Topic 2

Question 1

21. Is Sputum Cytology a good screening test for early lung cancer detection? (2marks)

Answer 1

Sputum cytology can be used for screening in conjunction with radiological imagining. However, screening is used to detect early lesions and initiate appropriate treatment, therefore it is not ideal because it is not cost effective and has a low detection rate. This low detection rates owes to the fact that pre-neoplastic cells are not exfoliative.

Question 2

22. Advantages of bronchoscopy

Answer 2

Decreased contamination with saliva and microorganisms present in buccal cavity.
It may be difficult to obtain a sample from peripheral lesion with sputum cytology but bronchoscopy can obtain a sample from desired lesion that was located on CT scan, therefore, it is more useful for peripheral lesions.
• The sample is directly sampled through a bronchoscope which contains a small camera with light to ensure that an adequate sample is obtained.
• The tumour subtype can be determined better by immunocytochemistry through the cell block technique. If a biopsy is obtained immunohistochemistry can be used to determine the subtype

Question 3

23. List the various types of cytological and histological specimens that can be obtained during bronchoscopy procedure, and briefly outline the sampling method for each. (12marks)

Answer 3

For cytology
a. Bronchial washings/lavage: a saline solution is aspirated onto an area of interest and fluid is re-collected with the exfoliated cells.
b. Bronchial alveolar lavage (BAL): a larger saline solution is aspirated onto the area and then collected in small aliquots into trap bottles.
c. Bronchial brushings: a brash is extracted from the bronchoscopy tube and the area of interest is brushed to exfoliate cell from the suspected lesion.
d. Transbronchial FNA: if the tumour has not invaded trough the mucosa, a needle may be passed through the bronchoscope tube and negative pressure is applied to obtained a sample from a solid tumour.
For histology:
e. Transbronchial biopsy – small forceps are inserted through bronchoscope and a piece of tissue is obtained with preserved architecture.

Question 4

24. List the two types of lung specimen that can be obtained from FNA and why this isn’t used.

Answer 4

Transbronchial FNA - if the tumour has not invaded trough the mucosa, a needle may be passed through the bronchoscope tube and negative pressure is applied to obtained exfoliated cells.
Transthoracic FNA – if lesion is radiologically visible, a needle can be inserted into the lesion through the skin without the need to insert a bronchoscope.
This is a very invasive procedure and rarely used because BAL or bronchial brushings are preferred since this provides increased risk on the patient.

Question 5

25. Sputum Cytology is the simplest and most cost-effective investigation for the laboratory detection and diagnosis of lung cancer.
    List characteristics and requirements for a good quality sputum specimen, evaluate its clinical use and effectiveness, and its potential suitability as a screening test for early lung cancer. (20 marks)

Answer 5

Sputum needs to be collected 3 times in the early morning in three consecutive days. In order to increase the chance of obtaining an adequate sample the mouth is rinsed with water to minimise contamination form food and microorganisms. The specimen is obtained from the early morning, and needs be induced to ensure that it comes from deep inside the lung tissue. If the patient cannot induce a cough, saltwater mist may be given to do so. The sputum is collected in a clean jar that is not opened before needed.
The macroscopic evaluation should be mucoid or mucopurulent and not contaminated with saliva. When observed microscopically it should contain carbon-laden macrophages and bronchial epithelial cells to indicate that it was a deep lung tissue specimen.
Sputum sample is useful for diagnosing mediastinal lesions which are usually squamous cell carcinoma. They possess minimised if any, risk to the patient, and the procedure is non-invasive, so it can be repeated for confirmation.
Screening for lung cancer requires that the lesion is detected as early as possible to start treatment before malignancy progresses. Sputum cytology can be used for screening in conjunction with radiological imagining to determine the location. However, this is better for individuals that are at risk because it has a high rate of false negatives and low detection rates for early lesions. This low detection rate is because early lesions are not so exfoliative, in addition, they may be so small they are radiologically occult.

Question 6

Cancer is a Genetic Disease.

26. What is the above statement based upon? (4 marks)

Answer 6

Cancer is a genetic disease because malignant cells are a result of variations in the genes that encode for proto-oncogenes (induce differentiation and proliferation) and tumour suppressor genes (cell cycle repair and apoptosis). This will lead to uncontrolled proliferation of cells even if there is DNA damage. As DNA mutations increase over time they lead to clonal proliferation of cells which become a neoplasm. These variations in the DNA can be induced by:
Inherited factors – inherited genetic mutations in these genes increase the risk of developing cancer. Especially those related to proto-oncogenes because in tumour suppressor genes the individual needs to acquire another mutation to result in cancer.
Environmental factors can sometimes implement genetic damage such as UV-rays which induce pyrimidine dimers. However, some genetic factors in certain racial and geographical areas provide a susceptible or resistant role to the damage from these environmental factors.
Oncogenic viruses - certain viruses are capable of integrating with the DNA to create oncogenes such as Human papilloma virus (HPV) which induces E6 and E7 proteins that lead to continuous proliferation of the cells.

Question 7

27. What are Proto-oncogenes and what are their functions? (4 marks)

Answer 7

Proto-oncogenes are genes that are transcribed and translated into proteins that are required for normal cellular proliferation and differentiation, and they also inhibit apoptosis.

Question 8

28. What Factors are thought to induce genetic mutations? (4 marks)

Answer 8

Genetic mutations can arise from:
• Environmental and life-style factors induced by carcinogens (e.g. benzene, asbestos, solar radiation)
• Hereditary genetic mutations
• Mutations induced by oncogenic virus that are typical retroviruses that carry oncogenes in their DNA.

Question 9

29. What are primary chromosomal and secondary chromosomal changes?

Answer 9

Primary chromosome aberration which are involved in carcinogenesis which are mutations in genes that control the cell cycle, these are protooncogenes and tumour suppressor genes. Variations that occur in the proto-oncogenes that control proliferation and differentiation of cells cause continuous expression of the proto-oncogenes or produce proteins which are resistant to degradation. While tumour suppressor genes are suppressed and cannot induce apoptosis if there is DNA damage. The is non-random and specific and leads to increased proliferation of cells.
Secondary chromosomal aberrations are random mutations that occurs in cells that are continuous being proliferated. Therefore these cells may develop mutations with every proliferation leading to neoplastic cells. These neoplastic cells eventually form a clonal population which can be detected clinically as a tumour.

Question 10

30. What are Gene Products? (3 marks)

Answer 10

Gene products are RNA or protein molecules obtained after transcription and translation of a gene sequence. An abnormal or absent gene product may give rise to neoplastic changes.

Question 11

31. List five different chromosomal abnormalities that may lead to oncogenic expression. (5 marks)

Answer 11

1. Point mutations – if this mutation occurs in the promoter region there will be overexpression of the protein but in the gene itself it may give rise to hyperactive protein or protein resistant to degradation.
2. Large Deletions – detached segment without reattachment e.g. deletion of chromosome 13, band q14 leading to a variation in the retinoblastoma gene which produces retinoblastoma. This retinoblastoma normally suppresses the effects of E2F which is a transcription factor required to produce proteins needed for cell proliferation.
3. Large Insertions – a segment breaks off and attaches onto a homologous chromosome, this gives rise to a chromosome without the DNA sequence and the other with duplicate DNA sequences.
4. Translocations: a segment of DNA breaks off and attaches onto a non-homologous chromosome e.g. Philadelphia chromosome, that results from translocation of chromosome 9 and 22 resulting in BCR-ABL on chromosome 22 which is an oncogene most commonly found in patients with chronic myeloid leukaemia.
5. Gene amplification – multiple copies of the gene all producing the same protein leading to uncontrolled proliferation of gene expression.

Question 12

32. Distinguish between Proto-oncogenes and Oncogenes. (6marks)

Answer 12

Proto-oncogenes are genes that are transcribed and translated into proteins that are required for normal cellular proliferation and differentiation, and inhibit apoptosis. These genes are under the normal homeostasis of the body that are required for cell cycle continuation to produce new cells without any DNA damage or other defects.
If a mutation arises in these genes it is transformed into an oncogene. This oncogene will produce abnormal qualitative or quantitive amounts of proteins that are needed for normal cell cycle regulation leading to uncontrolled cell proliferation and cell differentiation which may eventually lead to malignancy if other aberrations in the genes arise. The mechanisms by which oncogenes are formed are translocations, deletions, point-mutations, insertion or other mutations such as gene amplification and increased mRNA stability.

Question 13

33. List six types of mutagenic events which may take place in the cellular genome. (6marks)

Answer 13

1. Gaps – when one strand of the DNA helix has missing nucleotide bases complimentary to the secondary strand.
2. Inversions – a segment is detached and reverses its orientation but reattaches the original chromosome, no genes lost. This is common of the tropomyosin receptor kinases (trk) found on chromosome 1.
3. Large Deletions – detached segment without reattachment e.g. deletion of chromosome 13, band q14 leading to a variation in the retinoblastoma gene.
4. Large Insertions – a segment breaks off and attaches onto a homologous chromosome, this gives rise to a chromosome without the DNA sequence and the other with duplicate DNA sequences.
5. Translocations: a segment of DNA breaks off and attaches onto a non-homologous chromosome e.g. translocation of chromosome 9 and 22 which gives rise to the Philadelphia chromosome (chromosome 22) due the presence of BCR-ABL which is an oncogene.
6. Ring formations – when the terminal ends of the chromosomes are joined together, they typically occur in foetuses or new born with developmental abnormalities.

Question 14

34. What is the possible mechanism of retroviruses.

Answer 14

When retroviruses infect a host they can use the enzyme reverse transcriptase to produce complementary DNA from their RNA. This cDNA integrates with the host cell and the retrovirus gains a proto-oncogene in its sequence during transcribing, it may modify this gene producing an oncogene. This oncogene can then be inserted into a new host cell leading to uncontrollable proliferation. If the virus is slow-transforming it may carry promoter regions that once inserted into the genes can induce overexpression of protooncogenes.

Question 15

35. Mention three situations involving gene products which may lead to uncontrolled cell growth and proliferation. (6marks)

Answer 15

• A gene product that is not produced from tumour suppressor genes cannot provide the necessary mechanisms that are required for apoptosis and the cell may continue to grow even if it has several mutations.
• The presence of an abnormal altered gene product from a proto-oncogene has oncogenic properties and can be hyperactive or resistant to degradation, therefore, it stimulates the cell to continue growing and proliferating uncontrollable.
• Abundance of normal gene product as a result of overexpressed oncogene.

Question 16

36. Give THREE main functions of the lymph node. (3marks)

Answer 16

1. Phagocytosis – macrophages are present in the medullary region, that may engulf a pathogen by phagocytosis. Phagocytosis may be enhanced if B-cells release antibodies which opsonise the pathogen.
2. Antibody-dependent B-cell differentiation – in the lymph node the naïve B-cell can encounter a T-helper cell that has been activated by a dendritic cells and this will initiate the production of high-affinity antibodies.
3. Filters lymph fluid from microorganisms, malignant cells and other foreign particles.

Question 17

37. What is meant by Lymphadenopathy? (2marks)

Answer 17

Lymphadenopathy is the generalised enlargement of a lymph node that results from benign conditions which commonly lead to activation of naïve B-cells and these differentiate leading to increased number of B-cells (hyperplasia). However, it may also result as a clonal proliferation of malignant cells such as non-Hodgkin lymphoma.

Question 18

38. List the B-cell differentiated lymphoid cell types found in a reactive lymph node aspirate and give ONE distinguishing morphological feature of each. (10marks)

Answer 18

When a naïve B-cell encounters an antigen it undergoes a differentiation process to transform into a plasma cell and start producing antibodies.
• Lymphocytes – dark nuclei with high nucleus to cytoplasmic ratio.
• Centroblasts – contain two to three prominent nucleoli in the nucleus.
• Centrocyte – the dark nucleus is cleaved
• Immunoblasts – large eccentric nucleus with one large prominent nucleolus
• Plasma cell – large with a pale region in proximity to the nucleus due to abundance of golgi bodies.

Question 19

39. What are Tingible body macrophages?

Answer 19

Large cells that are multi-nucleated and may contain several phagocytic material in their cytoplasm in vesicles known as phagosomes that fuse with lysosome to destroy the pathogen.

Question 20

40. How is FNA processed?

Answer 20

When an FNA is obtained in most cases there is rapid onsite evaluation (ROSE) to give feedback on the adequacy of the sample and patient re-call. This procedure involves a cytopathologist viewing the sample near the patient during aspiration. The cytopathologist ensures that a satisfactory amount of intact cells with minimal peripheral blood was obtained. ROSE improves the quality and quantity of the sample and can be useful to decrease the TAT as it gives indication on how the sampled needs to be processed so it’s placed in the appropriate fixative.
At least three slides are prepared directly from FNA for routine cytology and together with the rest of the aspirate are sent to cytopathology. One slide is fixed by air drying, for Romanowsky for observing cytoplasm and extracellular matrix, another slide fixed in 95% ethyl alcohol for staining with Papanicolaou which provides nuclear and intranuclear details. Other slides may be prepared for repeated staining or according to the requirements.
If an FNA is sent to cytology in a tube this needs to be sent in a special fixative and not in alcohol. Smears are prepared using a Cytospin, and if the sample is very cellular it may need to be diluted using phosphate buffer solution. If the sample is bloody it is prepared using density gradient centrifugation to remove haemorrhagic cells that can occlude vision. In addition, to Giemsa and pap smears, the aspirate can be used to prepare a cell block which is used for FISH, flow cytometry and immunocytochemistry.

Question 21

41. What are the roles of FNA?

Answer 21

FNA can be used for diagnosis and differentiation of reactive lymphadenopathy, some primary and metastatic malignancies, sometimes it is also used to monitor treatment.

• In reactive lymph nodes, the percentage of large lymphocytes are increased and is a mixed population of lymphocytes that are at different differentiation stages since they are slowly being activated to defend against the invading pathogen. Neutrophilia, eosinophilia or increase plasma cells depending on the infection is common. If tingle- body macrophages are abundant it is indicative of follicular hyperplasia. In infectious mononucleosis the aspirate is usually hypercellular with atypical immunoblasts and tingle macrophages are few. If there is granulomata and necrosis, it could be suggestive of tuberculosis (TB) but if there isn’t, TB still cannot be excluded. Other causes of granulomatous lymphadenopathy include leprosy, cat scratch disease and leishmaniasis etc.
• Primary lymph node malignancy - if it’s query lymphoma in addition to preparation of giemsa and PAP smears, some of the aspirate should be placed in RPMI solution for cell block and flow cytometry. The lymphomas are classified in Hodgkin’s (HL) and non-Hodgkin’s lymphoma (NHL). NHL is characterised by a monomorphic population of cells and is subdivided into low grade or high-grade lymphoma. High-grade lymphomas are more aggressive and highly pleomorphic and atypical, therefore, they are more easily detected than low-grade NHL. Low-grade NHL may resemble a reactive lymph node and can lead to false-negative results. Since centroblasts are more fragile than normal lymphocytes, an increased number of ruptured cells should be noted as a falsely low percentage of centroblasts will be calculated resulting in misdiagnosis. A biopsy may need to be obtained if the FNA is equivocal, requires subtyping by immunohistochemistry, unless the patient is unfit for surgery or lymph nodes are inaccessible. It should be noted that hodgkin’s lymphoma is difficult to diagnose based on cytology alone and requires further investigations, however, FNA is often used to monitor treatment and minimal residual disease.
• Metastatic malignancy - Metastasis is suspected when there are cells presents that aren’t part of the lymph node such as melanocytic cells. A cell block is prepared from the aspirate so that tumour markers can be adopted to identify the site of origin. When it’s query metastasis, the original site of neoplasia needs to be found in order to confirm diagnosis. Common metastatic tumours include small cell carcinoma, squamous cell carcinoma adenocarcinoma, and melanoma. Melanoma usually present with large dyscohesive cells, ovoid eccentric pleomorphic nuclei and confirmed by staining with May Grunwald Giemsa (MGG) as the melanin pigment appears bluish black.

Question 22

42. What is the general cytological picture of a Non-Hodgkin’s Lymphoma? (5marks)

Answer 22

The smear is usually composed of a monotonous and monomorphism cell depending on the carcinoma present, with features of malignancy including nuclear pleomorphism. moulding and crowded cells. This is subdivided into low-grade and high-grade, and the former may be difficult to distinguish from reactive and infective lymphadenopathy. However, the latter is more easily differentiated as cells have more dysplastic features and mitotic cells are often increased.
This requires differential diagnosis often made by flow cytometry, immunocytochemistry and FISH. In addition, an excisional biopsy may be obtained.

Question 23

43. What is the general cytological picture of Hodgkin’s lymphoma?

Answer 23

This is more difficult than NHL to diagnose by cytological means but is often used for monitoring of disease and therapy given. There are two main types, the classical Hodgkin’s lymphoma identified from the presence of Reed-Sternberg cells and the nodular lymphocyte predominant is a rare type that where R-S cells are absent.

Question 24

46. What are the specimen types that can be obtained from breast?

Answer 24

Nipple discharge – discharge is rarely seen unless individual is pregnant but may be induced by tranquilising drugs, oral contraceptives, herpes, Paget’s disease, neuroendocrine disorders, and intraductal papilloma’s.
Breast cyst aspiration – cysts are commonly seen in women reaching menopause due to fluctuations and imbalances in hormones, particularly increased oestrogen and decreased progesterone levels. These changes lead to hyperplasia of epithelial cells that line the ductules, lobules and stroma. The cavity of this thickened epithelial lining is filled with fluid which can be sampled for diagnostic confirmation and is also therapeutic.
Fine needle aspiration – this has been replaced by core needle biopsies because they are more accurate and precise since the architecture can be observed and they also have similar complications to FNAs. However, ultrasound guided FNA can be used to differentiate palpable and non-palpable lesions from being cystic or solid and benign or malignant. This will indicate what further tests or treatments are necessary. In addition it can be used to confirm metastasis or recurrence of a disease and may be used for aspiration of breast cysts which is therapeutic.

Question 25

47. What are the advantages of Breast FNA? Why is a core needle biopsy preferred over FNA.

Answer 25

• Safe – less complications and invasiveness than biopsy and can be done as an outpatient procedure.
• Accurate – if sampled correctly the diagnosis has a high specificity
• Efficient – may be therapeutic when cyst fluid is removed.
• Fast – this has a fast turn around time and can provide diagnosis quickly to initiate treatment faster and relief the patients anxiety.
A core needle biopsy is preferred because the architecture can be investigated which is useful for differentiating between in situ and invasive carcinoma which may be difficult by cytology alone.

Question 26

50. How does breast cancer screening help to detect early lesions? What is the optimal method.

Answer 26

In situ lesions are asymptomatic but can be detected by a mammogram and therapy is very effective if initiated this early in the disease. The morbidity is reduced if malignancy is detected at an early stage, thus screening programmes are implemented worldwide, in fact in Malta it starts at the age of 50.
The most common method used for screening is a mammography, and this has a low false-positve and negative rates in patients over 50. If a lesion is found it can be sampled by FNA or core needle biopsy. An alternative approach is digital breast tomosynthesis, which is far more accurate and efficient at detecting breast lesions. However, this uses a higher radiation dose and requires more expertise for interpretation, therefore, it may only be useful for individuals with dense breasts since mammography may not detect lesions.
Screening before the age of 50 can be done in patients that have a strong family history of breast cancer but they should be informed of the increased risk of having false-positive and false-negative results.