19_Hematological Malignancies Flashcards
1
Q
How does Clonal Hematopoiesis (CHIP) develop? Which biopsy samples are required for diagnosis?
A
- Some of the hematopoietic stem cells develop mutations that are also seen in hem cancers (particularly MDS).
- As we age, blood represents fewer clones and some clones with these muts can become predominant and make us think that the patient carries a germline mut! Skin biopsy is needed for confirmation of germline status in these cases
2
Q
What mutated genes are involved CHIP?
A
- Most mutated genes involved in CHIP are indeed assoicated with epigenetic regulatory which include TET2, DNMT3A, JAK2, and ASXL1, as well as SF3B1 and TP53.
- The same mutations can occur as pre-leukemic events
3
Q
Why mutation screening is not used for MDS detection in CHIP cases?
A
CHIP is associated with increased risk of hem malignancy, but most do not develop malignancy.
As such, mutation screening is not used for MDS detection
4
Q
Schematic description of Hematopoeisis in humans:
A
5
Q
Depict development of different blood cells from haematopoietic stem cell to mature cells.
A
6
Q
Some terminologies in hematology and blood disorders:
A
- Penia means loss of cells;
- cytosis mean increased count, as in leukopenia and leukocytosis;
- ia means increased number of granular cells such as neutrophilia, eosinophilia, etc.
7
Q
How leukocytes (WBCs) are classified in terms of granularity?
A
- Granular (neutrophil, basophil, and eosinophil)
- Non-granular (monocyte and lymphocyte)
8
Q
Where does CML originate from in the hierarchy of Haematopoiesis
A
CML originates somewhere between the hematopoietic stem cell (first top cell in the haematopoiesis diagram) and the next two levels, but most often takes the path to become a lymphoid tumor.
Note: Depending on the original stem cell the tumor can be myeloid or lymphoid
9
Q
What is the Cyto analysis guideline for heme cancers?
A
- The specimen of choice is BM.
- However, if adequate BM can’t be collected, unstimulated peripheral blood can yield good results when circulating blast count is > 10-20%.
10
Q
List the Myeloid (chronic) hematological malignancies.
A
- Myelodysplastic syndromes (MDS)
- Myeloproliferative neoplasms (MPN)
11
Q
Describe the cyto features, associated cancer ristks, and age for diagnosis in Myelodysplastic syndromes (MDS).
A
- Dysplasia in ≥1 myeloid lineage [cells look strange; BM can be hyper- or normo-cellular];
- increase in blast but <20% (>20% is called AML);
- cytopenia & ineffective hematopoiesis [because cells look abnormal so there is more apoptosis and peripheral blood counts is low with ≥1 cytopenia];
- increased AML risk;
- diagnosis at old age.
12
Q
What are the recurrent, MDS-defining, and non MDS defining cyto cahnges in MDS?
A
- Half cases have recurrent cyto, including +8, -7/7q, -5/5q, -20q, -Y, i(17q)/t(17p) (most common ones, each ~5-10%).
- -5/5q, -7/7q/t(7q), -11q, -12p/t(12p), -13/13q, i(17q)/del(17p)/t(17p), idic(X)(q13), and complex karyotype are MDS-defining [Dx of MDS in the absence of morphology].
- +8/+15/-20q/-Y are common in elderly and not MDS-defining.
13
Q
Explain the relavence of cyto changes and prognosis in MDS.
A
- -Y / -11q alone have ‘very good’ px.
- NL karyotype, -5q, -12p, -20q have good px.
- -7q, +8, +19, i(17) are intermed.
- -7, inv(3), t(3), del(3q) are poor.
- Complex karyotype (>3) is very poor.
14
Q
Describe the MDS Cytogenetic Scoring System.
A
15
Q
What informatoion is provided by Revised international prognostic scoring system (IPSS-R)?
A
- MDS-defining changes: 5/5q-, 7-, 11q-, i(17q), 13/13q-, complex (≥3 abnormalities)
- Microarray has been found useful to further stratify risk.
- IPSS-R low/intermediate risk group had worse px if they had abnl CMA;
- Also, some 5q dels can’t be found by karyotype alone and you need CMA;
- CMA is also needed when karyotype fails or is non-informative, and for detection of LOH, determining clonality (using LOH).
- As such, NCCN recommends Karyotype first for MDS, and if you can’t get 20
16
Q
Which genes are mutated in MDS? What is the relevance of these mutations to disease severity, prognosis, and progression?
A
- SF3B1, TET2, RUNX1, ASXL1, and DNMT3A are the most common mutated genes (each >10%);
- Muts in ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2,
U2AF1, ZRSR2 are MDS-defining. - TP53 means aggressive disease;
- SF3B1 (involved in RNA splicing) means “MDS with ring
sideroblasts”. - The only gene mut associated with good px is SF3B1. All others have poor/conflicting px.
- Mutations in RAS, FLT3, JAK2, NF1, RUNX1, ETV6, SETBP1 are involved in disease progression.
Note: Seq variants in MDS should be dealt with caution due to CHIP.
17
Q
What are WHO MDS categories are defined by genetics?
A
- MDS with SF3B1 muts: ring sideroblasts >15%; good px
- MDS with biallelic TP53 inactivation
- MDS with isolated del(5q): the only category defined by
cyto; It responds to Lenalidomide with good px, but
should test for TP53 before administration, because this
medication leads to expansion of pre-leukemic TP53-mutant clones due to selective degradation of Ck1α.
18
Q
Other than MDS with SF3B1 muts, biallelic TP53 inactivation, and isolated del(5q), what other categories fall under MDS?
A
- Chronic myelomonocytic leukemia (Myelodysplastic CMML [MD-CMML])
- Therapy induced MDS
19
Q
Which muts are associated with Chronic myelomonocytic leukemia (Myelodysplastic CMML [MD-CMML]?
A
- muts of epigenetic control (e.g., TET2, ASXL1 [45%,
histone modifier]), - muts of pre-mRNA splicing (e.g., SRSF2, U2AF1)
- muts of cell signaling (e.g., NRAS, KRAS, CBL or JAK2).
20
Q
What are the recurrent and most common cyto abnormalities associated with therapy induced MDS?
A
In therapy-related MDS:
* -7/7q, -5q, and -Y each happen in 25-50%.
* del(5q) is seen in about 50% of cases.
* i(17q) is a recurrent abnormality that is much more common (25-30% vs 3- 5%).
21
Q
Among therapy-related MDSs, what cyto abnormalities are the typical of alkylating agent-induced MDS?
A
Complex karyotypes with loss/deletion of chromosomes 5 and/or 7 together with deletions of 6p, 12p, and/or 16q are typical of alkylating agent-induced MDS
22
Q
Among therapy-related MDSs, what cyto abnormalities are associated with preceding therapy with DNA topoisomerase II inhibitors.
A
Balanced translocations involving 11q23 (MLL) and
21q22.3 (RUNX1) or t(3;21)(RUNX1-MECOM)
23
Q
What are the hematologic features of Myeloproliferative neoplasms (MPNs)?
A
- clonal proliferation of ≥1 myeloid lineage [granulocyte, erythrocyte, megakaryocyte];
- relatively mature cells and terminally differentiated but issue is you have too many of them and they accumulate in BM, liver, spleen, etc.;
- high blood counts and hepatosplenomegaly;
Note: most in 5-7th decade
24
Q
What are the different types of Myeloproliferative neoplasms (MPNs)?
A
- Chronic myeloid leukemia (CML), BCR-ABL positive
- Polycythemia Vera (PV)
- Primary myelofibrosis (PMF)
- Essential thrombocytopenia (ET)
- Chronic neutrophilic leukemia (CNL)
- Chronic Eosinophilic Leukemia (CEL)
- Juvenile MyeloMonocytic Leukemia (JMML)
25
Are MPNs distinguishable from MDS or MDS/MPN spectrum? why?
They are distinguishable from MDS or MDS/MPN spectrum, since morphologic dysplasia [abnormal cell shape] is not prominent in MPN unlike in MDS or MDS/MPN spectrum.
26
Which muts are associated with MPNs?
* Most MPN’s either have BCR-ABL or JAK2/CALR/MPL.
* 5-10% of BCR-ABL neg MPN is also triple neg for JAK2/CALR/MPL.
* Many non-driver muts are also present in MPN (genes in methylation pathway, splicing, TFs, cell signaling).
27
What is the most common form of MPN? What are its molecular/cyto/clinical features?
**Chronic myeloid leukemia (CML), BCR-ABL positive**
* 15% of all leukemias;
* mostly in older adults;
* increased WBC and decreased Hb;
* BM biopsy is performed;
* leukemogenesis occurs at the pluripotent stem cell level;
28
What are the three phases of Chronic myeloid leukemia (CML), BCR-ABL positive?
1. Chronic
2. Accelerated
3. Blasic
29
Describe the **chronic** phase of Chronic myeloid leukemia (CML), BCR-ABL positive.
* clonal expansion but mature cells;
* asymptomatic if treated;
* duration>25 years;
* usually CML is diagnosed at this stage;
* BCR-ABL1 is present in the very early hematopoietic
stem cells before division to lymphoid or myeloid (initiating
event).
* The fusion forces the stem cell to produce more
granulocytes using the myeloid line [mostly Neutrophils].
30
Describe the **accelerated** phase of chronic myeloid leukemia (CML), BCR-ABL positive.
* progressively impaired cell maturation;
* acquiring of new muts;
* blasts >15%,
* duration 4-5 yrs.
31
Describe the **blastic** phase of chronic myeloid leukemia (CML), BCR-ABL positive.
* blasts ≥20% in blood or BM, 6-12 mo. duration;
* the blasts are either myeloid [2/3, AML] or lymphoid [1/3, ALL]);
* Acquiring new muts in the stem cell, which occurred in
accelerated phase, stops the cells from maturation.
* Proliferation occurs in both lines of myeloid and
lymphoid this time. That’s why you can have either AML or ALL.
32
Cyto abnormalites in at different phases of CML:
In the chronic phase of CML,
we only see t(9;22), but as the
disease transforms into
accelerated and blastic phases
other changes are required,
like +8, i(17)(q10), double Ph,
+19, indicating clonal evolution
(80% of cases).
33
What are the features of Philadelphia Chr [der(22)t(9;22)(BCR on chr22 :: ABL1 on chr9)]?
* is seen in almost all cases,
* often M-BCR (p210 – major breakpoint)
and rarely m-BCR (p190) or μ-BCR (p230).
* Occasionally due to 3-way translocation or cryptic [not seen by cyto – like a small ins of 9 into 22];
* Ph Chr is also seen in AML but there it is rare and has bad px.
* Minor breakpoint (p190) is the predominant form in ALL
* BCR-ABL fusion has higher TK phosphorylation activity [ABL tail] and is treated with TKI drugs, like imatinib, nilotinib, etc. [good px].
34
What are the detection methods for BCR-ABL fusion?
* FISH
* Karyotype
* RT-qPCR
35
Which methods are used to measure the response to treatment in BCR-ABL fusion?
* molecular
* cyto
* hemato methods (reduction in fusion transcript [3 log reduction by qPCR, meaning you go from 1.5 to 0.0015 for ratio of BCR-ABL to a control gene], absence of translocation in cyto, and reduction in blast);
Note: For RT-PCR, sample should
arrive within 24hrs to avoid RNA degradation.
36
What is the instruction for Sanger and NGS performed to find resistance mutations in BCR-ABL fusion?
* Sanger and NGS are performed to find resistance mutations in the kinase domain of ABL (T315I) of the BCR-ABL fusion.
* First, LR-PCR of the reverse-transcription of the fused gene is performed, followed by PCR amplification of the kinase domain of the ABL1 (not BCR!) before sanger/mutation analysis.
* Based on the mutation, the medication can be changed.
37
What is the response rate to TKI in Ph chr? What is the ultimate cure?
* Response rate by TKI is 80% in chronic phase but lower/shorter in accelerated/blastic phase.
* The ultimate cure is stem cell transplant.
38
Why multiple PCRs are required for BCR-ABL detection?
For BCR-ABL reverse transcriptase PCR, A couple of pairs of primers usually are used to yield PCR products of different sizes to cover different breakpoints. Therefore the test needs multiple PCRs.
39
Describe cyto and molecular features of Polycythemia Vera (PV).
* increased RBC;
* hypercellular marrow;
* JAK2 V617F (ex14, 95%) or JAK2 ex12-15 GOF muts (2%; only in PV), disrupting autoinhibitory loop, resulting in constitutive activation of JAK2-STAT pathway;
40
What is the testing guildeline for Polycythemia Vera (PV)?
For testing usually, ppl do allele specific PCR (for V617) + Sanger of ex12 for different muts; Rx: JAK2 inhibitor
41
What are the cyto and molecular features of Primary myelofibrosis (PMF):
* Increased MKcytes (platelet) and granulocytes with
fibrosis and scar in BM and impaired normal cell production;
* JAK2 V617F (60%), CALR ex9 indel (20%), MPL ex10 W515 (5%).
Note: M=F
42
What are the cyto, molecular, and clinical features of Essential thrombocytopenia (ET)?
* increased MKcytes (platelet/thrombocytosis) without fibrosis [fibrosis can happen over many years thou];
* marrow proliferation;
* JAK2 V617F (60%), CALR ex9 indel (20%), MPL ex10
W515 (5%).
* recurrent anemia, stroke, DVT, PE, hemorrhage;
43
What do you know about CALR mutations and their relavence with ET prognosis?
* CALR is a quality check pr in endoplasmic reticulum that binds misfolded proteins and stops their export to Golgi.
* The indels are type I (52bp del, c.1092_1143del, p.L367fs*46, 50%) and type II (5bp ins, c.1154_1155insTTGCC, p.K385fs*47,
30%).
* They are both frameshift but don’t lead to NMD since they are in the 3’ end of the gene.
* They result in an open reading frame [poz charge] and
remove the repetitive KDEL seq (proline rich, neg charge) in 3’ leading to higher affinity to MPL, leading to JAK/STAT activation.
* Detection is done using fluorescent PCR followed by capillary
electrophoresis to measure PCR size of exon 9.
* Note that a 9bp Del is a common polymorphism in
this region which is detected in Het status (50-50 signal intensity) and is not reportable but confounds your results!
***CALR muts have better px and lower risk of thrombosis compared to JAK2.***
44
What is the biologic roles of MPL?
MPL encodes thrombopoietin receptor (aka TPOR), regulator of megakaryocyte growth and survival.
45
What do MLP mutations lead to?
* Its mutations (W515L/K/R) lead to constitutive activation of TPOR in a cytokine independent fashion and activation of JAK-STAT pathway.
* Germline MPL muts lead to “Congenital Amegakaryocytic Thrombocytopenia”.
46
What is the prognosis for different categories of MLP?
* There is a triple negative category in ET/PMF (JAK2/CALR/MPL), which has worse px.
* cnLOH happens commonly around MPL and JAK2.
* Other ET/PMF muts occurs in ASXL1, EZH2, IDH1/2, SRSF2, TP53, U2AF1, alongside triple genes or alone, and most have poor px.
47
How it could be ensured that 90% of MLP cases are detected?
To ensure > 90% of cases are detected, the adequate analytical sensitivity of a clinical JAK2 (or other) assay must be at least 1% (mutation rate).
48
What are the current methods in use for MLP detection?
* Current methods in use include: RFLP, dHPLC, high res melting curve analysis, pyrosequencing, and various allelespecific PCR systems with electrophoretic analysis of the products.
* Most of these methods typically do not achieve sensitivities of less than 5% of alleles.
49
Compare the clinical, molecular, and cytogenetic features of PMF, ET, PV.
* Cyto abnormalities are recurrent but are not pathognomonic.
* JAK2/MPL/CALR muts are mutually exclusive.
* MPL muts happen in 8% of PMF, half of that in ET, but never in PV.
* CALR muts happen in 20-25% of ET/PMF and never in PV.
* JAK2 muts happen in 60% of PMF/ET and 97% of PV.
50
Which muts drive Chronic neutrophilic leukemia (CNL)? What is the treatment approach?
* Pathognomonic activating CSF3R ex17(14?) muts in transmembrane domain including T618I/T615A in >80% of cases;
* targeted therapy
51
What are the most relevant genetic abnormalities associated with Chronic Eosinophilic Leukemia (CEL)?
* PDGFRA/B, and FGFR1 rearrangements, or PCM1-JAK2;
* discussed in “other heme cancers”.
52
What are the genetic features of Juvenile MyeloMonocytic Leukemia (JMML)
* RAS pathway activation driven (90%);
* most commonly muts in PTPN11 (35-40%), NRAS (15-25%) and KRAS (15-20%), etc.
* Different gene mutations are mutually exclusive in JMML.
* Origin of mutations [when somatic] can be traced back to utero.
53
Testing algorithm for myeloproliferative neoplasms:
54
How does the Acute myeloid leukemia (acute - AML) develop and what are the clinical signs?
* Acute myeloid leukemia is clonal expansion of myeloid precursor cells with reduced capacity to differentiate (≥20% blasts), leading to impaired hematopoiesis and bone marrow failure.
* Anemia,
* Thrombocytopenia,
* Neutropenia,
* bone pain;
* occurrence at older age
55
What is the sequence of mutational events in developing acute AML?
* initial mutation(s) freeze a myoblast in an immature state (undifferentiated)
* subsequent mutation(s) dysregulate proliferation.
56
What is the underlying reason for AML heterogeneity?
AML heterogeneity is explained by differentiation arrest occurring at different steps along the pathway
57
What are the classes of genes
mutated in AML?
1) signaling [RTK-RAS],
2) 2) TFs,
3) Epigenetic regulators,
4) Tumor suppressors
5) RNA maturation and splicing factors.
**Different combinations of these can be present in AML.
58
How are mutations classified in AML?
AML Mutations can be classified into two classes:
* Type I) survival advantage: RAS, PTPN11, FLT3ITD;
* Type II) Differentiation arrest: PML-RARA, AML1-ETO, NPM1, and CEBPA.
59
What is the testing protocol for every AML patient?
Every patient with AML gets tested by
FISH for core-binding factor genes and three MDS defining genes.
60
What FISH probes are used in AML?
* KMT2A (seen rarely amplified by double minutes),
* RUNX1T1-RUNX1,
* CBFB-MYH11,
* NUP98,
* PML-RARA,
* 7/5/20,
* EVI1/MECOM,
* case-dependent probes
61
What molecular testings could be used for AML diagnosis?
* Molecular testing can include **NGS panel** but **single gene testing** of FLT3, NPM1, CEBPA can be done for quick TAT [IDH1/2, TET2, and DNMT3A can also be included].
* NGS panels can be used for both first dx and relapse to study clonal evolution, tumor heterogeneity, finding therapeutic
targets, and eligibility for clinical trials.
* NGS is not used for disease monitoring for clinical remission due to high background noise, inability to detect low level variants, and high cost for repeated use.
62
According to WHO 5th edition, presence of which AML-defining genetic changes is considered AML with no need for blast count?
Exception is AML with BCR::ABL1, CEBPA mutation, and MDS-related which require at least 20% blasts:
1. Good px: APL with PML::RARA fusion: t(15;17)(PML-RARA)
2. Good px: AML with RUNX1::RUNX1T1 fusion: t(8;21)(RUNX1-RUNX1T1 [AML1-ETO]) – Core binding factor AML
3. Good px: AML with CBFB::MYH11 fusion: inv(16)(p13;q22)/t(16;16)(p13;q22)(CBFB-MYH11) – Core binding factor AML
4. Poor px: AML with DEK::NUP214
5. Poor px: AML with RBM15::MRTFA
6. Poor px: AML with BCR::ABL1
7. Intermed. px: AML with KMT2A rearr.
8. Poor px: AML with MECOM rearr.
9. Poor px: AML with NUP98 (11p15.4) rearr.
10. Good px: AML with NPM1 mutation
11. Good px: AML with CEBPA mutation
12. Poor px: AML, MDS-related
13. AML with other defined changes
Note: Only PML-RARA, RUNX1-RUNXT1, CBFBMYH11,
and CEBPA/NPM1 muts have good px.
63
What are the features of AML with t(15;17); PML::RARA; aka acute promyelocytic leukemia (APL/APML)?
* occurs due to differentiation stop at promyelocyte step;
* slide with hyper granular promyelocytes and bundles of Auer rods;
* good px;
* This is STAT since it causes disseminated intravascular
coagulation (DIC);
* treatment with all-trans retinoic acid (ATRA) once confirmed.
* ITD and tyrosine kinase domain muts also frequently occur in APL.
* Detection of CD56 expression means less favorable;
* FISH is used for PML-RARA;
* RT-PCR must be used with different primer sets (there are both short and long forms).
64
Wher other fusions with RARA can be seen in APL?
* Other fusions with RARA can be seen if t(15;17) is absent (~2% of times) with NPM1, ZBTB16, NUMA1, STAT5B, PRKR1, ECOR, FIP1L1.
* The underlined ones are resistant to ATRA, so if you don’t see the expected pattern in dual fusion dual color FISH for PML-RARA, be cautious that RARA may be fused with something else [there is no yellow fusion color for PML-RARA, but you see 3 RARA signals];
* in these cases, you can use break-apart FISH or do karyotype.
65
What are the biological roles of RARA (Retinoic Acid Receptor Alpha)?
* RARA is Receptor for retinoic acid,
* it functions in cell growth, differentiation, and the formation
of organs in embryonic development.
66
What are the features of AML with t(8;21)(q21;q22), RUNX1::RUNX1T1 (AML1-ETO)?
* good px;
* contains additional chr abnormalities;
* NRAS/KRAS muts are common (30%);
* KIT muts (25%);
* presence of CD56 expression or KIT muts means poor px.
* The fused protein leads to suppression of RUNX1 target genes by recruitment of incorrect TFs
67
Interpret depicted test results.
The first two are typical t(8;21), but the last one is atypical.
Board will ask about next step. Answer is metaphase FISH and karyo.
This was a 3-way t(8;17;21).
68
How does the fused protein in AML with t(8;21)(q21;q22), RUNX1::RUNX1T1 (AML1-ETO contribute to disease formation?
* RUNX1 (aka AML1) is on chr8 and RUNX1T1 (aka ETO) is on chr21.
* The fused protein on der(8) is the driver part which associates into the CFB complex, resulting in abnormal regulation of target genes.
* RUNX1T1 is a core-binding factor (CBF).
69
What are the molecular and prognotsic features of AML with inv(16)(p13q22)/t(16;16)(CBFB-MYH11)?
* good px;
* Poor px when KIT is mutated.
* CBFB (core-binding factor beta) break apart probe is used.
* Other mutations: NRAS (in 45%), KRAS (in 13%), FLT3 (in 14%). CBFB is another Core binding factor.
70
What is the contribution of mutant KIT in forming CBF AMLs features?
* CBF AMLs have worse prognosis when KIT is mutated.
* KIT is mutated in 8% of AML, primarily in exon 17, the activation loop of the kinase domain.
* KIT mutations fall into class I of the “two-hit” theory of leukemogenesis as a tumor suppressor gene.
* In GIST, unlike AML, somatic KIT mutations are spread across the gene.
Note: Inv/t(16) and t(8;21) are CBF AML
71
What are the the cyto, molecular, and prognistic features of AML with NPM1 mutation?
* good px;
* prognosis gets bad if FLT3-ITD is present.
* Mostly 4bp dup/ins fs seen in 30% of AML [often cytogenetically NL];
* occurring later in disease process, and not present in preleukemic state;
72
Through which pathogenic mechanisms NPM1 muts contribute to AML?
* NPM1 is a nuclear chaperone and mutations result in loss of its nuclear localization signal, so it ends up in cytoplasm and interacts and mis-localizes with transcription factor PU1 (gain of function mechanism);
* Often it co-occurs with muts in methylation machinery genes [73%];
* during clonal evolution, in up to 10% of time the NPM1 subclone is gone;
* Dx can be done by PCR and fragment size analysis using capillary electrophoresis.
* NPM1 does not have TK domain.
73
What are the cyto, molecular, and prognistic features of AML with CEBPA?
* good px;
* 20% blast is required for this category.
* Often cytogenetically NL;
* Previously one truncating in 5’ and one missense in 3’ in trans was needed;
* Now only the 3’ mutation (missense in bZip domain, monoallelic or biallelic) is included in WHO guidelines as a prognostic entity.
* CEBPA has no intron! Mutations in bZip domain (C-Terminal) are often in-frame or missense. The N-terminal muts are instead usually frameshift and lead to usage of a shorter isoform (30 kDa instead of 42 kDa) which has dominant negative effect on the 42-kDa isoform.
74
What are the molecular, prognostic and diagnostic feature of AML with translocations of KMT2A (11q23)?
What is the prognosis of AML in a patient with the presented karyotype?
* poor px;
* KMT2A has >100 partners [MLLT3>MLLT10>ELL>AFDN>MLLT1>…] of which
only t(9;11)(p22;q23)(MLLT3-KMT2A[MLL]) has intermed. px; others are poor;
* Use breakapart FISH since fusions are cryptic
75
Is MLL (KMT2A) necessarily fused with a partner gene in all AML patients?
In some patients with AML, MLL (KMT2A) is not fused with a partner gene but rather is elongated with a partial tandem duplication (PTD, in frame) of exons 11-5 or 12-5 (former exon designations were 6-2 and 8-2).
76
How Leukemia-cell RNA with the MLL-PTD can be detected?
* With RT-PCR using primers that flank the duplication repeat.
* This RT-PCR method has a greater sensitivity compared to genomic DNA amplification
and Southern blot.
* Note that this is not considered AML with KMT2A rearrangements!
77
What are the features of AML with t(1;22)(RBM15-MKL1 [MRTFA])?
* Megakaryoblastic;
* more in kids;
* rare (<1% of AML’s);
* poor px
78
What are the features of AML with t(6;9)(p23;q34); DEK-NUP214?
* poor px;
* FLT3-ITD is common (~70%)
79
What is the most common chromosomal abnormality in AML with MECOM (3q26.2) rearrangements?
* most common (40%) is inv(3)(q21.3q26.2)/t(3;3)(q21.3;q26.2), both leading to GATA2-MECOM (bringing GATA2 enhancer near MECOM, causing MECOM overexpression and GATA2 haploinsufficiency);
* In other cases t(3;v)(q26.2;v) can happen;
PS: poor px; mostly adults; RAS pathway mutations are found.
80
Which specific chromosomal abnormality has a high frequency in AML with MECOM (3q26.2) rearrangements?
Chr abnormalities include monosomy 7 (>50%), followed by 5q
deletions and complex karyotypes.
81
What are the features of AML with t(9;22)?
* poor px;
* <5% of AML;
* 20% blast is needed.
* M-BCR like in MDS
82
What are the features of AML with NUP98 (11p15.4) rearrangements?
* poor px;
* >40 partners;
* 2-5% of kids AML;
* cryptic since located at the end of 11p15.4
83
What are the WHO and ICC requirements for the diagnosis of AML with MDS-related changes?
* >20% blast by WHO and >10% by ICC is needed for
Dx.
* poor px
84
What are the MDS defining findings for AML with MDS-related changes?
The MDS defining findings include cyto, gene mutations, or TP53 muts (see MDS section).
85
?Which specific treatments are most effective for patients with AML with MDS-related changes?
They respond better to VYXEOS (liposomal Daunorubicin and cytarabine) [liposomal formulation of standard chemotherapy].
86
What are the features of Therapy-related AML (t-AML)?
* poor px,
* VYXEOS is administered;
* most have TP53 muts
87
What chromosomal abnormalities and mutations are mainly detected in AML associated with Alkylating agents?
* loss of 5, 7, 17, 18, 21,
* mutations in RUNX1, RAS, TP53
88
Whar are the main features of AML with no cytogenetic changes?
* up to 50% of all AML – most common type;
* very heterogeneous and can be due to many mutations with variable px;
* as we don’t know the drivers, we call them intermediate px.
89
What are the main features of AML with complex cytogenetic changes (≥3)?
* poor px;
* associated with TP53 muts.
90
What are the main features of AML with monosomal karyotype?
* 10% of cases;
* at least 2 autosomal monosomies (sex Chr doesn’t count) or 1 autosomal monosomy
+ structural abnl;
* poor px.
91
What are the main features of AML with Trisomy 8?
* usually older male;
* lower WBC count;
* often with somatic ASXL1 (ex12) and RUNX1 muts but less FLT3-ITD or
CEBPA muts;
* ASXL1 alone has poor px.
92
What are the msin features of AML with somatic mutations in ASXL1?
* Somatic mutations in ASXL1 have been described in all types of myeloid malignancies.
* They occur in exon 13 (referred to as exon 12 in many publications) and are frequently frameshift or nonsense mutations that result in C-terminal truncation of the protein
upstream of the plant homeodomain finger region.
They are associated with poor px
93
Is RUNX a proto-oncogene or tumor suppressor gene?
* It is unclear whether RUNX1 is a proto-oncogene or tumor suppressor gene.
* In human leukemia, RUNX1 is involved in various chromosomal translocations. Moreover, structurally intact RUNX1 is also oncogenic when overexpressed. For example, RUNX1
duplication/amplification has also been reported in AML.
* AML patients with isolated trisomy 8 more often harbor acquired pathogenic variants in the ASXL1 and RUNX1 genes.
* RUNX1 mutations are associated with poor px.
94
How is the prognosis of AML with mutations in ASXL1, TP53, RUNX1, DNMT3A, WT1?
poor px.
95
How many percent of AML cases are impacted by Mutated chromatin or RNA-splicing genes occur and what are the most common genes from this group?
* Mutated chromatin or RNA-splicing genes occur in 18% of AML.
* RUNXT1, MLL, and SRSF2 are the most common from this group.
96
AML in Down syndrome and DS-related myeloid proliferation
* read in inherited cancer section;
* good px.
97
How is FLT3 engaged in driving AML with FLT3 (ITD and tyrosine kinase domain [TKD] muts)?
* FLT3 is a receptor TK that is highly expressed in heme stem cells.
* It is mutated in 1/3 of cytogenetically normal AML;
* the TK domain muts are activating;
* ITD’s suppress the inactivating domain of the protein (so eventually they are also activating). ITD is a tandem dup that occurs in juxtamembrane domain and leads to ligand independent phosphorylation [causing growth]. For Dx, the ratio of ITD size to WT is reported.
* FLT3 is monitored throughout disease for diagnostic clones or new clones;
* at the time of Dx you can have multiple sizes of ITDs, meaning there are different clones.
* During relapse u can have different ratios or new ITD sizes, indicating new clones;
* Rx: TKI
98
What is the prognosis of AML with FLT3 in cases of either ITD or TKD mutations?
* poor px for ITD;
* unclear for TKD
99
How ITD detection can be done?
* ITD detection can be done using PCR and capillary electrophoresis;
* peak heights are used for measuring ratios of each clone
100
What is the max ratio for IDTs?
* The max ratio is 1 [het/het] commonly, but in cases of LOH of 13q you can have very high rations [like 5], which has worse px.
Note: For measuring ITD ratios you need to dilute samples before analysis, so your peaks become in range.
101
How is the prognosis for the following conditions:
AML with both FLT3 and NPM1 muts
AML with both FLT3 and CEBPA
AML with IDH1/2 muts
AML with both FLT3 and NPM1 muts: still poor px
AML with both FLT3 and CEBPA: unclear px
AML with IDH1/2 muts: unclear px
102
What is the biological role of IDH2?
IDH2 is a mitochondrial NADP-dependent isocitrate dehydrogenase that catalyzes oxidative decarboxylation of isocitrate to alphaketoglutarate, producing NADPH. It is associated with D-2-hydroxyglutaric aciduria 2.
103
What is the subcellular localization of IDH1 and IDH2?
* IDH1 is the cytoplasmic enzyme
* IDH2 is the mitochondrial enzyme
Note: IDH inhibitor drugs are available.
104
What is the incidence rate of myeloid sarcoma in AML and in individuals who undergo an allogeneic stem cell transplant?
* Myeloid sarcoma occurs in 2-9% of patients with AML and in 5-12% of those with allogenic stem cell transplant;
* most often occurs as AML relapse;
Note: can have any of the abnormalities seen in AML and listed above. Px depends on the underlying cause.
105
What does systemic mastocytosis refer to?
* Release of numerous vasoactive cell mediators due to excessive activity of mast cells, which results in a wide variety of symptoms including anaphylaxis, flushing, nonspecific GI as well as neuropsychiatric complaints.
* Mast cells are derived from myeloid linages and are in the connective tissue.
* Disease can be triggered by factors like food, alcohol, medications, etc.
106
What are the key mutations in systemic mastocytosis?
* A somatic mutation in KIT (D816V) is a characteristic of disease.
* TET2 muts are seen in 25% and corelate with more severe symptoms.
107
What genes are included in MPN panel testing?
JAK2, CALR, MPL, CSF3R, ASXL1, TET2, EZH2, IDH1/2, DNMT3A, TP53, SF3B1, SRSF2, U2AF1, CBL, N/KRAS, SETBP1, and others.
108
What is most commonly indicative of acute lymphoblastic leukemia (ALL)?
Accumulation of immature lymphoid cells in BM or PB
109
Which one is more common in children and adults: B-ALL or T-ALL ?
B-ALL is more common than T in both children/adults
110
What types of abnormalities usually B-cells and T cells go through ?
* B-cells have many chomosomal translocations,
* but T-cells usually result from rearrangements of T-cell receptor (TCR) loci with oncogenes.
111
What are the different types of ALL?
B-lymphoblastic leukemia/lymphoma (B-ALL)
T-lymphoblastic leukemia/lymphoma (T-ALL)
112
What is the most common type of ALL?
* B-lymphoblastic leukemia/lymphoma (B-ALL)
* 85% of pediatric ALL and 75% of adult ALL
113
What cell/lineage is the origin of B-ALL?
B-ALL is Neoplasm of precursor lymphoblasts committed to the B-cell lineage.
114
What is the incidence frequency and survival rate of B-ALL in children and adults?
* B-ALL is considered a pediatric disease, but can occur in adults;
* >90% survival in kids but 20-40% in adults
115
What are the predominant genetic findings in B-ALL for children, infants, and adults? How does the disease prognosis differ among the three groups?
Adult B-ALL has a different genetic makeup and is a different disease;
* In kids, t(12;21)(ETV6-RUNX1) and high hyperdiploidy
constitute most cases, both with good px;
* In infants, KMT2A rearrangement is most common (80%).
* In adults, these are rare; instead, t(9;22)(BCR-ABL1) is the predominant finding which has poor px.
116
What FISH probe are used for B-ALL?
CEP4/10, BCR-ABL1, KMT2A, ETV6-RUNX1, CEP8/MYC/IGH, CRLF2, ABL1, ABL2, PDGFRB.
117
What are the isk factors for B-ALL?
* Down syndrome
* Germline muts in PAX5 and ETV6
118
What are the different classifications of B-ALL?
1- B-ALL with t(9:22)(m-BCR [p190])
2- B-ALL With t(v;11)(v;q23)(MLL [KMT2A] rearranged)
3- B-ALL with t(12;21)(p13;q22)(ETV6-RUNX1)
4- B-ALL with hypodiploidy
5- B-ALL with (high) hyperdiploidy
6- B-ALL with intrachromosomal amplification of chromosome 21 (iAMP21)
7- B-ALL with t(1;19)(q23;p13.3)(TCF3::PBX1):
8- B-ALL with t(17;19)(HLF-TCF3)
9- B-ALL with t(5;14)(q31;q32)(IL3-IGH or IGH::IL3)
10- B-ALL with BCR-ABL1-like (Ph-like) features
11- B-ALL with NL cyto
12- B-ALL (Burkitt leukemia variant)
119
What are the features of B-ALL with t(9:22)(m-BCR [p190])?
* worst px
* 25-30% of ALL in adults, 5% in kids;
* increased TK activity;
* Rx: Imatinib
120
What are the three BCR breakpoint cluster regions?
* The ABL1 breakpoints are intronic.
* The breakpoints of BCR are in one of the three possible breakpoint cluster regions: the major (M) bcr, the minor (m) bcr, or the micro (μ) bcr.
**In most cases** of CML, the fusion occurs between BCR exon b2/3 (Major) and ABL1 int1/2, giving rise to either the b2a2 or b3a2 variant (aka e13a2 and e14a2, respectively; both translate to p210 [M-BCR]).
**Less common** breakpoints are those in the minor BCR region leading to an e1a2 fusion gene (p190 [m-BCR]), which is seen in most cases of Ph-positive ALL. The p190 protein has higher tyrosine kinase activity than p210 and is associated with a more aggressive leukemia.
**Micro** bcr breakpoints resulting in the e19a2 (c3a2) fusion gene leading to the p230 protein are also occasionally found.
121
What genetic conditions are *never seen* in CLL but in other hematological malignancies?
* BCR-ABL fusion is never seen in CLL.
* Ph Chr is seen in MPN, CML, AML, B-ALL, T-ALL, but never in CLL.
122
If you find t(9;22) together with another cause (like hyperdiploidy), how would you classify the malignancy?
you would still classify the malignancy as t(9;22) because most likely this is the driving event and the rest are secondary changes.
123
Board exam Q: patient has relapsed after imatinib therapy. What do we do next?
Answer: sequence TK domain (cDNA of the BCR-ABL1 gene) and look for resistance muts before changing medication.
124
What are the features of B-ALL With t(v;11)(v;q23)(MLL [KMT2A] rearranged)?
* ‘v’ means variable partners;
* Second worst px;
* MLL rearrangement is the most common (~80%) leukemia in infants <1-YO;
* frequently associated with overexpression of FLT3;
* same events in AML
* Half of the time t(4;11)(q21;q23)(KMT2A-AFF1 aka MLL-AF4) is seen in infants (1% in older kids).
125
What are the features of B-ALL with t(12;21)(p13;q22)(ETV6-RUNX1)?
* 15-25% of all B-ALL;
* Cryptic;
* FISH needed;
* pediatric mainly;
* very good px.
126
What are the features of B-ALL with hypodiploidy?
* ≤43 chr;
* poor px;
* can double and represent pseudo-(hyper)diploid.
127
What are the subtypes of B-ALL with hypodiploidy?
1-Near haploid: 24-31 Chr; 1-2% of ped B-ALL and rare in adults; associated with RAS and Tyrosine Kinase mutations.
2- Low hypodiploid: 32-39 Chr; 1% of B-ALL in kids, 5% in older Kids, and 10% in adults; half have TP53 muts.
3- High hypodiploid: 40-43 Chr
128
What are the features of B-ALL with (high) hyperdiploidy?
* 51-65 Chr;
* 25-30% of ped B-ALL (most common class);
* very good px if IKZF1 is not deleted [9% of cases];
* chr 21, X, 14, and 4 are the most common gains;
* presence of T4,10,17 together (triple trisomy) is the best px (regardless of other chr gains);
* note that this is different from plasma cell hyperdiploidy;
* can be confused with pseudo-hypodiploid (poor px)
129
What are the features of B-ALL with near tetraploidy?
* near 92 Chr;
* 1% of ped B-ALL;
* exclusively happens in those with ETV6-RUNX1 fusion;
* good px.
130
Interpret the images and mention the relavent nomenclature?
These images show a hypodiploid clone which is endoreduplicated in some cells. The nomenclature is:
34<2n->,X,-X,-2,-3,-4,-5,-7,-9,-13,-
15,-16,-17,-20[11]/63~64,idemx2,-
1,-6,-11,-12,-13[cp8]/46,XX[6]
131
What are the features of B-ALL with intrachromosomal amplification of chromosome 21 (iAMP21)?
* poor px;
* intensive chemotherapy is needed;
* high occurrence in germline carriers of rob(15;21) (2700 fold higher);
* defined using FISH by ≥5 copies of RUNX1 probe or ≥4 copies of RUNX1 probe on a single abnl chr21;
* ~2% of childhood ALL;
* rare in adults
132
What are the features of B-ALL with t(1;19)(q23;p13.3)(TCF3::PBX1)?
* ~6% of ALL;
* poor px (good px if treated with intensive chemo)
133
What are the features of B-ALL with t(17;19)(HLF-TCF3)?
* rare;
* poor px
134
What are the features of B-ALL with t(5;14)(q31;q32)(IL3-IGH or IGH::IL3)?
* <1% of ALL;
* eosinophilia;
* IL3 overexpression;
* poor px
135
What are the characteristics of B-ALL with BCR-ABL1-like (Ph-like) features?
* translocations involving tyrosine kinases or cytokine receptors;
* poor px;
* 10-15% of kids and 20-25% of adults;
* similar gene expression profile to B-ALL with BCR-ABL1;
* the TK’s genes involved can be CRLF2 [50%, poor px], EPOR (erythropoietin receptor or MPL), ABL2, CSF1R, NTRK3, JAK2, PDGFRB, TYK2, etc. For some of them there is TKI.
136
How CRLF2 rearrangements are associated with ALL?
* CRLF2 [PAR1 chrX] rearrangements (wt. IGH/P2RY8) are common in Down syndrome ALL (>50% of cases) and BCR-ABL-like ALL.
* This is often associated with IKZF1 deletion/mutations and JAK1/2 mutations.
* It has poor px, except in Down syndrome.
* FISH probe for CRLF2 rearrangements should be used for ALL.
137
What are the features of B-ALL with NL cyto?
* 30-40% of kids and 15-34% of adults
* intermediate-good px
138
What is the genetic hallmark of B-ALL (Burkitt leukemia variant)?
IGH/K-MYC fusion
139
What are the features of T-lymphoblastic leukemia/lymphoma (T-ALL)?
* 15% of peds and 25% of adult ALL;
* better px than B-ALL in adults;
* opposite in kids (worse than B-ALL).
140
Mention some T-ALL associated abnormalities.
1. Clonal rearrangements of TCR loci (Alpha/Beta/Delta/Gamma – TRA/B/D/G; mostly on chr7/14) with other genes [enhancer
hijacking] are very common. These are most common partners: HOX11 [TLX1] (10q24), HOX11L2 [TLX3] (5q35), MYC (8q24.1),
and TAL1 (1p32).
=> t(1;14)(p32;q11.2)(TRA::TAL1)
=> t(11;14)(p13;q11.2)(LMO::TRD): 5-10% of pediatric T-ALL
2- IgH rearrangement (20% of cases)
3- t(10;11)(p12;q14)(P1CALM::MLLT10[AF10]): 10% of T-ALL; Not TCR related; it impairs differentiation; poor px.
4- TAL1-STIL fusion due to interstitial del at 1p32
5- t(11;18)(p15;q12)(NUP98::SETBP1)
6- Mutations in NOTCH1 (activating), hCDC4, CDKN1/2, and PHF6 (LOF)
7- 9p21 del involving CDKN2A/B (30% of cases): seen in both B and T-ALL; unclear px; used for clonality detection by FISH.
8- MLL-ENL fusion due to t(11;19)
9- Abnl karyotype in 50-70%
141
In what order G-banding and FISH should be performed for T-ALL diagnosis?
In T-ALL, G-banding should be done first, and FISH is optional
142
What FISH probes are used in T-ALL?
BCR-ABL1, KMT2A, ETV6-RUNX1,
CEP8/MYC/IGH, CRLF2, and RANBP17/TLX3
143
What is common about mature lymphoid tumors?
* Ig rearrangements (IGH [14q32], IGK [2q12], and IGL [22q11]).
* These can derive from mistakes in normal recombination that should occur in Ig loci in B-cells, leading to oncogene activation using an Ig promoter.
* Unlike hybrid proteins, these types of rearrangements can’t be detected by NGS fusion panels or PCR since they only result in overproduction of the intact protein
144
What are the common genetic translocation partners in different types of lymphomas and multiple myeloma?
145
Do lymphoid and myeloid tumor cells grow in culture?
* For lymphoid tumors, because the tumor cells are mature they won’t divide on their own and you need stimulants like PMA, LPS, IL4, Pokeweed, and oligos (for B-cell) and PHA
(for T-cell). [Oligo stimulation works by adding oligos with CpG motifs, stimulating Toll-like receptor 9 on B-cells.]
* You do not need stimulation for myeloid disorders, because the cells were already dividing on their own. Oligo stimulation
works by adding oligos with CpG motifs, stimulating Toll-like receptor 9 on B-cells.
146
What are the features of Chronic lymphocytic leukemia (CLL)?
* mature B-cell Neoplasm;
* most common adult leukemia;
* elderly population;
* progressive accumulation of incompetent mature lymphocytes in blood/marrow/spleen (often B-cell monoclonal);
147
How is cytogenetic analysis of CLL is done?
with culture stimulation
(important for exam)
148
What are the most common cytogenetic findings in CLL?
* del(13q) is most common finding (55%), followed by del(11q)(ATM-)(18%), and 12/12q+ (16%)
* Using these in FISH will assist with dx since by morphology and flow cytometry it is hard to distinguish CLL from mantle cell
lymphoma [u need CCND1/IGH FISH also for this purpose];
149
How does the presence of del(13q14) alone with mutated IgVH, trisomy 12 or normal cytogenetics, and other cytogenetic abnormalities affect the prognosis in CLL?
* del(13q14) alone and mutated IgVH: good px;
* 12+ or normal cyto:intermediate px;
* all other are poor px (complex cyto, ATM(11q)/TP53(17p) dels, muts in NOTCH1, TP53, BIRC3, SF3B1, ATM, lack of mutation in IgVH genes [Ig heavy chain variable region – assessed by FISH for dels and sequencing and is compared with germline specimen for muts])
150
When treatment will be done for CLL?
What is the treatment?
* Treatment is only done when symptoms develop, regardless of cyto findings.
* Ibrutinib (a Bruton's tyrosine kinase [BTK] inhibitor) is used for Rx
151
Which mutations cause therapy resistance in CLL?
mutations in BTK/PLCG2 (encode B-cell receptors) induce resistance (test if resistance occurs – boardq).
152
How is CLL prognosis wit del(13q)?
* del(13q) has good px if it doesn’t involve RB1 and is not present in >70% of cells. There is no difference between Het/Hom for px.
153
How does the presence of mutations in the variable region (VDJ) affect prognosis in CLL?
* CLL arising from cells with mutations in the variable region (VDJ) have better px. They are hypermutated (2% divergence from control in mut number).
154
What are the IgH translocations in CLL?
IgH translocations in CLL include t(14;19)(q32;q13)(IgH-BCL3), t(14;18)(q32;q21)(IgH-BCL2), and t(11;14)(q13;q32)(CCND1-IGH)(more diagnostic of Mantle cell lymphoma).
155
Why is chronic lymphocytic leukemia (CLL) traditionally challenging to analyze cytogenetically?
* due to low mitotic index and lack of response to traditional mitogens
* However, new methods like IL-2 and DSP30 are being used to overcome these limitations.
156
What is the acute transformation of CLL called?
Richter’s syndrome
157
What are the features of Multiple Myeloma (MM)?
* aka plasma cell neoplasm
* in bone it’s called plasmacytoma
* clonal expansion of malignant plasma cells (terminally differentiated Ab producing B-Cells) in BM
* old age at dx
158
How does MM initiate and progress?
* the condition begins as monoclonal gammopathy of undetermined significance (MGUS – MYD88 p.L265P and CXCR4 muts), 25% of whom progresses to MM within 20 yrs [and some to LPL].
159
How is MM diagnosed?
by elevated Monoclonal (M) pr in serum/urine, high serum plasma cells (NL is <1%), and presence of organ damage [CRAB:
hyperCalcemia, Renal insufficiency, Anemia, Bone lesions].
160
Why is it difficult to study cytogenetics of MM?
* Due to low mitotic index and low levels of plasma cells, cytogenetic study is difficult, and Karyotype alone is not sufficient because you’ll never grow the right cells, so FISH is always needed [Karyotype can give some prognostic info, but its Dx rate is only 30%].
* Even FISH always gets back NL if you don’t enrich and separate plasma cells.
* FISH+Karyo has Dx rate of 90%.
161
How is cell enrichment performed in MM?
* Cell enrichment is performed using Ab’s targeting CD138 antigen on the cell surface;
due to enrichment, the ratios in cyto studies are not
* accurate and the FISH estimated tumor burden is not real.
162
Why observed abnormalities may not represent the true
nature/composition of malignancy in MM?
since not all malignant cells represent CD138
163
What is the process of Chr analysis in MM?
* Chr analysis includes 3 cultures [24hr unstimulated, 72hr unstimulated, and 4-5 days stimulated with cytokines and growth factors [IL4, IL6].
164
How does chromosomal analysis serve as a surrogate for plasma cell proliferation in MM, and why is FISH insufficient for this assessment?
Chr analysis is a surrogate for how proliferative the plasma cells are [since you can’t tell that from FISH due to enrichment].
165
What does Abnl karyo in MM indicate:
Abnl karyo means your plasma cells are proliferative and less differentiated, indicating active disease and higher tumor burden.
166
How is MM risk classified?
Those with no hyperdiploidy, IGH fusion wt. other than CCND1/3, 1q+, 13q- (RB1), and 17p- (TP53) are high risk and everything else has standard risk (good px).
167
What are the features of Hyperdiploidy in MM?
* Hyperdiploidy and t(CCND1/3-IGH) together account for 60% of changes.
* Hyperdiploidy here is different from kid’s BALL and includes odd number chr’s (3,5,7,9,11,15,19,21- excluding 1&17).
* Non-hyper-diploidy includes hypodiploid, pseudodiploid,
near tetraploid [4n dup of cells that are hypodiploid or hypodiploid].
* Hypodiploidy has frequent loss of 13, 14, 16, and 22
* Hypodiploidy and t(IGH) tend to co-occur.
168
How does hypodiploidy and t(IGH) co-occurence contribute to MM development?
* t(IGH) is necessary for tumorigenesis but not sufficient.
* Their freq increases during transition from MGUS to MM.
* They highjack IGH enhancers for oncogenes [directly or indirectly associated with dysregulation of cyclin genes – CCND1/2/3].
169
What are the IGH partner genes?
IGH partners include CCND1 (11q13), FGFR3 (4p16), MAF (16q23), or MAFB (20q12).
170
What are the most common types of chromosomal abnormalities in MM, and how does the prognosis differ for each?
t(11;14)(CCND1-IGH) is the most common [20%; good px; also happens in Mantle cell lymphoma and CLL, followed by t(4;14)(FGFR3) [15%, intermed px], and others (each less than
5% and all poor px).
Other abnl include -1p, +1q (CSK1B, poor px, rare in MGUS but 40% of MM and 70% of relapse), MYC (8q24)
rearrangements, 12p-, 13/13q-, and 17p- (TP53). Most of these have poor px.
171
Are there targeted therapies available for chromosomal abnormalities in MM?
There are targeted therapy for some of these, like t(4;14).
172
What should Fish testing include at minimum for MM diagnosis?
FISH at minimum should include 17p (TP53), t(4;14)(FGFR3-IGH) [cryptic by cyto], and t(14;16)(IGH/MAF).
173
What is the association between CCND1 rearrangement and hematologic malignancies?
* CCND1 is a G1/S gate keeper.
* Its rearrangements are present in MM, CML, and Mantle cell lymphoma.
* It is associated with good px in MM but poor px in Mantle cell lymphoma!
174
How is RB1 involved in MM?
* del(13q) involves RB1 here (unlike in CLL). That’s why it’s good in CLL but bad in MM (only when detected by Cyto, not FISH).
* By FISH it has no meaning; because only detection by cyto means that abnl cells are dividing and disease is proliferative!
* FISH finding of RB- still provides a clonal target for future monitoring of patient’s disease.
175
When you have both hyperdiploidy and 1q+ in MM, which one determines the px?
When you have both hyperdiploidy and 1q+, it’s the latter that determines the px (bad)!
176
What are the most frequent gene rearrangements in lymphomas?
* Most have gene rearrangements with Ig heavy chain Ig (IGH, 14q32); light chains (IGK/IGL on chr2/22) are also
occasionally used.
* Other recurrent changes are trisomy 3, 12, 18.
177
What is the relavence between lymphomas and Hodgkin?
* Lymphomas are classified as Hodgkin’s (Reed-Sternberg cell; ~10% of lymphomas) and non-Hodgkin (NHL).
* Of NHL, about 90% are of B-cell origin, and 98% of NHL cases show IGH changes.
178
What are the diffenet classes of mature B-cell lymphomas?
1- Follicular lymphoma
2- Burkitt’s lymphoma (BL)
3- Large B-Cell lymphomas
4- Mantle cell lymphoma (MCL)
5- Marginal Zone lymphomas
6- Splenic B-Cell lymphoma/leukemia
7- Lymphoplasmacytic Lymphoma (LPL)
179
What are the features of Follicular lymphoma?
* 22% of all lymphoid cancers;
* t(14;18)(q32;q21)(IGH-BCL2) is hallmark (90%);
* t(3;v)(BCL6-Ig) is also seen;
* 25-35% of patients transform to Large B-Cell lymphomas.
180
What is the association between t(14;18)/BCL2 overexpression and B-cell lymphomas/leukemias?
* t(14;18) results in BCL2 overexpress
* BCL2 (B-cell lymphoma/leukemia-2) is an integral outer
mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes (specifically B cells)
181
What gene is the only driver of Burkitt’s lymphoma (BL)?
Myc
182
How Myc translocation initiates BL?
* t(8;14)(MYC-IGH) is the hallmark (85-90%) and initiating event.
* MYC is translocated near enhancer of a highly active gene like IGH;
* In 10-15%, MYC is translocated to 2 or 22 (IGK/L);
* MYC is a transcription factor. Its activation occurs on der(14) of t(8;14) but on der(8) of t(2;8)/t(8;22), so breakpoints are different [important for FISH].
183
What are the guidelines for BL FISH testing?
* For FISH testing most labs use both MYC break-apart and MYC-IGH dual color/fusion probe.
* The 5’ and 3’ probes are ~2Mb apart and the cutoff should be looser for probe distance when validating FISH compared to other genes, because of the variable breakpoints in three translocations.
* For detecting MYC rearrangements you need to use both break-apart and fusion probe design.
184
What are the different subclasses of Large B-Cell lymphomas?
A- Diffuse large B-cell lymphoma (DLBCL), NOS
B- DLBCL (or high-grade B-cell lymphoma) with MYC and BCL2 rearr
C- ALK-positive large B-cell lymphoma
D- Large B-cell lymphoma with IRF4 rearrangement
E- High grade B-cell lymphoma with 11q aberrations
185
What are the features of Diffuse large B-cell lymphoma (DLBCL), NOS?
* 30% of adult lymphoma;
* Rearrangements of BCL2 (25%), BCL6 (40%), and MYC (10%) happens frequently (same ones seen in FL/BL). Cases with
double fusions of BCL2+MYC are not in this class (see next class).
* Those with isolated MYC fusions or dual fusions are called “DLBCL, NOS with MYC or ‘MYC and BCL6’ rearrangement”.
186
What are the features of DLBCL (or high-grade B-cell lymphoma) with MYC and BCL2 rearr?
* Aggressive disease;
* Dual rearrangements one involving MYC, and the other involving BCL2 are needed. This was previously known as double hit lymphoma since it contained the same translocations seen in both Follicular/Burkitt! According to the new WHO book, presence of BCL6 rearrangement is optional in this category (previously known as triple hit lymphoma).
187
What are the features of ALK-positive large B-cell lymphoma?
* <1% of large cell lymphomas;
* mostly t(2;17)(p23;q23)(ALK-CLTC[clathrin]).
188
What are the features of High grade B-cell lymphoma with 11q aberrations?
* exclude MYC rearr
* 11q should have either gain, loss, or telomeric LOH.
189
A complex case of double hit lymphoma:
190
An example of how atypical rearrangments can be clinically important:
191
What are the features of Mantle cell lymphoma (MCL)?
* t(11;14)(q13;q32)(CCND1-IGH) is the hallmark [95%], leading to overexpression of Cyclin D1(CCND1);
* poor px;
* cell morphology overlaps CLL;
* FISH is needed for diff; t(11;14)(CCND1-IGH) is the same as in MM.
Note: BCL1 is a different name for CCND1 (Cyclin D1). BCL1 promotes passage of cells from G1 to S.
192
What are the different subclasses of Marginal Zone lymphomas?
A-Extra-nodal marginal zone B-Cell [aka mucosa-associated lymphoid tissue (MALT)] lymphoma
B-Nodal Marginal Zone lymphoma
C-Primary cutaneous marginal zone lymphoma
D-Pediatric nodal marginal zone lymphoma
193
Which organs are involved in Extra-nodal marginal zone B-Cell lynphoma? Which cyto abnormalities are frequently found in depending on the involved organs?
* This can involve many organs and has various forms. Gastric, intestine, skin, ocular adnexa, lung, and salivary gland can be involved
* All can have +3 and +18 in cyto.
* t(11;18)(q21;q21)(API2-MALT1)(gastric) is the most common finding. Others include: t(14;18)(q32;q21)(IGHMALT1;
orbital/salivary; same cyto band as in FL/DLBCL but different gene), t(1;14)(BCL10-IGH), and FOXP1-IGH
t(3;14)(p14.1;q32)(Thyroid/orbital/skin).
194
What are the features of Nodal Marginal Zone lymphoma?
Similar lymphoma to the previous two, but with limited involvements outside lymph nodes.
195
196
What are the different subclasses of plenic B-Cell lymphoma/leukemia?
A-Hairy cell leukemia (HCL)
B-Splenic marginal zone lymphoma
C-Splenic diffuse red pulp small B-cell lymphoma
D-Splenic B-cell lymphoma/leukemia with prominent nucleoli
197
What is the cell of origin for Hairy cell leukemia (HCL)? What is a key diagnostic and therapeutic target for this disease?
* HCL is an indolent neoplasm of small mature lymphoid cells;
* BRAF V600E exists in ~100% of cases and serves as a key diagnostic and therapeutic target
198
What is the main chromosomal abnormality detected in Splenic marginal zone lymphoma?
30% have loss of 7q which is absent in B-cell lymphoid disorders (more seen in myeloid).
199
What are the main features of Lymphoplasmacytic Lymphoma (LPL)?
* neoplasm of small B-cell, plasmacytoid lymphocytes, and plasma cells;
* Waldenström macroglobulinemia (WM) is commonly found.
* 90% of cases have MYD88 p.L265P and up to 40% CXCR4 mutation. Del(6q) is seen.
Note: These mutations are also present in MGUS (see MM section). MGUS can later progress to LPL or MM.
200
What are the different calsses of Mature T-cell lymphomas?
1-Anaplastic large cell lymphoma/leukemia (ALCL)
2-Hepatosplenic T-cell lymphoma (HSTCL)
3-T-cell Large Granular Lymphocyte Leukemia (T-LGL)
4-Follicular T cell lymphomas
5-T Prolymphocytic leukemia (T-PLL)
201
What are the key features of Anaplastic large cell lymphoma/leukemia (ALCL)?
Rare (3% of adult and 15% of pediatric NHL); positive IHC for CD30.
202
How ALK protein expression is correlated with chromosomal abnormalities and prognosis in ALCL?
ALK negative: good px when rearrangements at 6p25; poor px when TP63 rearrangements.
ALK positive: half cases; t(2;5)(p23;q35)(NPM1-ALK) and other ALK rearr. incl t(1;2)(ALK-TPM3); good px; ALK+ stain
203
Which organs are mainly involvd in Hepatosplenic T-cell lymphoma (HSTCL) and what is the most common cyto abnormality present in this disease?
* HSTCL is a TCL involving the liver, spleen, and bone marrow with a gamma-delta T-cell phenotype.
* i(7q) is a very common abnormality seen in HSTCL and often used to confirm the diagnosis.
204
Which mutant genes are associated with T-cell Large Granular Lymphocyte Leukemia (T-LGL)?
* STAT3 muts
* STAT5B mutation (poor px)
205
Which mutant genes are frequently observed in Follicular T cell lymphomas?
RHOA, CD28, TET2, DNMT3A, IDH2
206
What are the recurrent chromosomal rearrangements observed in T Prolymphocytic leukemia (T-PLL)?
How is the general prognosis of T-PLL?
* Translocations that cause activation of the TCL1A (14q32.1) or MTCP1 (Xq28) [these are homologous genes];
* inv(14)/t(14;14)(TCR::TCL1A), t(X;14)(q28;q11.2)(TCR::MTCP1)[5%];
* poor px
207
Which chromosomal abnormalities are shared between MM and Lymphomas?
MM and Lymphomas share the presence of many IGH translocations, with t(11;14) being the most common.
208
What key considerations should be noted about the IGH break-apart probe during FISH analysis?
IGH break apart probes cover 5’ and 3’ of IGH. One is a constant Ig region (3’) and the other is a variable region (5’). It is possible that due to somatic rearrangements, the binding site of one probe is lost and instead of two fusions you get one yellow (fusion) and one green (or red) signal alone. This is a normal variation and should be considered during FISH analysis.
209
In what spectrum do the Myeloid/lymphoid neoplasms with eosinophilia with rearrangement of PDGFRA, PDGFRB, or FGFR1, or PCM1-JAK2 fall?
And why they can not be placed why under MPN category?
* They fall in the spectrum of Idiopathic hyper-eosinophilic syndrome (HES).
* They can present as chronic myeloproliferative neoplasms (MPNs), but the frequency of manifestation as lymphoid vs myeloid leukemia varies. That’s why they can’t be placed under MPN.
210
What is the WHO classification of myeloid/lymphoid neoplasms?
* Myeloid/lymphoid neoplasms with PDGFRA rearr.: TKI; Cytogenetically Cryptic; interstitial del in 4q causing FIP1L1::PDGFRA
* Myeloid/lymphoid neoplasms with PDGFRB rearr.: TKI
* Myeloid/lymphoid neoplasms with FGFR1 rearrangement: No response to TKI; Poor px
* Myeloid/lymphoid neoplasms with t(8;9)(p22;p24.1)(PCM1-JAK2): may respond to JAK2 inhibitors.
Note: Their identification is important for targeted therapies.
211
What is the current approach for distinguishing between benign and malignant lymphoid proliferations?
* Currently, distinguishing between benign and malignant lymphoid proliferations is based on a combination of clinical characteristics, cyto/histomorphology, immunophenotype, and the identification of well-defined chromosomal aberrations.
* However, such diagnoses remain challenging in 10%-15% of cases of lymphoproliferative disorders, and clonality assessments often help to confirm diagnostic suspicions.
* In such cases, molecular gene rearrangement studies
have proved useful as an additional diagnostic tool.
212
What is the basis of Molecular clonality analysis?
Molecular clonality analysis is based on the principle that all cells of a malignancy share a common clonal origin and thus exhibit identical rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes, distinguishing them from reactive lymphoproliferations which show polyclonally rearranged Ig/TCR genes
213
What is the clinical significance of molecular clonality analysis in diagnosing lymphoproliferative disorders?
The diagnosis of malignant B- and T-cell proliferations is supported by the finding of Ig/TCR gene monoclonality, whereas reactive lymphoproliferations show polyclonally rearranged Ig/TCR genes.
214
What is the purpose of using the EuroClonality/BIOMED-2 consortium?
* Euro clonality consortium BIOMED-2 assay is designed for the purpose of detecting immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements.
* This assay uses quantitative PCR measurement of IgH/K/M and TCR genes followed by capillary electrophoresis.
* Based on whether T cell receptors or Ig’s are monoclonal/polyclonal, different types of heme cancers can be detected.
215
In which diseases monoclonal B cells may be present?
Monoclonal B cells may be seen in B lymphocytic neoplastic diseases such as multiple myeloma and follicular
lymphoma, but also in other illnesses, such as amyloidosis and lupus
216
How can B-cell clonality identified at diagnosis aid in tumor monitoring after treatment?
As a tumor marker, specific DNA rearrangement (monoclonal B
cells) identified at diagnosis may be used to identify minimal residual disease after treatment.
217
How does IGH gene rearrangement contribute to the identification of clonal B-lymphoid processes?
IGH is the first rearranged gene in B-cell development with light chain genes for kappa (IGK) and lambda (IGL) occurring only after heavy-chain gene rearrangement has occurred (allelic exclusion). So IGH gene rearrangement is an early marker of clonal B lymphoid processes.
218
In cases of suspected B-cell clonality, which targets are chosen?
In cases of suspected B-cell clonality, generally the three different IGH V-J targets (not D-J) are chosen, in parallel to or
followed by the IGK targets.
219
Why false negative results are seen in the IGH or IGK PCR
assay of B-cell lymphoproliferative neoplasms?
False negative results are seen in the IGH or IGK PCR
assay alone in 5%-20% of B-cell lymphoproliferative neoplasms owing to intrinsic biologic mechanisms.
These include absent or incomplete IGH rearrangements in
immature B-cell neoplasms, such as lymphoblastic leukemia/lymphoma, and the presence of extensive somatic mutation in some mature B-cell lymphomas, particularly follicular lymphoma, and plasma-cell neoplasms.
Note: Technical issues, such as degradation of DNA samples, may also cause false negative results.
220
What strategy could overcome the emergence of false negative results in the IGH or IGK PCR assay of B-cell lymphoproliferative neoplasms?
The combination of IGH and IGK PCR can overcome these limitations and detect a clonal rearrangement in up to 99% of B-cell neoplasms. Thus, a B-cell clonality panel with both IGH and IGK is recommended for lymphoblastic neoplasms and in suspected follicular lymphoma.
221
Where are the chromosomal loci of TCR genes?
The TCR genes are located on chromosomes 7 (TCRβ/γ – TCRB/G) and 14 (TCRα/d – TCRA/D).
222
Which TCRG locus is the
most tested TCR and why?
The TCRG locus is the most tested TCR because most T cells have rearranged TCRG and because the TCRG locus is significantly less complex than TCRB.
223
Which method provides a 90% sensitivity for T-cell clonality detection?
Combination of TCRB and G using BIOMED-2
224
What are the potential underlying reasons for false negatives in T-cell clonality detection?
False negatives can happen due to:
* low ratio of malignant cells to NL at early stages of lymphoma/leukemia,
* somatic mutations at primer binding site,
* the involvement of V or J rearrangements outside the covered areas by primers,
* chromosomal translocations involving the testing region,
* poor DNA quality (FFPE).
225
What are the potential underlying reasons for false positives in T-cell clonality detection?
False positive can happen by activation of a non-cancer or non-representative population of lymphocytes following clonal Ig/TCR rearrangement.
Note: The presence of a B/T-cell clone is not always equivalent to the presence of a B/T-cell neoplasm.
226
In what occasions Pseudo-clonality may happen?
Pseudo-clonality may happen when only very few B/T cells are in the sample.
For cases with a low percentage of suspected B or T
cells, reproducibility of the profiles is essential. A low number of lymphocytes in, for example, skin or intestinal lesions can easily result in overinterpretation of coincidental dominant peaks.
227
What approach is recommended to prevent misinterpretation due to pseudo-clonality?
To prevent misinterpretation, assessment of the targets in duplicate as well as adjustment of the amount of DNA by increasing the DNA concentration, and hence the number of cells per PCR, are strongly recommended. When low amplitude peaks are seen, it is most appropriate to repeat the test with the same specimen first to rule out pseudo-clonality before reaching a conclusion.
228
What are the advantages and disadvantages of PCR vs. Southern blotting in detecting B/T-cell neoplasms?
* PCR/capillary electrophoresis is faster than Southern blot, and it works well with paraffin-embedded tissue. Southern blot needs a lot of high-quality DNA, which is hard to obtain from paraffin-embedded tissues.
* On the other side, PCR/capillary electrophoresis cannot detect all possible rearrangements because of the limitation of the primers. Southern blot can theoretically detect all rearrangements.
* “pseudo-clonality” may happen when only very few B/T cells are in the sample if PCR/capillary electrophoresis is used for the analysis. So, PCR/ capillary electrophoresis has a higher false positive rate than Southern blot.
229
Note about differences of clonal rearrangement in B vs T cell:
* T-cell receptor loci have roughly the same number of V gene segments as do the immunoglobulin loci, but only B cells diversity rearranged V region genes by somatic hypermutation.
* Somatic hypermutation does not generate diversity in T-cell receptors.
* The T-cell receptor loci comprise sets of gene segments and are rearranged by the same enzymes as the immunoglobulin loci.
230
At what age most of our T cells are produced?
Most of our T cells are produced when we are less than 12 years old.
231
Why are developing T cells more likely to productively rearrange β chains than developing B cells rearranging H chains?
Because there are two sets of D, J, and C β loci so that if rearrangement at the first locus fails, rearrangement can still occur at the second.
