It is now unquestionable that virtually all cases of cervical cancer are due to persistent infection by high-grade human papillomavirus (HPV) types, especially HPV types 16, 18, 31 and 45.⁴ As a result, HPV testing is arguably the most important cytology-specific molecular diagnostic test. HPV is now recognised as a precursor of both squamous and glandular cervical pathology.^5,6

The test is best suited for three main clinical scenarios: (1) for diagnostic triage of women with a conventional smear showing low-grade abnormalities, that is, low-grade dyskaryosis (low-dysk) or low-grade squamous intraepithelial lesion (LSIL);⁷ (2) as a primary screening tool for women older than 30 years of age;⁸ (3) as a follow-up test for patients after treatment of a high-grade abnormality.⁹ Either by itself or as a complementary approach to conventional cervical smear cytology, HPV molecular testing can increase diagnostic accuracy and, importantly, do so in a more cost-effective manner. Large studies have shown that implementation of HPV DNA testing in cervical screening leads to earlier and higher detection rates of high-grade lesions¹⁰ and greater sensitivity for detection of cervical intraepithelial neoplasia (CIN)^11,12 compared with conventional smear testing. Additionally, certain molecular HPV testing may be used as an effective triage for ASCUS or LSIL cervical smears, in detecting subsequent high-grade lesions during follow-up.¹³ Further considerations on how to integrate HPV DNA testing in the routine diagnostic setting are discussed in Chapters 22 and 23.

There are various HPV detection modalities, based on different molecular techniques, such as signal amplification, target amplification, genotyping or in situ hybridisation, among others.¹⁴ A literature meta-analysis¹⁵ reveals two methods which are more commonly employed for routine diagnostic use, namely PCR-based studies using MY09/MY11 primers^16,17 and hybrid capture techniques.^18,19 However, these test modalities will only be useful if they achieve a practical balance between sensitivity and specificity to minimise unnecessary colposcopic intervention in high-risk HPV-positive patients without detectable lesions.²⁰

Another approach is the identification of hypermethylated genes using quantitative methylation-specific PCR.²¹ Aberrant promoter methylation of selective tumour suppressor genes has been detected in squamous intraepithelial lesions and invasive cervical cancer. Identification of methylation profiles of these genes in liquid-based cytology specimens can therefore be potentially useful to differentiate low-grade from high-grade lesions.²² This is also discussed later in this chapter.

Finally, detection of p16(INK4A), a cyclin dependent kinase inhibitor, via immunocytochemical analysis has also been shown to increase the sensitivity of HPV testing. Compared with other biomarkers, p16(INK4A) was the most reliable marker for CIN.²³

Although there is little doubt about the clinical utility of HPV molecular testing in routine diagnostic practice, its precise use may vary in the socioeconomic context of individual countries. This use may also be modified in the not-so-distant future once we see the impact of HPV vaccination in both developed and developing societies.

The main applications of molecular and cytogenetic testing in soft tissue tumours are:

Fig. 34.1 Molecular analysis of synovial sarcoma in a cytology sample (cell block). (A) H&E of a patient with monophasic synovial sarcoma showing tightly packed interlacing fascicles of spindle cells. (B) Diff-Quik stained smears of the fine needle aspirate demonstrate densely packed spindle cells with overlapping nuclei and interlacing fascicles. (C) Diff-Quik and (D) Papanicolaou stained smears showing relatively monomorphous spindle, oval and epithelioid-looking cells at higher power. (E) The spindle or epithelioid cells stain focally for cytokeratin 7. (F) A fluorescent in situ hybridisation study using a break apart probe demonstrates rearrangement of the SYT gene (indicated by the separation of the red and green signals). (G) Sequencing showing the site of translocation involving SYT exon 10 and SSX exon 5.

In the diagnostic work-up of certain problematic tumours, the presence of recurrent or specific chromosomal translocations in many soft tissue tumours (Table 34.1) can be detected by various molecular testing modalities, including FISH, PCR, DNA sequencing and conventional cytogenetics. As well as conventional cytogenetic testing, which can be labour-intensive and slow, PCR and FISH are both suitable as first-line molecular investigations and nearly all forms of cytological preparations can be utilised for these tests, each with their inherent strengths and limitations.

Table 34.1 The commonest molecular diagnostic targets associated to sarcomas

In every case, interpretation of these molecular results should always be correlated with the clinical and pathological assessment as, very rarely, unrelated sarcomas (such as, for example, angiomatoid fibrous histiocytoma versus clear cell sarcoma), may possess similar translocations, that is, show chromosomal promiscuity²⁵ or aberrant chromosomal abnormalities including widely dispersed breakpoints and the variant translocations. Additionally, the use of molecular testing should be limited to a specific group of tumours, for instance recurrent translocations, and should be used only after appropriate initial work-up, so as to avoid unnecessary and costly testing or false results due to inherent problems with molecular tests such as false positive amplifications in PCR testing. Please also refer to Chapter 33 for further discussion on the cytogenetics and corresponding ancillary techniques of paediatric sarcomas and solid tumours.

The main applications of molecular and cytogenetic testing in the clinical context of lymphoma that are readily used in diagnostic cytopathology,²⁶ include: (1) establishing monoclonality to facilitate the initial diagnosis of a lymphoid malignancy; (2) identification of distinct entities within specific lymphoma subtypes to enable precise classification, prognostication and targeted personalised therapy; (3) detection and monitoring of minimal residual disease; (4) study of the clonal evolution during disease progression. Numerous analytical techniques are available for molecular testing and they can be applied on a variety of cytological specimens, including FNA, effusions, imprint smears and archival cytological material.^14,27 Importantly, each of the techniques has its limitations and the data gleaned from them should be interpreted in the appropriate clinical context with consideration of the morphology and the immunophenotype.

Flow cytometry (FC) and immunocytochemistry (ICC) are complementary tests and the preference to utilise either one of them depends on the laboratory’s expertise and experience and the resources available.¹⁴ The uses of ICC in the diagnosis of lymphoma are well characterised^14,28,29 and the reader is referred to Chapter 13 for discussion of the immunophenotype of specific lymphoma subtypes. The primary advantage of FC over ICC is the multiparametric evaluation of single cells and the small sample requirement. In particular, the ability to evaluate antigen co-expression on the same cell, and simultaneous analysis of cytoplasmic and surface antigens confer distinct advantages to ICC. Other advantages include the determination of B-cell and T-cell clonality by analysis of immunoglobulin kappa and lambda light chains, and antibodies against the variable region of the TCR beta (V beta) chain, respectively.¹⁴

Polymerase chain reaction (PCR)-based assays are now the preferred tests in the assessment of clonality to differentiate between reactive and neoplastic lymphoid proliferations (Fig. 34.2). Although clonality assays are powerful tools, it is important for clinicians and cytopathologists to be aware of the limitations, sensitivity and specificity of the assays. First, clonality does not necessarily equate with malignancy and the failure to detect clonality does not imply benignity. Second, false positive and false negative results can arise due to a combination of biological and technical factors and errors of interpretation. Third, IgH and TCR gene rearrangements are not necessarily restricted to B and T-cell lineages, respectively.³⁰

Fig. 34.2 Tonsil biopsy with imprint smears of a patient with EBV-positive post transplant lymphoproliferative disorder, polymorphic subtype. The biopsy (A) and smear imprint (B) revealed a polymorphic lymphoid population including immunoblasts, small and intermediate-sized lymphocytes showing plasmacytic differentiation, and plasma cells. Many of the lymphoid cells in the biopsy were positive for EBER (C). Polymerase chain reaction for IgH gene rearrangement (D), performed using tissue scrapings from the imprint smears, identified a monoclonal B-cell population (top panel: monoclonal control; middle panel: polyclonal control; bottom panel: patient case).

The most common primary recurring genetic aberrations of non-Hodgkin lymphomas (NHL) are balanced chromosomal translocations leading to deregulation or activation of associated oncogenes (Table 34.2).^2,31 A variety of methods can be used to detect the translocations in cytological specimens and they include Southern Blot (SB), PCR and RT-PCR, conventional cytogenetics (CC), FISH and ICC. Of these, CC and SB techniques require fresh tissue for analysis. PCR-based assays are highly specific but are unable to detect the translocations with widely dispersed breakpoints and the variant translocations, unless multiple primer sets are used. Hence, in many pathology laboratories, interphase FISH and ICC are the preferred modalities because they can be readily applied on archival and imprint cytological preparations (Fig. 34.3).¹⁴

Table 34.2 Recurrent chromosomal abnormalities associated with specific non-Hodgkin lymphomas

Fig. 34.3 Follicular lymphoma FISH analysis. Fine needle aspiration cytology of the cervical lymph node of a 54-year-old man with follicular lymphoma and diffuse large B-cell lymphoma transformation. Diff-Quik smear (A) and cell block preparation (B) of the FNA cytology revealed a predominant population of large lymphoid cells resembling centroblasts and a minor population of centrocytes with grooved or folded nuclei. Immunohistochemistry was performed on the cell block. The tumour cells expressed CD20 (C) and bcl2 (E), and were negative for CD3 (D). Fluorescent in situ hybridisation performed on the cell block using IGH/BCL2 Dual Color, Dual Fusion Translocation Probe Set detected two orange/green (yellow) fusion signals in the cells, confirming the presence of t(14;18) (q32;q21) and hence the diagnosis of follicular lymphoma (F).

With the abundant information related to the molecular basis of gastrointestinal pathology, cytology specimens are readily utilised for molecular testing in colorectal cancer (CRC) and gastrointestinal stromal tumours (GIST), as well as in pancreatic and pancreato-biliary oncology (see Chs 7 and 10).

CRC is arguably the neoplasm that is better understood from a molecular viewpoint. Of all the genetic information that is relevant to CRC, there are four tests that are of molecular diagnostic importance, namely APC mutations for the diagnosis of familial adenomatous polyposis, microsatellite instability and BRAF mutations as part of the molecular screening of hereditary non-polyposis colorectal cancer (HNPCC) and KRAS mutations for the use of cetuximab in the treatment of metastatic CRC. While the first test is primarily analysed in peripheral blood, the other three can be detected on various cytology samples and, on occasion, this may be the only sample available.

The microsatellite instability (MSI) test is used for both HNPCC diagnosis and for prognostication in the context of conventional therapy.³² MSI analysis is possible on cytology samples³³ and, with the use of a mononucleotide repeat-only panel, no normal counterpart is needed for the analysis. Thus cytology samples with a good representation of neoplastic cells, that is at least 20–30% according to personal experience, are suitable for this analysis.³⁴ The MSI testing is complemented with the BRAF test for HNPCC diagnosis.³⁵ Mutation analysis of BRAF, easily done on cytology specimens in our experience, also offers additional information on sensitivity to newly developed MEK inhibitors.

One of the better established examples of personalised medicine is the detection of KRAS mutations in metastatic colorectal cancers.³⁶ Patients with wild-type KRAS are more likely to have disease control on Cetuximab therapy, while those with KRAS mutation may not benefit from this treatment. Our sensitivity studies show that a minimum of 20% of malignant cells in a cytology sample is sufficient to give a reliable analysis by direct sequencing.

GIST is molecularly characterised by mutations in the c-kit and PDGFRA genes. The detection of these mutations in cytology samples has diagnostic value when conventional immunocytochemistry does not allow a confident diagnosis as well as prognostic significance, since exon 11 mutations indicate a worse prognosis and also a better response to imatinib mesylate (Fig. 34.4, lower panels). This analysis can be demonstrated in material from cytology cell blocks, with prior careful confirmation of sufficient cells in the conventional H&E stain, see Ch. 7).³⁷

Fig. 34.4 Detail of the cell block cytology of a FNAC lung sample showing an adenocarcinoma (A) and the corresponding mutation in EGFR exon 19 (B); detail of the cell block cytology of a FNA gastric sample showing CD117 expression in a gastrointestinal stromal tumour (C) and the corresponding mutation in c-kit exon 11 (D).

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