BRCA mutation. The lifetime risk of breast cancer in women is 10% and the lifetime risk in BRCA1 or BRCA2 is 70%.
Three BRCA mutations appear with high frequency in persons of Ashkenazi Jewish descent, together accounting for over 90% of the BRCA mutations in this population. What are they?
A two base pair deletion in codon 23 (185delAG) in BRCA1, a 5832insC mutation in BRCA1, and a 6174delT mutation in BRCA2.
MEN1 manifests with pituitary adenomas, parathyroid adenomas, and pancreatic islet cell tumors. What are nonendocrine lesions associated with MEN1?
Facial angiofibromas, collagenomas, lipomas, and meningiomas.
There are 3 subtypes of MEN2: MEN 2A, MEN 2B, and FMTC. All are autosomal dominant, all are due to a mutation in RET, and all have a high risk for medullary thyroid carcinoma. While the histology of the medullary thyroid carcinoma in the syndrome is not distinctive, the appearance of the background thyroid is. What is seen?
C-cell hyperplasia and numerous small foci of medullary carcinoma.
What tumors occur in Carney complex?
Cutaneous lentigenes (simple lentigos). Blue nevi, particularly the cellular blue nevus. Cardiac myxomas (as well as myxomas of breast, female genital tract, and skin (especially on eyelid and external ear)). Endocrine tumors including thyroid follicular adenomas, pituitary adenomas (GH-secreting), and the so-called primary pigmented nodular adrenocortical disease (a form of multinodular hyperplasia of the adrenal cortex that causes Cushing syndrome). Large-cell calcifying Sertoli cell tumor. Psammomatous melanotic schwannoma.
Large cell calcifying Sertoli cell tumor of the testis is associated with what 2 syndromes?
Peutz-Jeghers syndrome and Carney complex.
Large cell calcifying Sertoli cell tumor of the testis is a unique variant of Sertoli cell tumor that can be sporadic (__%) but can also be part of Peutz-Jeghers and Carney syndromes (__%).
Large cell calcifying Sertoli cell tumor of the testis is a unique variant of Sertoli cell tumor that can be sporadic (60%) but can also be part of Peutz-Jeghers and Carney syndromes (40%). This variant tends to occur in young individuals with an average age of 16 and can be bilateral in 40% of cases.
Hereditary hemochromatosis is an autosomal recessive disorder resulting from mutations in the HFE gene. What is the most common HFE mutation (seen in 70-100% of patients diagnosed with the disease)?
C282Y.
In what metabolic disorder is urine black, connective tissue grossly blue-black, and connective tissue microscopically brown or ochre?
Alkaptonuria, an autosomal recessive disorder of amino acid metabolism. Increased homogentisic acid is secreted in urine and polymerized homogentisic acid deposits accumulate in connective tissue. The pigment is Fontana-Masson positive and Prussian blue negative.
In CLL/SLL, do cases with trisomy 12 and cases with 13q14 abnormality tend to have mutated or unmutated immunoglobulin variable gene regions?
Cases with trisomy 12 have predominantly unmutated Ig variable gene regions and therefore have a more aggressive course and worse prognosis. Cases with 13q14 abnormalities usually have mutated immunoglobulin and better prognosis.
What is the diagnostic translocation for acute promyelocytic leukemia?
t(15;17).
Burkitt lymphoma is characterized by the translocation of the c-MYC gene on chromosome 8q24 and one of three locations on Ig genes. What are the 3 translocations and their relative frequencies?
t(8;14), Ig heavy chain gene, ~80%. t(2;8), kappa light chain gene, ~15%. t(8;22), lambda light chain gene, ~5%.
The genetic defect implicated in the pathogenesis of Wilson disease is localized to chromosome 13, and involves the gene encoding ___.
The genetic defect implicated in the pathogenesis of Wilson disease is localized to chromosome 13, and involves the gene encoding the protein ATP7B. ATP7B is an ATP-dependent copper transport protein that plays a pivotal role in the biliary excretion of copper and in the incorporation of copper into apoceruloplasmin to form ceruloplasmin, the major circulating copper carrying protein.
What is analbuminemia? What are clinical and laboratory manifestations?
Analbuminemia is a rare AR disorder manifested by the absence or severe reduction of circulating serum albumin. Analbuminemic individuals usually have surprisingly minimal findings, since the absence of albumin is partially compensated for by an increase of other serum proteins, such as globulins, transferrin, and coagulation factors. The main clinical symptoms include mild edema and
occasionally chronic fatigue or lipodystrophy, especially in women, while the most common
biochemical signs are gross hyperlipidemia with hypercholesterolemia and elevated LDL-cholesterol
levels. The finding of a low albumin concentration during routine SPEP, with normal liver function and no GI or renal protein loss, suggests the clinical diagnosis. This needs to be confirmed by the molecular diagnosis, based on the identification of the causative mutation within the albumin (ALB) gene by DNA sequence analysis.
What genetic disorders can cause hypokalemia?
Congenital adrenal hyperplasia (11-beta hydroxylase or 17-alpha hydroxylase deficiency). Glucocorticoid-remediable hypertension. Bartter syndrome. Gitelman syndrome. Liddle syndrome. Gullner syndrome. Glucocorticoid receptor deficiency. Hypokalemic periodic paralysis. Thyrotoxic periodic paralysis (TTPP). Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome).
What are the classic translocations seen in alveolar rhabdomyosarcoma?
t(2;13) with PAX3-FOXO1 (formerly known as FKHR) fusion and t(1;13) with fusion of PAX7 and FOXO1. As many as 20% of cases of alveolar rhabdomyosarcoma are negative for either translocation, and usually the solid variants.
t(12;15) resulting in ETV6-NTRK3 fusion is seen in what tumors?
Mammary analogue secretory carcinoma of the salivary gland, secretory carcinoma of the breast, infantile fibrosarcoma, cellular congenital mesoblastic nephroma, and some types of acute myelogenous leukemia.
What genetic abnormalities are seen in chondroblastoma?
Chondroblastoma is not associated with recurrent translocations or specific genetic abnormality.
What translocation is seen in extraskeletal myxoid chondrosarcoma?
The EWSR1-NR4A3 translocation t(9;22) is seen in about half of cases.
What genetic abnormalities have been identified in mesenchymal hamartoma of liver?
Aneuploidy in some, as well as balanced translocation involving a common breakpoint on the long arm of chromosome 19 (band 19q13.4) with alternative partners on chromosomes 11 and 15.
What is the most common form of skeletal dysplasia, and what is the most common lethal form of skeletal dysplasia?
Most common form of skeletal dysplasia: achondroplasia (1 per 10-30,000 live births). Most common lethal form of skeletal dysplasia: thanatophoric dysplasia (1 per 35-50,000 births).
Achondroplasia is an autosomal dominant disorder. It is due to mutation in what gene? What % are caused by sporadic new mutations?
Fibroblast growth factor receptor 3, encoded by the FGFR3 gene on chromosome 4p16.3. 75% of cases are caused by sporadic new mutations.
Lipoblastoma is a tumor of infancy and early childhood composed of lobules of mature adipocytes and lipoblasts in different stages of development within peripheral zones of myxoid stroma. M:F = 2:1. Genetic abnormality?
Lipoblastomas are characterized by rearrangements of 8q11-13 involving the PLAG1 gene.
What are the most common genotypes for complete and partial moles?
The most common genotype of complete moles is 46,XX; a small number are 46,XY. Triploid and tetraploid complete moles are much less common. Triploid conceptions which have duplication of paternal chromosomes are partial moles, typically with a 69,XXX or 69,XXY genotype. Triploid conception resulting from a duplication of maternal chromosomal material (also 69,XXX or 69,XXY genotype) represent non-molar triploidy and lack the trophoblastic proliferation which is the hallmark of a molar pregnancy.
Both partial hydatidiform mole and non-molar triploid conceptions can have 69,XXX or 69,XXY genotypes. What differentiates them?
Triploid conceptions which have duplication of paternal chromosomes are partial moles, typically with a 69,XXX or 69,XXY genotype. Triploid conception resulting from a duplication of maternal chromosomal material (also 69,XXX or 69,XXY genotype) represent non-molar triploidy and lack the trophoblastic proliferation which is the hallmark of a molar pregnancy.
p57 IHC is useful for distinguishing complete hydatidiform mole from hydropic abortus and partial hydatidiform mole (HA and PHM have nuclear staining of villous cytotrophoblast and stromal cells, while CHM does not). What IHC stain distinguishes HA from PHM?
IHC cannot distinguish HA (biparental diploidy) from PHM (diandric triploidy). Short tandem repeat genotyping, which determines the parental source of polymorphic alleles, is useful.
What is the mutation seen in Alexander disease AKA fibrinoid leukodystrophy?
Alexander disease has 3 clinical phenotypes: infantile (63%), juvenile (24%), and adult (13%). The majority of infantile and juvenile types are caused by a heterozygous (dominant) gain of function mutation in the gene encoding GFAP, located on chromosome 17q21. These mutations are typically sporadic and de novo. In Alexander disease, a heterozygous missense mutation in the GFAP gene appears to interfere with polymerization of the protein, and results in abnormal protein folding with overexpression and accumulation of the abnormal GFAP protein within Rosenthal fibers.
What is the mutation seen in Alexander disease AKA fibrinoid leukodystrophy?
A
There are >20 known human diseases of intermediate filaments involving the keratins, desmin, lamin A/C, neurofilament, phakinin, and GFAP. Which disease differs from all other known intermediate filament disorders in that there is a gain of function mutation, rather than a loss of function mutation?
Alexander disease AKA fibrinoid leukodystrophy.
What is Alagille syndrome (AGS) AKA syndromic paucity of interlobular bile ducts AKA arteriohepatic dysplasia?
AGS is a multisystem disorder with AD inheritance and variable penetrance and expressivity. The traditional diagnostic criteria have consisted of paucity of the interlobular bile ducts in association with 3 of 5 major findings, including chronic cholestasis, cardiac disease (most frequently peripheral pulmonary artery stenosis), skeletal manifestations (short stature, butterfly vertebrae), ocular abnormalities (most commonly posterior embryotoxon, a circular opacity of the posterior peripheral cornea), and characteristic facial features (triangular face with a prominent forehead, deep-set eyes, a pointed chin, and a straight nose with a bulbous tip). Mutations are in the JAGGED1 (JAG1) gene.
What is the mutation in Alagille syndrome?
Mutations are in the JAGGED1 (JAG1) gene, which encodes a cell surface protein that functions as a ligand in the Notch signaling pathway regulating cell proliferation and differentiation. The precise mechanisms by which JAG1 mutations cause AGS is still incompletely understood. JAG1 mutations have been identified in >90% of clinically diagnosed probands; additionally, NOTCH2 mutations have been detected in the small minority of AGS patients lacking JAG1 mutations.
In Alagille syndrome, abnormalities in which organ system causes the most mortality?
Hepatic complications do contribute significantly to the morbidity of AGS but it is the cardiovascular sequelae that are most directly responsible for the early mortality. Specifically, intracardiac anomalies, which affect ~25% of AGS patients (most commonly tetralogy of Fallot).
Only a few of almost 100 genes identified in the EBV genome are actively transcribed in immortalized B-cells, the so-called latent genes. What are they?
These include six EBV nuclear antigens (EBNA-1, 2, 3A, 3B, 3C, and LP), three latent membrane proteins (LMP1, 2A, 2B) localized in the plasma membrane of the infected B cells, and two small non-polyadenylated nuclear RNAs, EBER1 and EBER2 (which far often used as sensitive markers for the presence of EBV within a cell). Three patterns of latency (I, II, and III) are identified depending on the expression of these genes.
EBV belongs to the gamma-herpesvirus subfamily of herpes virus along with HHV-8. The virus gains entry into the body by infection of B-lymphocytes by the interaction between the major viral glycoprotein ___ and the complement receptor ___ which is expressed on B-cells. Following primary infection there is the expression of lytic cycle proteins resulting in the release of infectious virus and generalized seeding of B-cells throughout the body.
EBV belongs to the gamma-herpesvirus subfamily of herpes virus along with HHV-8. The virus gains entry into the body by infection of B-lymphocytes by the interaction between the major viral glycoprotein gp350 and the complement receptor CR2 which is expressed on B-cells. Following primary infection there is the expression of lytic cycle proteins resulting in the release of infectious virus and generalized seeding of B-cells throughout the body.
What brain tumors are associated with the following syndromes? Turcot syndrome (recently renamed Crail syndrome)/APC gene mutations. Tuberous sclerosis. Von Hippel Lindau disease. Li-Fraumeni syndrome. NF type 1. NF type 2. Gorlin syndrome.
Turcot syndrome (recently renamed Crail syndrome)/APC gene mutations: medulloblastoma, rarely ependymoma. Tuberous sclerosis: subependymal giant cell astrocytoma. Von Hippel Lindau disease: hemangioblastoma. Li-Fraumeni syndrome (with germline TP53 mutations): medulloblastoma, astrocytic tumors, meningioma, schwannoma, choroid plexus tumors, and central PNET. NF type 1: optic glioma, astrocytoma, glioblastoma multiforme. NF type 2: multiple meningiomas, bilateral vestibular schwannomas, spinal ependymomas. Gorlin syndrome: desmoplastic/nodular variant of medulloblastoma.
What are the most frequent genetic abnormalities seen in giant cell tumor of bone? What genetic syndrome predisposes to giant cell tumor of bone?
While no specific translocation or chromosomal anomalies are found in GCT, normal karyotypes are rarely encountered. The most frequent genetic abnormality seen in giant cell tumor of bone is reduction in telomere length (11p, 13p, 14p, 15p, 19q, 20q, 21p). Telomeric fusion is detected in ~70% of GCT. A higher incidence of chromosomal anomalies have been reported for recurrent benign GCT or metastatic GCT than for GCT cured after initial surgery. There are no known genetic syndromes predisposing to giant cell tumor.
What are some genetic syndromes associated with bone lesions?
Ollier disease (enchondromas, chondrosarcoma). Mafucci syndrome (enchondromas, chondrosarcoma). Bloom syndrome (osteosarcoma). Li-Fraumeni (osteosarcoma). Rothmund-Thompson syndrome (osteosarcoma). Werner syndrome (osteosarcoma). McCune-Albright syndrome (polyostotic fibrous dysplasia, osteosarcoma). Mazabraud syndrome (polyostotic fibrous dysplasia, osteosarcoma).
What bone lesions are the following syndromes associated with? Ollier disease. Mafucci syndrome. Bloom syndrome. Li-Fraumeni. Rothmund-Thompson syndrome. Werner syndrome. McCune-Albright syndrome. Mazabraud syndrome.
Ollier disease (enchondromas, chondrosarcoma). Mafucci syndrome (enchondromas, chondrosarcoma). Bloom syndrome (osteosarcoma). Li-Fraumeni (osteosarcoma). Rothmund-Thompson syndrome (osteosarcoma). Werner syndrome (osteosarcoma). McCune-Albright syndrome (polyostotic fibrous dysplasia, osteosarcoma). Mazabraud syndrome (polyostotic fibrous dysplasia, osteosarcoma).
Peutz-Jeghers syndrome is an autosomal dominant condition caused by inactivating mutations in the serine-threonine protein kinase 11 (STK11) gene, also called LKB1, on chromosome 19p13.3. Mutations in STK11 are found in ~__% of familial cases and ~__% of sporadic cases.
Peutz-Jeghers syndrome is an autosomal dominant condition caused by inactivating mutations in the serine-threonine protein kinase 11 (STK11) gene, also called LKB1, on chromosome 19p13.3. Mutations in STK11 are found in ~70% of familial cases and ~30-70% of sporadic cases.
Although most juvenile polyps in children are sporadic, a subset of these patients develop numerous polyps (juvenile polyposis) due gene to mutations in ___ or ___. In this syndrome, juvenile polyps may develop dysplasia and eventually carcinoma.
Although most juvenile polyps in children are sporadic, a subset of these patients develop numerous polyps (juvenile polyposis) due gene to mutations in SMAD4 or BMPR1A. In this syndrome, juvenile polyps may develop dysplasia and eventually carcinoma.
The PTEN hamartoma syndromes include ___ syndrome and ___ syndrome, both resulting from mutations in the tumor suppressor gene phosphatase and tensin homolog (PTEN) on chromosome 10q23.3. These syndromes are associated with increased risk of breast and thyroid carcinomas.
The PTEN hamartoma syndromes include Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome, both resulting from mutations in the tumor suppressor gene phosphatase and tensin homolog (PTEN) on chromosome 10q23.3. These syndromes are associated with increased risk of breast and thyroid carcinomas.
What are the 3 main subtypes of hepatic adenoma based on immunophenotypic/molecular studies?
Cellular atypia/beta-catenin mutated adenoma. Steatotic/hepatocyte nuclear factor 1-alpha mutated adenoma. Telangiectatic/inflammatory adenoma. Those HA not fitting these three main subtypes are labeled as “unclassified” HA.
In Ewing sarcoma, a t(11;22) producing EWSR1/___ is present, while in desmoplastic small round cell tumor a different t(11;22) producing EWSR1/___ is present.
In Ewing sarcoma, a t(11;22) producing EWSR1/FLI-1 is present, while in desmoplastic small round cell tumor a different t(11;22) producing EWSR1/WT1 is present.
What are the 2 most common translocations seen in alveolar rhabdomyosarcoma?
~75% of alveolar rhabdomyosarcomas have a balanced translocation involving chromosomes 2 and 13 or chromosomes 1 and 13. The most common, t(2;13)(q35;q14) generates a fusion gene and corresponding fusion protein involving the genes PAX3 and FKHR (AKA FOXO1). The second most common, t(1;13)(p36;q14), generates a fusion gene and protein involving PAX7 and FKHR. Several other translocations have been described involving the PAX3 and PAX7 genes. The remaining 25% of cases do not have translocations identifiable by karyotyping.
Loss of what protein/mutation of what gene is seen in atypical teratoid rhabdoid tumor?
ATRTs are highly malignant (WHO grade IV) CNS neoplasms seen in very young children. INI1 is a ubiquitously expressed protein encoded by the hSNF5/INI1 gene on chromosome 22q11.2. Mutations in this gene are seen in ATRT. Cytogenetic studies often show monosomy or deletion of chromosome 22; however, this is not as specific or sensitive as IHC, since other tumors with complex karyotypes may show loss of 22 in addition to other events, and the INI1 gene may be mutated by a mechanism other than deletion (i.e. point mutations) not detectable by FISH or LOH studies. The presence of an hSNF5/INI1 gene mutation or loss of the INI1 protein expression by IHC is sufficient to confer a diagnosis of ATRT.
What are common gene mutations seen in serous, mucinous, and endometrioid ovarian carcinomas?
Mucinous tumors have a high prevalence of KRAS mutations. Serous tumors commonly have TP53 gene mutations. CTNNB1 (beta-catenin) gene mutations and PTEN mutations are common in endometrioid adenocarcinomas.
What is “attenuated FAP”?
Attenuted FAP is a phenotype with fewer adenomas (<100), more proximal colonic location of polyps, delayed age of onset of carcinoma, and lower overall lifetime risk of carcinoma (69%) as compared to classic FAP. Patients with attenuated FAP also often lack family history of polyposis or extraintestinal manifestations. Attenuated FAP is typically caused by mutations in the most proximal or most distal portions of the APC gene.
Hereditary pyropoikilocytosis is currently classified as a subtype of common hereditary elliptocytosis by virtue of the documented coexistence of HE and HPP within the same families, and the presence of the same mutation affecting the gene for the RBC membrane protein spectrin. But what is one essential distinction?
The RBCs of HPP patients manifest a superimposed quantitative deficiency in spectrin, which has been termed a “thalassemia-like” defect of a-spectrin. Thus, the most common scenario leading to the HPP phenotype is the combination of an elliptocytogenic a-spectrin missense mutation within one parent and a silent “thalassemia-like” defect within the other parent; when this biochemically occult allele occurs in trans to the missense mutation, the expression of the mutant spectrin is promoted and the production of normal spectrin is significantly inhibited.
What is GM1 gangliosidosis?
An AR lysosomal storage disorder caused by deficiency of the beta-galactosidase enzyme due to mutations in the GLB1 gene on chromosome 3p. The function of the enzyme is to hydrolyze the terminal beta-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans. Without the enzyme function, GM1 gangliosides accumulate in lysosomes of various tissues, particularly the CNS. The 3 clinical phenotypes are: type 1 (infantile), type 2 (late infantile or juvenile), and type 3 (adult or chronic).
What is alpha beta-Crystallin and its significance with basal-like breast carcinoma?
Alpha beta-Crystallin is one of many heat shock proteins that function as inhibitors of apoptosis by preventing accumulation of denatured proteins, thus prohibiting a signal for cell death and enabling survival. It impedes the proteolytic activation of caspase-3, a protease involved in the apoptotic cascade. Alpha beta-Crystallin is preferentially expressed in BLBCs and rarely in other molecular subtypes.
MammaPrint (Agendia, Amsterdam, the Netherlands). What is it used for? How does it work?
Based on a 70-gene set profile using an oligonucleotide array. A cDNA microarray-based test. Initially could be performed only on fresh frozen tissue but now FFPE tissue with RT-PCR can be used as well. The test helps in deciding whether the pt should receive adjuvant chemo by separating breast cancers by gene signatures into those with good vs poor prognosis. Pts with gene signature showing good prognosis will get hormone therapy (if the tumor is ER+). The poor prognosis signature consists of genes regulating cell cycle, invasion, angiogenesis, and metastasis. Pts with a gene signature showing poor prognosis will get chemo and hormone therapy (if the tumor is ER+). The test is offered for patients with tumors <5 cm, with LN-neg disease and low clinical stage (stage I or II). The advantage of MammaPrint is that the test can be performed on both ER+ and ER- tumors. However, the clinical utility of MammaPrint with ER- breast cancers is limited because only 0-4% of pts with ER- tumors are considered to have a good prognosis by the gene signature.
Oncotype Dx (Genomic Health Inc, Redwood City, California). What is it used for? How does it work?
An RT-PCR-based assay performed on FFPE tissue of invasive breast cancer specimen (but also recently approved for testing DCIS) which provides predictive and prognostic information in ER+, LN- tumors. It gives a recurrence score based on a 21-gene panel, with RS given from 0 to 100 and divided into 3 risk groups: low risk, <18; intermediate risk, 18-31; high risk, 31 or greater. The low RS pts get no chemo, the intermediate RS pts may or may not get chemo, and the high RS pts do get chemo.
HOXB13/IL17RB Ratio (AviaraDx Inc, Carlsbad, California). What is it used for? How does it work?
An assay that determines the risk of recurrence in women with LN neg, ER pos breast cancers who have received Tx with tamoxifen. The assay analyzes the ratio of homeobox B13 and interleukin 17 receptor gene expression using RT-PCR on FFPE tissue. To improve the assay, a numerical score called the molecular grade index was created by selecting 5 cell-cycle-related genes (BUB1B, CENPA, NEK2, RACGAP1, and RRM2) to be used concurrently with the H/I index to improve risk stratification. The combined MGI and H/I index stratifies pts into 3 risk groups: low risk, low for MGI and low or high H/I index; intermediate risk, high MGI and low H/I index; high risk, high for both MGI and H/I index.
What genetic abnormalities are seen in sporadic MPNST and in NF1-associated MPNST?
Both typically have complex karyotypic abnormalities that are both numerical and structural, but no consistent karyotypic pattern has been identified. On a molecular level, homozygous deletions of the CDKN2A gene, which encodes the p16 cell cycle inhibitory molecule, occurs in the progression of neurofibromas to MPNST, being identified in ~50% of MPNST but not in neurofibromas.
What genetic mutations cause primary/familial forms of hemophagocytic lymphohistiocytosis (FHLH)? What are causes of secondary/acquired forms of HLH?
~30% of FHLH cases are due to mutations in the gene encoding perforin (PRF1, 10q21-22), and 20-25% of cases are due to mutations in Munc 13-14 (UNC13D, 17q25). Acquired forms of HLH are typically triggered by infections, especially viral infection such as EBV, CMV, other herpes viruses, and parvovirus B19. Other forms of infection which trigger HLH include bacterial, fungal, and protozoan infections, especially Leishmaniasis. It is important to note that genetic forms of HLH may also be triggered by infection, and isolation of a causative agent does not distinguish primary and secondary forms. Malignancies associated with HLH include primary lymphomas, an association that is more common in adults than in children. Specifically, ALCL is a frequent associated malignancy. HLH in the setting of autoimmune disease has been distinguished from other forms and is called “macrophage activation syndrome” or “ reactive hemophagocytic syndrome.” This is most often reported in association with systemic-onset juvenile idiopathic arthritis, but may also be seen with LE and other collagen vascular diseases.
The locus on chromosome 11 that, in germline form, leads to MEN1 has been found to contain somatic mutation in ~__% of sporadic parathyroid adenomas.
The locus on chromosome 11 that, in germline form, leads to MEN1 has been found to contain somatic mutation in ~25% of sporadic parathyroid adenomas.
The IDH1 gene is mutated in __% of primary GBMs and in __% of secondary GBMs.
The IDH1 gene is mutated in 80% of secondary GBMs.
The recombination frequency for 2 loci on the same chromosome is given by the Greek letter ___ and varies from (#) to (#).
The recombination frequency for 2 loci on the same chromosome is given by the Greek letter theta and varies from 0 (no recombination) to 0.5 (completely independent assortment, as if they were on different chromosomes).
The recombination frequency for 2 loci on the same chromosome is given by the Greek letter theta and varies from 0 (no recombination) to 0.5 (completely independent assortment, as if they were on different chromosomes). A smaller theta indicates that the 2 loci are closer and more tightly linked, while a theta of 0.5 indicates that the loci are completely unlinked, either by being on different chromosomes of by being so far apart on one chromosome that there will almost always be recombination. A theta of 0.01 means that there is roughtly a 1% recombination rate between the 2 alleles. This is referred to as 1 (unit).
A theta of 0.01 means that there is roughtly a 1% recombination rate between the 2 alleles. In honor of the geneticist Thomas Hunt Morgan, this is referred to as 1 centimorgan. Genetic distances are roughly additive, so if genes A, B, and C are on the same chromosome and A and B are 1 centimorgan apart, and B and C are 1 centimorgan apart, then A and C will be roughly 2 centimorgans apart and will recombine in about 2% of meiosis. However, there is only rough correspondence between genetic distance-the probability of recombination-and physical distance.
There is only rough correspondence between genetic distance-the probability of recombination-and physical distance. For long distances (high recombination rates) the observed frequencies underestimate the actual rate of recombination. Why?
Because there are situations in which a recombination is followed by a second recombination that returns the chromatids to their original state.
Genes that are within the same region, between 2 breakage-prone points, tend to be inherited as a unit called a ___.
Because crossing over is partially related to whether a region of DNA is recombination-rich or recombination-poor, some regions are not typically exchanged and other areas are exchanged with great frequency. Genes that are within the same region, between 2 breakage-prone points, tend to be inherited as a unit called a haplotype.
Do pedigrees depict genotypes or phenotypes?
Pedigrees typically depict phenotypes, not genotypes, with a few exceptions.
A genetic family history is called a pedigree and is depicted by vertical lines representing different generations and horizontal lines representing one generation. Squares represent ___, circles represent ___, and diamonds indicate ___. A diagonal line from the lower left to the upper right across a symbol indicates ___. Offspring and their mates are denoted by ___. Twins are indicated by ___. Successive generations are numbered by ___ numerals, while individuals within a generation are numbered by ___ numerals. A filled symbol indicates ___, while an open symbol indicates ___. An open symbol comtaining a dot indicates ___. A circle with a dot in the center often indicates ___. An arrow pointing to a symbol indicates ___.
A genetic family history is called a pedigree and is depicted by vertical lines representing different generations and horizontal lines representing one generation. Squares represent males, circles represent females, and diamonds indicate individuals of unknown or disguised gender. A diagonal line from the lower left to the upper right across a symbol indicates a deceased person. Offspring and their mates are denoted by a horizontal line (a sibship line) beneath a vertical line. In a sibship line, older children are depicted to the left of younger siblings Twins are indicated by diagonal lines connecting children to parents instead of a vertical line and a horizontal sibship line. A short horizontal connection between the diagonal twin line indicates that they are identical. Successive generations are numbered by Roman numerals, while individuals within a generation are numbered by Arabic numerals, starting on the left. A filled symbol indicates an affected person, while an open symbol indicates phenotypic normality, though complete openness of the symbol must not be construed as homozygosity for the wild-type genes because often, a heterozygous state will also be phenotypically normal. An open symbol comtaining a dot indicates known heterozygosity. A circle with a dot in the center often indicates a female carrier of an X–linked trait. For a dominant trait, a partially shaded individual is affected but is known to be heterozygous. An arrow pointing to a symbol indicates the proband (the person of principal study).
Congenital coagulation factor XIII deficiency. What is the genetic/molecular basis?
Congenital FXIII deficiency can be attributed to defects in both the FXIII-A and FXIII-B genes. It is an AR trait; patients with severe disease are homozygotes or compound heterozygotes. Congenital FXIII-A deficiency may be a quantitative defect (type I deficiency) or a qualitative defect (type II deficiency). Severe FXIII-B deficiency has been rarely reported.
At least 30% of paragangliomas and pheochromocytomas are associated with familial syndromes. Specific genotype-biochemical correlations highlight the importance of laboratory testing to characterize patterns of catecholamine excess. The biochemical profiles of tumors associated with mutations of genes encoding succinate dehydrogenase subunits, (collectively referred to as SDHx; x refers to all subunits, SDHA refers to subunit A, etc) are characterized by ___ and/or ___ production. VHL-related tumors are associated with ___ production. RET- and NF1-related tumors are associated with ___ production.
The biochemical profiles of tumors associated with mutations of genes encoding succinate dehydrogenase subunits, (collectively referred to as SDHx; x refers to all subunits, SDHA refers to subunit A, etc) are characterized by dopamine and/or norepinephrine production. VHL-related tumors are associated with norepinephrine production. RET- and NF1-related tumors are associated with epinephrine production.
Patients with multiple paragangliomas (including multiple pheochromocytomas in the same adrenal gland) should be evaluated for the possibility of underlying genetic susceptibility and thus genetic testing for ___ (7) mutations should be considered.
Patients with multiple paragangliomas (including multiple pheochromocytomas in the same adrenal gland) should be evaluated for the possibility of underlying genetic susceptibility and thus genetic testing for RET (rearranged during transfection), NF1 (neurofibromin 1), VHL (von Hippel-Lindau), SDHx (succinate dehydrogenase subunits, (collectively referred to as SDHx; x refers to all subunits, SDHA refers to subunit A, etc)), TMEM127 (transmembrane protein 127), MAX (MYC-associated factor-X), and KIF1Bbeta (kinesin family member 1B) mutations should be considered.
VHL-related pheochromocytomas/paragangliomas may have what unique histologic features?
VHL-related tumors often exhibit a thick vascular capsule, hyalinized and myxoid stroma, round tumor cells intermingled with small vessels, cells with predominantly amphophilic and clear cell cytoplasm, absence of intracytoplasmic hyaline globules, lipid degeneration, and lack of nuclear atypic or mitoses.
Adrenal medullary hyperplasia is a precursor lesion of pheochromocytomas arising in ___ syndrome and is characterized by a nodular and/or diffuse enlargement of the adrenal medulla.
Adrenal medullary hyperplasia is a precursor lesion of pheochromocytomas arising in MEN 2 syndrome and is characterized by a nodular and/or diffuse enlargement of the adrenal medulla. Other predisposing genetic syndromes are not usually associated with adrenal medullary hyperplasia.
Fragile X is the most frequent form of inherited mental retardation. What is the molecular basis of it?
Expansion of CGG triplet repeats in the FMR1 gene is the basis, with normal having 6 to 54 CGG repeats, 55-200 is permutation, and >200 is full mutation. Occasionally, point mutations can also cause a similar phenotype. The FMR1 gene produces the protein FMRP, which is a regulator of synaptic translation, selectively binding brain mRNAs and blocking their translation. Absence of FMRP allows unregulated translation of mRNAs, producing excessive internalization of the AMPA receptor, a signal of a weakened synapse.
What laboratory investigations are often undertaken following two or more spontaneous abortions?
Parental karyotyping (karyotyping of an abortus is often indicated as well). Endometrial bxs may be obtained to exclude luteal phase defect (endometrial histology that is 2 or more days discrepant with dates). Endometrial culture may be obtained to exclude subclinical infection with U. urealyticum or C. trachomatis. Thyroid function tests. Tests for lupus anticoagulants.
What is the CCND1 (cyclin D1) gene? Is it expressed normally in lymphocytes?
AKA BCL1 (B-cell leukemia/lymphoma 1) AKA PRAD1 (parathyroid adenomatosis 1). On 11q13.3. Encodes the cyclin D1. Mainly nuclear localization. No normal expression in lymphocytes.
t(11;14)(q13;q32) is mainly found in mantle cell lymphoma. What else is it seen in?
It is also found in B-prolymphocytic leukemia, plasma cell leukemia, and splenic lymphoma with villous lymphocytes. It is found rarely in chronic lymphocytic leukemia and multiple myeloma.
What is the BCL2 gene? Where is it expressed normally in the hematolymphoid system?
BCL2 (B-cell leukemia/lymphoma 2) is located on 18q21.33. In normal and reactive lymph nodes, it is expressed by mantle cells and the small number of mantle cells that normally permeate the follicle center. It is an inhibitor of apoptosis (although there are other members of the bcl-2 family that are pro-apoptotic), so its expression is inhibited in germinal centers, where apoptosis forms part of the B-cell production pathway.
What is the BCL6 gene? Where is it expressed normally in the hematolymphoid system?
BCL6 (B-cell Lymphoma 6) AKA BCL5, LAZ3, ZBTB27, ZNF51, and BCL6A is located on 3q27.3. The gene product is a critical transcription factor responsible for normal secondary follicle formation during the germinal center reaction in lymphoid tissues and for proper T-cell-dependent Ab responses upon exposure to Ag. Accordingly, BCL6 is strongly expressed by germinal center B cells (GCBs) but is not present in naive or post-GCBs. BCL6 is thought to protect normal GCBs during the affinity maturation process (i.e., somatic hypermutation) in part by downregulating proapoptotic stimuli elicited by physiologic DNA double-strand breakage.
How does copy-number neutral loss of heterozygosity or uniparental disomy occur?
It results from the loss of genetic material on one chromosome and subsequent duplication of the missing material from the other chromosome.
What is the most common cytogenetic abnormality seen in hepatosplenic T-cell lymphoma?
Isochromosome 7q.
What is 5q- syndrome?
The 5q- syndrome is a myelodysplastic syndrome characterized by a defect in erythroid differentiation. Patients have severe macrocytic anemia, normal or elevated platelet counts, normal or reduced neutrophil counts, erythroid hypoplasia in the bone marrow, and hypolobated micromegakaryocytes. It is seen mostly in middle-aged to older women. Survival is usually long and cytogenetic evolution is uncommon.
90% of AML have chromosomal abnormalities. What cytogenetic findings are favorable, intermediate, and unfavorable?
Favorable Cytogenetics: inv(16)(p13;q22), t(8;21)(q22;q22), t(15;17)(q22;q12). Intermediate Cytogenetics: +8, t(6;9)(p23;q34), t(9;11)(p22;q23) in children, normal cytogenetics. Unfavorable Cytogenetics: -7, -5, del 7q, t(11q23), inv(3q), t(9;22), complex abnormalities, post-chemotherapy or post-radiation therapy.
90% of AML have chromosomal abnormalities. What cytogenetic findings from the following are favorable, intermediate, and unfavorable? Complex abnormalities. t(9;11)(p22;q23). +8. t(8;21)(q22;q22). -7. Post-chemotherapy or post-radiation therapy. -5. t(6;9)(p23;q34). t(9;22). Normal cytogenetics. inv(3q). inv(16)(p13;q22). t(11q23). -del 7q. t(15;17)(q22;q12).
Favorable Cytogenetics: inv(16)(p13;q22), t(8;21)(q22;q22), t(15;17)(q22;q12). Intermediate Cytogenetics: +8, t(6;9)(p23;q34), t(9;11)(p22;q23) in children, normal cytogenetics. Unfavorable Cytogenetics: -7, -5, del 7q, t(11q23), inv(3q), t(9;22), complex abnormalities, post-chemotherapy or post-radiation therapy.
For precursor B-cell acute lymphoblastic leukemia, which chromosomal abnormalities are low risk, which are standard risk, and which are high risk in regards to prognostic significance? t(4;11)(q21;q23). t(12;21)(p12;q22). t(9;22)(q34;q11). t(1;19)(q23;p13). Hyperdiploidy >50.
Low risk: Hyperdiploidy >50 and t(12;21)(p12;q22). Standard risk: t(1;19)(q23;p13). High risk: t(4;11)(q21;q23) and t(9;22)(q34;q11).
What is the BCL6 gene? Where is it expressed normally in the hematolymphoid system?
BCL6 (B-cell Lymphoma 6) AKA BCL5, LAZ3, ZBTB27, ZNF51, and BCL6A is located on 3q27.3. The gene product is a critical transcription factor responsible for normal secondary follicle formation during the germinal center reaction in lymphoid tissues and for proper T-cell-dependent Ab responses upon exposure to Ag. Accordingly, BCL6 is strongly expressed by germinal center B cells (GCBs) but is not present in naive or post-GCBs. BCL6 is thought to protect normal GCBs during the affinity maturation process (i.e., somatic hypermutation) in part by downregulating proapoptotic stimuli elicited by physiologic DNA double-strand breakage.
What is the BCL2 gene? Where is it expressed normally in the hematolymphoid system?
BCL2 (B-cell leukemia/lymphoma 2) is located on 18q21.33. In normal and reactive lymph nodes, it is expressed by mantle cells and the small number of mantle cells that normally permeate the follicle center. It is an inhibitor of apoptosis (although there are other members of the bcl-2 family that are pro-apoptotic), so its expression is inhibited in germinal centers, where apoptosis forms part of the B-cell production pathway.
t(11;14)(q13;q32) is mainly found in mantle cell lymphoma. What else is it seen in?
It is also found in B-prolymphocytic leukemia, plasma cell leukemia, and splenic lymphoma with villous lymphocytes. It is found rarely in chronic lymphocytic leukemia and multiple myeloma.
What is the CCND1 (cyclin D1) gene? Is it expressed normally in lymphocytes?
AKA BCL1 (B-cell leukemia/lymphoma 1) AKA PRAD1 (parathyroid adenomatosis 1). On 11q13.3. Encodes the cyclin D1. Mainly nuclear localization. No normal expression in lymphocytes.
What are CD45 isoforms?
The CD45 family consists of multiple members that are all products of a single complex gene (PRPRC). Different subsets of hematopoietic cells express different CD45 isoforms due to variable exon splicing, which can change in response to cytokines. The gene contains 34 exons and three exons of the primary transcripts (these three exons generate the RA, RB and RC isoforms) are alternatively spliced to generate up to eight different mature mRNAs and after translation eight different protein products. The isoforms are: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC).
What is CD59? How is it related to hematology?
AKA protectin, complement regulatory molecule, MAC-inhibitory protein (MAC-IP), or membrane inhibitor of reactive lysis (MIRL). Regulates complement mediated cell lysis by inhibiting formation of membrane attack complex (MAC); binds to C8 or C9 components, preventing incorporation of multiple copies of C9 required for complete formation of osmolytic core. It is present on nearly all human cells. Decreased cell surface expression, along with decreased CD55 - DAF, is a feature of the affected clone in paroxysmal nocturnal hemoglobinuria.
Genetic defects that reduce both CD__ and CD__ on erythrocytes produce paroxysmal nocturnal hemoglobinuria.
Genetic defects that reduce both CD55 and CD59 on erythrocytes produce PNH.
Genetic defects that reduce both CD55 and CD59 on erythrocytes produce what disease?
Genetic defects that reduce both CD55 and CD59 on erythrocytes produce paroxysmal nocturnal hemoglobinuria.
What are the most common genetic abnormalities seen in T cell prolymphocytic leukemia?
Rearrangements involving TCL1 (at 14q32.1) are common and relatively specific for T-PLL. These may take the form of either inv 14(q11;q32) or t(14;14)(q11;q32). Another abnormality, t(X;14)(q28;q11), involves a homolog of TCL1, MTCP1 (mature T cell proliferation 1 gene) that is on Xq28. Overall, approximately 90 percent of cases demonstrate chromosome abnormalities involving chromosome 14. Also, abnormalities in chromosome 8 are seen in ~75% of cases, including idic(8p11), t(8;8), and trisomy 8q.
Alkylating agent-related acute myeloid leukemia is characterized by cytogenetic abnormalities similar to those seen in what hematologic disorder?
Alkylating agent-related acute myeloid leukemia is characterized by initial presentation as a myelodysplastic syndrome and cytogenetic abnormalities similar to those seen in myelodysplastic syndrome. The risk is related to total cumulative dose and survival is short.
For the 2 main categories of therapy-related myeloid neoplasms: Those caused by (1) alkylating agents or radiotherapy or (2) topoisomerase II inhibitor therapy, how soon do they present, what myeloid neoplasms do they present with, and what cytogenetic abnormalities are typically seen?
(1) Alkylating agents: Occurs median 5 years after initiation (range, 2-11 years). Presents as MDS, AML or MDS/MPN (CMML). Abnormalities of chromosomes 5 or 7 or complex cytogenetic abnormalities. (2) Topoisomerase II inhibitor therapy: Abrupt onset
The BCL1 gene is rearranged in virtually all cases of ___ lymphoma. The BCL2 gene is rearranged in up to 95% of ___ lymphoma. The BCL6 gene is rearranged in up to 30% of ___ lymphoma. BCL10 gene rearrangements have been reported in ___ lymphoma.
The BCL1 gene is rearranged in virtually all cases of mantle cell lymphoma and in some plasma cell neoplasms. The BCL2 gene is rearranged in up to 95% of follicular lymphoma. The BCL6 gene is rearranged in up to 30% of diffuse large B cell lymphoma. BCL10 gene rearrangements have been reported in extranodal MALT lymphoma, but are not characteristic of any lymphoma.
What cytogenetic abnormalities can be seen in marginal zone lymphoma, MALT type?
t(11;18)(q21;q21). t(14;18)(q32;q21). Trisomy 3. Trisomy 18. t(1;14)(p22;q32). t(1;2)(p22;p12).
In the germline configuration, what are the 4 segments of DNA that will encode the IgH chain called, what are the 3 segments that will encode the kappa light chain called, and what are the 3 segments that will encode the lambda light chain called?
The segments of DNA that will encode the IgH chain are V (variable), D (diversity), J (joining), and C (constant). The segments that will encode the kappa and lambda light chains are V, J, and C.
