Applications and limitations of oncogene mutation testing in clinical cytopathology

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Available online at www.sciencedirect.com

www.elsevier.com/locate/semdp

Applications and limitations of oncogene mutation testing in clinical cytopathology Claudio Bellevicine, MDa, Giulia De Vita, MDb, Umberto Malapelle, PhDa, Giancarlo Troncone, MD, PhDa,c,n a

Dipartimento di Sanità Pubblica, Università di Napoli Federico II, Naples, Italy Anatomia Patologica, I.R.C.S. CROB, Rionero in Vulture, Potenza, Italy c CEINGE–Biotecnologie Avanzate, Naples, Italy b

article info

abstract

Keywords:

In an increased number of settings, cytology represents the only source of sampling and it

Cytology

often substitutes histology as an independent diagnostic modality. Thus, DNA molecular

Aspiration

targets to stratify patients for targeted therapy are often evaluated on cytology. In addition,

PCR

DNA mutational tests may refine indeterminate thyroid and pancreas cytology. This review

c-KIT

discusses the applications and limitations of DNA mutational testing on cytology. With

BRAF

respect to histology, most cytological samples have the advantages of a purer population of

EGFR

tumor cells, with low stromal component, a better preserved DNA, and assessing at the

KRAS

same time of sample collection cellular adequacy for DNA testing. However, since in vitro

NRAS

diagnostic tests are licensed only for paraffin-tissue, all mutational assays on cytology are “home brew,” requiring a rigorous validation process. This should take into account not only the performance characteristics of the molecular assay but also features inherent to any given cytological samples, such as its source, preparation type, fixation and staining modalities, and the most effective tumor cell enrichment methods. This calls for a change of cytotechnologists and cytopathologists mentality to collect and process the cytological samples not only for microscopy but also to assess clinically relevant molecular markers. & 2013 Elsevier Inc. All rights reserved.

Cytopathology is a reliable screening tool for early detection of neoplastic disease and for monitoring disease recurrence; in many settings it is also a diagnostic modality that can independently guide therapy. In particular, fine-needle aspiration (FNA) biopsy is a safe, rapid, affordable, specific, and accurate tool to achieve a tissue diagnosis for superficial lesions or with image guidance for deeper-seated masses. FNA effectiveness can tremendously be improved by the application of several ancillary modalities that should be adapted to smaller specimens, such as those commonly encountered in cytopathology practice. Immunocytochemistry has long been widely and routinely used on FNA specimens for many applications; conversely, still in the year 2000, n

in a timely review on the applications of molecular techniques to cytology Rimm1 noted that “the practicing cytopathologist and cytotechnologist have been hearing for years how molecular biology will change their lives.” However, he further observed, “we still find relatively limited practical use for molecular techniques but still unlimited promises regarding what this technology will do in the future.”1 Today, after more than 10 years, the application of molecular techniques to clinical cytopathology is much more than a vague future prospective; while complex and multi-analyte mRNA-based tests still await clinical validation, the use of DNA molecular targets to stratify patients for targeted therapies has increasingly moved into routine clinical practice

Corresponding author at: Dipartimento di Sanità Pubblica, Università di Napoli Federico II, via Sergio Pansini 5, I-80131 Napoli, Italy. E-mail address: [email protected] (G. Troncone).

0740-2570/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.semdp.2013.11.008

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with the implementation of specific DNA-based molecular tests.2 Testing cytological samples for somatic mutations of a number of oncogenes, such as c-KIT, BRAF, EGFR, KRAS, NRAS, and PIK3CA has in certain carcinomas an unquestionable diagnostic and/or predictive significance2; moreover, although earlier data suggested that cytological samples were not adequate for mutation analysis,3 the current DNA testing methodology on cytological material is sufficiently robust and reproducible in multiple laboratories to be suitable for widespread implementation.4 This review focuses on oncogene mutation testing in clinical cytopathology that can either refine indeterminate diagnosis, in particular in thyroid and pancreas aspirates, or predict the response to targeted therapy in adenocarcinoma of the lung colonic adenocarcinoma, gastrointestinal stromal tumor, and malignant melanoma.

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limiting the length of the DNA fragments that can be successfully amplified and increasing the number of cells required for accurate testing. Conversely, fixative solutions used in liquid-based cytology do not have the same problems with DNA degradation that are seen in formalin-fixed paraffin-embedded tissue. Since preservative solutions have been designed for optimal preservation of nucleic acids, the DNA extracted from liquid-based cytology (LBC) samples has higher yield, long-term stability, optimal 260/280 ratios, and fragments not o400 bp. The stability of PreservCyt (Cytyc Corporation, Boxborough, MA), a methanol-based medium used in liquid cytology, is high and samples can be stored for 4–5 years before the specimens accrue significant losses in DNA integrity as detected by PCR.12

Preparation type

Biospecimen issues Cytology as a molecular biospecimen Some features, inherent to any type of cytological sample, underline the very great potential of cytology as a molecular biospecimen. Any cytological slide is fixed within seconds of being obtained, whereas surgical tissues may frequently be maintained at room temperature for lengthy periods before being processed and fixed. Because 5-μm tissue sections usually divide nuclei, most sectioned nuclei on a histology slide will contain only a portion of the genome. In comparison, each nucleus is preserved intact in most cytological preparations, so fewer cells are required for similar quantities of DNA. The cellular composition and in particular the ratio between normal and neoplastic cells is dependent on the source of the cytological specimen. Differences exist between exfoliative and aspirative cytological specimens. Effusions, brushings, and washings frequently contain numerous nonlesional cells, making the selective isolation of neoplastic cells difficult. FNA ensures a more selective sampling; a single FNA pass yields up to 1 million neoplastic cells,5 with a minimal stromal contamination.6 Symmans et al.6 evaluated neoplastic and stromal cell components in paired FNA and breast core needle biopsy (CBX) and showed that the FNA samples contained more tumor cells (80% vs. 50%) and fewer stromal cells (5% vs. 30%) than the CBX samples. In such a pure population of tumor cells with intact nuclei, a gene mutation can be detected even from the extremely small specimens, as those that are used in routine may form a residue after extensive diagnostic workup with special stains. In fact, a very low amount (o0.5 ng/μl) of extracted DNA, a scant-sized (40.5–2 mm2) cell block,7 or a needle rinse may be sufficient for mutation detection.8–10

Fixation To fully exploit the potential of the cytological sample, beside high standard cellular morphology, care should also be taken to preserve DNA quality within the sample.11 Alcohol-based fixatives are significantly better than formalin; the latter reduces the quality of extracted nucleic acids by causing widespread cross-linkage between nucleic acids and proteins,

Besides fixation effects, the type of cytological biospecimen from which DNA is extracted also matters. Sample collection and processing may greatly differ leading to many different cytopreparation types. Direct smears, CBs, and monolayer preparations2 have advantages and drawbacks in mutational assays. Owing to the high quality of nucleic acids that can be extracted and the ease of immediate assessment for specimen adequacy, direct smears have extensively been exploited for BRAF13–16 and EGFR testing17–20; in most studies, the design was retrospective and the results obtained on either Diff-Quik or Papanicolaou direct smears were compared to those obtained on matched histological samples.2 Also CBs were employed for mutational testing.7,21–25 As a general rule, direct smears are a more robust and valuable DNA source than matched cell blocks,26,27 and Diff-Quik-stained smears yield better preserved DNA than the Papanicolaou ones.28 Furthermore, cellular adequacy of direct smears may be assessed on site at the same time of the FNA procedure, and the most representative slide can be selected for DNA extraction. However, the policies that could allow to “sacrifice” a smeared slide for DNA extraction still need a clear understanding.26 Killian et al.28 scanned the cytologic slides on a digital scanner to preserve the cytomorphology prior to whole-slide scraping. CB processing is useful when the aspirated material, clotted after the prolonged echoendoscopic bronchial (EBUS) or digestive (EUS) procedures, cannot be smeared.24 The tissue collection for CB processing is easy, as the operator simply places the material aspirated by a dedicated pass in formalin; however, with respect to direct smears, the CB adequacy for tumor cells is not assessed immediately, requiring H&E staining of the paraffinembedded sections. CB cellularity is dependent on the number of dedicated FNA passes and on the post-procedural handling of the needle rinse specimen.26 In a recent FNA thyroid study of ours, the residual material from 1 or 2 passages by the lesion was processed as a CB.29 The criteria for CB adequacy were the presence of 3 or more groups of follicular cells or 2 or more tissue fragments according to Sanchez and Selvaggi.30 Only 27/84 (67.8%) cases had a contributory CB and were adequate for further molecular assessments. A cytopathologist's on-site evaluation of the harvested material, confirming the presence of a sizeable number of

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neoplastic cells, may be crucial. However, in a busy pathology department, it may not be possible to provide a cytology team for the assessment of adequacy because it can take up to 45 min for a single procedure.31 Liquid-based cytology (LBC) makes the processing of material easier and eliminates the need for slide preparation by clinicians. Instead of being smeared, cells are rinsed into a liquid preservative collection medium and processed on automated devices to prepare a monolayered LBC slide. With respect to the smear methods, LBC has the advantages of increased cellularity, a smaller examination area, a shorter examination time, reduced obscurement by blood or inflammatory cells, and more preserved nuclear details. Because only a portion of the collected cells is typically needed for the preparation of a liquid-based cytologic slide, the remaining specimen can be used for repeat cytology or mutational testing. LBC is an emerging solution to the application of molecular techniques to cytology. Liquid-based collection is currently the mode of choice for HPV testing in cervical cytology.32 Residual liquidbased preparation of cytologic samples from the thyroid33 and lung34 have successfully been used for DNA-based mutational testing.

DNA mutational assays on FNABs Laboratory-developed test validation Mutation assays approved for in vitro diagnostic (IVD) use by the European Community or by the U.S. Food and Drug Administration (FDA) are labeled only for routinely processed paraffin-embedded specimens.2 Given the lack of validated IVD tests for cytological material, all mutational assays on cytology are laboratory-developed tests (LDTs). Before being offered clinically, a rigorous validation process of any LDT is required and validation data should illustrate how the laboratory established assay performance characteristics such as specificity, sensitivity, and reproducibility; these, in turn, are influenced by the combination of individual specimen types (e.g., direct smear, CB, and monolayer preparation) and the analytical sensitivity of the selected assay (e.g., direct sequencing and real-time PCR). In recent studies by our group,34,35 we showed that for EGFR and KRAS mutation detection in lung cancer LBC samples, direct sequencing requires the selective isolation of the neoplastic cells on Papanicolaou-stained smears, whereas more sensitive nonsequencing methods directly performed on CytoLyt-derived DNA. In mutation testing, analytical sensitivity corresponds to the lowest limit of detection of mutant allele. An assay with a 10% analytical sensitivity can confidently detect a mutation if it is present in at least 10% of all alleles (including normal and mutant). To reach the threshold of 10% mutant alleles of a given gene, assuming the mutation is heterozygous, at least 20% of tumor cells are required. The amplification of mutant allele can increase the sensitivity of mutant detection.4 The assay sensitivity drops when the input level of tested material decreases; thus, the minimal input level of nucleic acid should be established for each sample/assay combination and only those specimens that meet this condition may be reliably processed. In addition,

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the comparison between cytological and paired histological samples from the same patient is an important step to evaluate the suitability of cytologic specimens for molecular testing, as it was shown for c-kit,33 EGFR,17 NRAS,36 BRAF,13,14 and KRAS37 processes validation. This comparison is feasible in thyroid molecular diagnostics. Our group validated BRAF and RET/PTC gene alteration retrospectively on papillary thyroid carcinoma (PTC) archival cytological slides and matched histological sample and then applied these markers by a FNA dedicated pass on prospectively collected samples.14,36,38 In contrast, in non-small cell lung carcinoma, a surgical resection specimen is often not available for direct comparison. Thus in many settings, the reported literature data may represent the only available benchmark.

Direct sequencing-based assays on cytology A variety of methods can be applied to cytological samples for somatic gene mutation analysis. From the technical viewpoint, the decision of which molecular assay should be performed depends on many factors. These include the mutation target (a limited number of known mutations vs. a wide variety of potential mutations); the type of the preparation, the prevalence, and distribution within the sample of the neoplastic cells; and, last but not the least, the equipment, experience, and personnel available in each testing institution. A simple and streamlined test may achieve a widespread clinical implementation; Sanger sequencing (SS) of polymerase chain reaction (PCR) products is robust and reproducible in multiple laboratories. SS relies on the incorporation of a chemically modified nucleotide (dideoxynucleotide) that terminates extension of the DNA strand at the point of incorporation. This results in a mixture of DNA fragments of different lengths. Each dideoxynucleotide (A, T, C, or G) is labeled with a different fluorescent dye, allowing their individual detection. The newly synthesized and labeled DNA fragments are separated by size through capillary gel electrophoresis. The fluorescence is detected by an automated sequence analyzer and the order of nucleotides in the target DNA is reported as a sequence electropherogram (Fig. 1). A gene point mutation will appear as the presence of both mutant and wild-type gene alleles (two overlapping peaks) at the particular nucleotide that is mutated. Similarly, an in-frame deletion gives rise to two overlapping sequences (Fig. 1). SS has extensively been applied on FNABs for BRAF9,10,13,16,39–42 and EGFR and/or KRAS19,20,34,43–46 testing, and it is mostly indicated in those diseases that have a wide variety of detectable mutations. Its main limitation is the low allelic sensitivity (15%); thus, in samples with o30% of neoplastic cells, the mutant burden does not reach the threshold of detection of SS, typically yielding false-negative results.23,47 Tumor cell enrichment by microdissection, either manually or by laser capture microdissection (LCM), makes direct sequencing more sensitive. Manual microdissection may be performed by scraping off the cells with a tip of an insulin syringe needle, a scalpel point, or hollow bore pipette while examining the cells under the microscope.13–15 However, this approach has intrinsic limitations because of the stochastic distribution of malignant and nonmalignant cells and the

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Fig. 1 – (A) Papanicolaou-stained smear before and after laser capture microdissection of a group of lung adenocarcinoma cells. (B) Electropherogram of exon 19 EGFR gene PCR products of cycle sequencing showing a deletion (arrow).

resulting technical difficulties of manually isolating a pure population of malignant cells.23 LCM makes direct sequencing highly sensitive even on a cytological preparation containing only a few cells (Fig. 1).17,19,21,34 Savic et al. used a laser pressure catapulting system to selectively dissect under visual control cancer cells from Papanicolaou-stained exfoliative and aspirative cytological specimens. This approach had a high (93%) success rate. The authors also estimated the minimal number of cells needed for EGFR gene sequencing. Although they were able to identify mutations even with only 30 cells, the most reliable results were obtained in samples from which at least 100 cells were analyzed.19 Chowdhuri et al.17 used LCM to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from lung adenocarcinoma cell block sections They were also able to consistently detect the mutation from as few as 50 microdissected tumor cells, while whole-slide scraping failed. Molina-Vila et al.21 applied material obtained by LMC directly in PCR buffer, thus eliminating the need for DNA purification. In a recent study, we consistently detected EGFR and KRAS mutations on 100, 50, and 25 microdissected cells from Papanicolaou-stained ThinPrep slides by LCM and direct sequencing.34

HRMA. Restriction fragment length polymorphism (RFLP) testing is based on mutation-specific restriction endonuclease sites, which give rise to size alteration of the PCR amplification product when treated with the enzyme.49 The PCR amplification process can be visualized in “real time” through fluorescence-labeled dyes, primers, or probes only when the tested mutation occurs. There is increase in signal intensity with increased quantity of amplification product, and it can be readily applied to the detection of specific mutations or to quantify a specific analyte. The EGFR, KRAS, and BRAF TheraScreen kits (DxS Ltd., Manchester, United Kingdom)50 based on the scorpion amplification refractory mutation system (S/ARMS) detected on cytology as few as 1% of mutant cells in a wild-type background.44,51,52 Similarly the peptide nucleic acid-locked (PNA-LCA) PCR clamp allows for selective amplification of the mutant alleles in the presence of 100-1000-fold wild-type alleles.53 Next-generation sequencing methods are attractive for mutational analysis since they are sensitive and can detect all mutations. However these techniques are expensive and are not yet readily available for routine clinical testing.54

Non-sequencing PCR assays on cytology

Applications of gene mutation assay to refine uncertain diagnoses

Although LCM enables mutation detection on a few cells, LCM is tedious, time-consuming, costly, and limited by the requirement of both special instruments and expertise. Thus, several highly sensitive non-sequencing methods that do not require the LCM step have more recently been described. High-resolution melting analysis (HRMA) is a highly sensitive and cost-effective screening method that allows rapid in-tube detection of DNA sequence variations based on specific sequence-related melting profiles of PCR products. HRMA identifies gene somatic mutations even in a small fraction of alleles in a background of wild-type DNA, as shown on ethanol-fixed and air-dried smears by Nomoto et al.48 and by Smith et al.,46 respectively. In a recent study, we showed that HRMA detects EGFR mutation directly on the DNA extracted from the LBC sample cell pellet overcoming the need of the time-consuming LCM.35 Although, HRMA is a reliable, sensitive, and rapid procedure for mutation detection, it should be kept in mind that positive results need confirmation. Mutation-specific methods are even more sensitive than

The use of DNA mutational testing on samples from superficial nodules, such as those of the thyroid, or from deep seated mass, such as those of the pancreas, is a rapidly expanding practice currently useful to refine indeterminate FNA diagnosis.

Thyroid The extremely large number of benign thyroid nodules and the small number of admixed malignant ones require an accurate screening tool. FNAB cytology efficiently identifies those nodules whose treatment unequivocally requires surgery. However, FNAB performance is highly dependent on the operator's experience, on accurate cytopreparation methodology, and on effective communication between physicians. The Bethesda system for reporting thyroid cytopathology (BSRTC) has been proposed at the 2007 National Cancer Institute (NCI) state-of-art thyroid FNA conference.55 This

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classification scheme divides adequate cases (containing at least six groups of thyreocytes) into a five-tiered risk-based system.55 At the end of this spectrum are those classes termed as “benign” or “malignant”; here, diagnosis is certain and the related post-FNA options are clear. Between “benign” and “malignant,” there are three diagnostic categories relative to indeterminate cytology; each features a different probability of malignancy.55 The associated risk is low (5– 10%) for “follicular lesion of undetermined significance” (FLUS), intermediate for “suspicious for follicular neoplasm” (15–30%), and high for “suspicious for malignancy” (60–75%).55 The final goal of molecular testing on thyroid FNABs is to further stratify into high-risk and low-risk categories the indeterminate cytology classes identified by the BSRTC.56 The BRAF mutation is the most common (up to 45%) genetic alteration in papillary carcinoma (PTC).57 It exclusively occurs in PTC and PTC-derived anaplastic thyroid carcinoma, whereas it does not occur in follicular thyroid carcinoma (FTC) and other types of thyroid tumors.57 Our group extensively tested BRAF and RET/PTC either on archival cytological slides or prospectively on a FNA dedicated pass14,36,38,58; in these studies and in several other series, all BRAF-positive FNA samples studied prospectively and retrospectively were papillary carcinomas. The high specificity of BRAF mutation for PTC underlines its potential clinical utility to refine undetermined cytological diagnosis (Fig. 2).57 However, BRAF testing cost-effectiveness is high only when PTC is strongly suspected on cytology.57,59 Deveci et al.,59 in a detailed analysis relative to the histopathologic follow-up of “follicular neoplasm” (n ¼ 339) and “suspicious for papillary thyroid carcinoma” (PTC) (n ¼ 120) FNAs, showed that these BSRTC classes have a very different malignancy rate (MR). The MR of “follicular neoplasms” was 22%, whereas that of “suspicious for PTC” was much higher (72%). In both classes, most of the malignant cases were follicular variants of PTC that represented 11% and 54% of cases, respectively. Conversely, classic PTC was seen in 2% and 12% of the cases.59 Since follicular variants of PTC do not or only rarely harbor the V600E and only in rare instances the K601E mutation, the diagnostic sensitivity of BRAF mutation testing alone is very

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low for “suspicious of follicular neoplasms” thyroid nodules and only slightly higher for “suspicious for PTC” classes.60 In an earlier study on archival smears, we were able to refine an undetermined diagnosis in 27% of cases14 and subsequently a similar rate (25%) was observed also in a study of prospectively collected FNAs.36 Although only a limited portion of patients within the gray-zone FNA diagnoses who need surgery may be identified,56,61 molecular testing has a very high positive predictive value (PPV).56 The high specificity of BRAF for PTC has led some institutions to consider BRAFpositive FNA, even in the absence of microscopically observed atypical, as the sole indication for initial thyroidectomy.62 Thus, BRAF is a low sensitive but 100% specific marker; in a recent analysis, we used BRAF together with a less specific, but more sensitive, immunocytochemical marker of thyroid cancer–galectin-3 (Gal-3).63–65 This is rarely detected in normal thyroid tissue and benign nodules, while its expression has frequently been demonstrated in malignant thyroid tumors. While Gal-3 sensitivity is high, there is some concern about its specificity because Gal-3 expression has been found in adenomatous goiter and Hashimoto's thyroiditis by immunohistochemistry.66 The BRAF/Gal-3 combined analysis yielded a modest improvement of the positive predictive value (from 73.9% to 78.6%), while the negative predictive value increased from 70.8% for Gal-3 alone to 89.5% for Gal-3/ BRAFV600E.38 Thus, a reduction of false-negative cases was achieved.38 The presence of only one “hotspot” (codon 600 in exon 15) renders PTC-associated BRAF mutations easily detectable on a technical point of view.67 Moreover, as a stable DNA molecular marker, BRAF mutation can be easily detected on common DNA specimens, even in low quantities, such as those obtained by FNA needle rinsing.8 Our method of preparation of FNA to harvest material sufficient for both tests was recently validated on a series of 128 routinely performed FNAs.8 The rationale behind our sample collection method was to ensure first an adequate cytological diagnosis and, then, to exploit part of the diagnostic material for molecular testing.8 Thus, two passes from different areas of the lesion are performed. A representative air-dried Diff-

Fig. 2 – (A) Diff-Quik-stained smear from a solitary thyroid nodule; note crowded and overlapping nuclei whose atypia were suspected but not conclusive for PTC. (B) The corresponding electropherogram showed the V600E mutation in the BRAF gene (arrow), refining into positive for PTC the uncertain cytological diagnosis.

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Quik-stained smear is prepared within few minutes and reviewed onsite.8 In 44 cases, the cytological evidences were sufficient for morphological assessment and the third pass was directly collected in RNA or DNA buffer extraction. Conversely, in 84 cases, the specimen either was deemed inadequate by the on-site evaluation or required an additional ethanol-fixed Papanicolaou-stained smear to better evaluate nuclear morphology. Thus, a third pass was dedicated to the preparation of an additional smear and only needle rinsing was collected for BRAF testing. Higher average of extracted DNA concentration was observed in the dedicated pass group (25.9 vs. 7.95 ng/ml). However, the rate of successful exon 15 BRAF amplification was similar with (43/ 44, 97.7%) or without (79/84, 94%) the dedicated pass. Thus, our protocol is suitable for both tests. When necessary, BRAF testing may also be performed on the residual samples of thyroid nodules, without interfering with routine cytology. Similarly, as far as mRNA markers are concerned, we have shown that in most samples, qRT-PCR analysis does not interfere with cytology.29 In fact, in a recent study on UbcH10 expression including 84 cases with a cytological diagnosis of either follicular neoplasm (n ¼ 57) or suspicious for malignancy (n ¼ 27), we found that most (73.8%) cases were adequate for both tests.29

Pancreas Endoscopic ultrasound (EUS)-guided FNAB has replaced histology in the workup of most pancreatic lesions.68 The Pancreatic Ductal Adenocarcinoma (PDA) cytological diagnostic criteria are well established.69 However, in some instances, microscopic features overlap with benign and reactive processes.70,71 This prompted many efforts to identify reliable molecular markers. The KRAS oncogene is the most promising one. Activating point mutations in its codon 12 occur in over 90% of cases, representing an early genetic alteration.72,73 The KRAS mutation, detected in a number of different cytologic specimens, including duodenal fluid,74 pancreatic duct brushing,75 and EUS-FNA,76,77 is a sensitive PDA marker, useful in undeterminated samples showing only few atypical cells. However, KRAS mutation has also been detected in the absence of cancer, in chronic pancreatitis and ductal hyperplasia, with or without atypias,78–80 questioning the specificity of KRAS as single molecular diagnostic marker. This has well been pointed out by Sturm et al.81 who on brush specimens that were obtained ex vivo from 57 consecutive pancreaticoduodenectomy resection specimens reported two mutations in pancreatic ductal hyperplasias. Thus to enhance specificity, rather than a single marker, a panel of gene alterations have been tested. Panel component candidates are TP53 and Smad4/Dpc4 whose mutations have been detected in 75%82 and 55%82 of PDAs, respectively. Indeed, by combining these three molecular markers (KRAS, p53, and DPC4), the diagnosis of atypia can be refined into adenocarcinoma in a third of cases.82 Cystic pancreatic lesions evaluated by FNA offer additional challenges. Since cyst epithelial lining may not be adequately sampled, malignant pancreatic cysts cannot always be differentiated from benign cysts by cytomorphology alone. Thus, clinical management must be based on a multidisciplinary

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approach83 including biochemical cyst fluid analysis and genetic markers. Among many different biochemical tumor markers,83 CEA better distinguishes between non-mucinous and mucinous cysts.84 However, its cut-off value is not well established and, in addition, CEA levels do not distinguish benign from malignant mucinous neoplasms reliably.83 This underscores the importance of interpreting information from biochemical analysis in the context of the clinical and radiological information, which may also be supplemented by molecular markers. Neoplastic mucinous cysts and PDA share some oncogenetic features, including earlier KRAS mutation occurrence followed by p53 mutation, and loss of p16 and Dpc4.85,86 One of the largest multi-institutional prospective studies published (the PANDA study) found that mucinous cysts harboring increased levels of DNA, the presence of KRAS mutation, and two loci of allelic imbalance (loss of heterozygosity) were predictive of malignancy,87 leading to the commercialization of several molecular tests.83,87,88

Applications of gene mutation assays in cytology to predict targeted treatment response The development of targeted therapies reflects the increased understanding of the pathways that have been mutated and deregulated in cancer.2 The principle is to block the growth and spread of cancer by interfering with specific molecules involved in carcinogenesis. Thus, the product of any gene frequently altered in neoplasia represents a target for drug development. In particular, a mutated gene product that is either constitutively active or active under conditions in which the normal gene product is inactive, is an ideal target. Identifying those patients carrying the alteration targeted by the drugs is crucial. This matters in cytology too.4 Targeted treatments are mostly effective in patients with locally advanced or metastatic disease; thus, performing a gene mutation test on a cytological sample is crucial. Many of the molecular targets are “driver mutations,” occurring early in carcinogenesis and being present throughout the tumor; thus cytological samples, even if representing only a very small portion of the lesion, are adequate for mutation detection.37

Anti-epidermal growth factor receptor therapy response prediction Two activated classes of EGFR antagonists, anti-EGFR monoclonal antibodies and small molecule tyrosine kinase (TK) inhibitors, are currently available for the treatment of the advanced forms of non-small cell lung cancer (NSCLC) and of colorectal cancer (CRC).89 EGFR antagonists are administered on the basis of gene mutation assays.89 Many studies have applied gene mutational assays in cytological samples of NSCLC and of CRC.2

EGFR testing on cytological samples of advanced NSCLC EGFR mutations are rare or absent in CRC tumors; conversely, they occur in lung cancer, preferentially being found in women, east Asians, never smokers, and adenocarcinomas.

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Activating mutations include in-frame deletions and amino acid substitutions clustered around the ATP-binding site in exons 18, 19, and 21 in the EGFR catalytic domain.2 These mutations correlate with a good response to gefitinib or erlotinib. A considerable fraction of NSCLCs are diagnosed solely by cytology, and o30% of patients with lung cancer at the time of presentation have an indication for surgery. In fact, advanced staging precludes the use of surgery as a first therapeutic approach in a large number of patients, limiting the availability of tissue samples for this kind of analysis. Therefore, applying EGFR mutational testing to cytology is of pivotal relevance. Literature data show that assessment of EGFR mutations in exons 18–21 mutation status from NSCLC patients' cytological samples is feasible on direct smears, CBs, and monolayer preparation by a number of molecular technique slides, and the results are comparable with those obtained from biopsies.2,4,90 Identification of EGFR mutations in cytological samples predicts clinical response to targeted therapies. In a recent study, the response rate and the median progression-free survival interval of patients whose mutations were detected on cytology were similar to those of patients diagnosed by histology.47 Recently, for the first time, a recommendation for good practice on lung cancer states that cytological specimens

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should be managed not only for diagnosis but also to maximize the amount of tissue available for molecular studies.91 In fact, EGFR testing on cytological samples needs to be integrated into a wider diagnostic algorithm. EGFR mutational testing cost-effectiveness strongly depends on the reliability of NSCLC subtyping.2,90 Although the distinction between small cell and non-small cell carcinoma on the basis of cytomorphology is highly accurate, refining the cytological diagnosis of NSCLC to squamous cells carcinoma (SQCC) or ADC can be difficult. However, NSCLC subtyping is important because patients with squamous carcinoma should not undergo EGFR testing.91 In addition, these patients cannot receive bevacizumab (Avastin) because of a 30% mortality rate due to fatal hemoptysis observed in patients with squamous cell histology.91 Most ADC cases display a positivity to thyroid transcription factor-1 (TTF-1), and a negativity for p63 and cytokeratin 5/6- immunohistochemical markers, whereas most SQCC cases show reverse expression for those markers (Fig. 3).52 In a recent prospective study, Nicholson et al.52 first subtyped 32 consecutive cases of NSCLC by this antibody panel, thus selecting eight cytological cases of ADC whose matched cell pellets were adequate for EGFR mutation analysis. Rather than manually aliquoting the harvested material, the LBC method may better preserve the “informativeness” of

Fig. 3 – (A) Papanicolaou-stained smear from a right lung peripheral mass FNA showing a neoplastic cell group suspicious for adenocarcinoma. (B) TTF1 immunostaining showing a strong nuclear signal. (C) Capillary electrophoresis of cycle sequencing exon 19 EGFR gene PCR products showing a deletion (arrow).

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Fig. 4 – (A) Diff-Quik-stained FNA smear of CRC metastasis to the thyroid; note the peripheral palisaded cigar-shaped nuclei typical of CRC. (B) On cell block, CK20 immunoreactivity confirmed the colonic origin of the cells. (C) The KRAS gene was mutated, as indicated by a double peak on the electropherogram (arrow).

the aspiration for morphology, immunostaining, and EGFR testing.34,35 LBC may capitalize the material to enable, besides morphology, DNA and RNA extractions and other ancillary techniques, such as immunostaining that may be required before EGFR testing to refine the cytological diagnosis of NSCLC to squamous cell carcinoma or adenocarcinoma. Our series included eight LBC samples that had previously been immunostained by the referring pathologists and were still adequate for EGFR and KRAS genotyping.34,35

KRAS testing on cytological samples of metastatic CRC CRCs harboring a mutation in exon 2 (codons 12 and 13) of the KRAS gene (40% of cases) do not derive benefit from

treatment with cetuximab, a chimeric IgG monoclonal immunoglobulin, and panitumumab, a fully human monoclonal IgG2 antibody.49 Typically, the samples tested for KRAS are tissue blocks of surgical specimens. However, it is estimated that 20% of the target patient population (mostly patients with rectal tumors undergoing neoadjuvant therapy) present with metastatic disease and do not have tissue archival material from the primary tumor.49 In these cases, cytological samples from the metastatic lesions may be exploited for KRAS mutation testing.37,51,92 In fact, KRAS mutation is an early event in colorectal carcinogenesis that marks the transition from early to late adenomas. In fact, even before malignant transformation, 30–35% of benign colorectal adenomas bear a KRAS mutation, a proportion similar to that

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observed in invasive cancer.2 Thus, KRAS testing could be indifferently performed on the primary tumors or on the corresponding metastases. This is relevant to the practicing cytopathologist. In fact, a similar KRAS mutational status was found both in primary tumors and in metastases for 490% of the patients with CRC. Only few cases of KRAS mutations in metastases arising from wild-type KRAS primary tumors have been documented.93 Recently, we aspirated a CRC metastasis to the thyroid harboring the G12D KRAS mutation; the same mutation was evident in the primary cancer of the colon and in its liver and lung metastases, which was in line with the notion of KRAS mutation being a stable feature of CRC (Fig. 4).94

Prediction on cytology of the response to targeted therapy of the GISTs GISTs are the most common mesenchymal neoplasms of the gastrointestinal tract, occurring more frequently in stomach (50–60%).95 EUS-FNA has demonstrated an overall good sensitivity in the preoperative diagnosis of GIST, although the cytologic yield is influenced by the size and location of the lesion.83 Cytological diagnosis is based on microscopic criteria supplemented by the immunohistochemical demonstration

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of KIT, a transmembrane receptor tyrosine kinase (TK), reactivity.96 However, less frequently, KIT-negative GIST may be encountered.97,98 In the latter case, an immunohistochemical panel including DOG1, can allow for the diagnosis.16 Gene activating mutations, found in over 80% of GISTs, induce KIT protein GIST overexpression and also predict positive response to the TK inhibitor imatinib treatment.99 A subset of GISTs lacking KIT immunostaining may instead harbor exon 18 PDGFRA activating mutations. The most common PDGFRA mutation, the D842V substitution, is associated with primary imatinib resistance while other less common mutations may induce treatment responsiveness.100 The cytological samples obtained by EUS-FNA have been exploited for KIT and PDGFR molecular analysis (Fig. 5),33,46,101,102 which allowed for neoadjuvant imatinib administration in the locally advanced stage or metastatic settings.103,104

Prediction on cytology of the response to targeted therapy of metastatic melanoma Since advanced melanoma is largely refractory to conventional systemic therapies, the recent introduction of

Fig. 5 – (A) Cell block preparation from EUS-FNA of a submucosal gastric mass; note the GIST spindle-shaped interlacing cells. (B) The latter showed a strong CD117 immunostaining. (C) Electropherogram of exon 11 c-KIT gene PCR products showing a deletion (arrow).

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targeted therapy has changed the clinical management of patients with metastatic melanoma.26 The impressive efficacy of BRAF inhibitors in unresectable or metastatic melanomas harboring the V600E or the V600K mutation led to the recent accelerated approval of Zelboraf (vemurafenib) for Stage IV melanoma.105 Since vemurafenib carries a significant risk of cutaneous squamous cell carcinoma and has the potential to increase disease progression in BRAF wild-type tumors, BRAF testing for therapeutic eligibility is warranted. In addition, although the standard of care is not yet established, off-label use of imatinib for melanoma is not uncommon and, therefore, also KIT testing is currently relevant.105 The cobass BRAF Mutation Test kit (cobass test) has recently obtained the FDA approval as a companion test BRAF for Zelboraf. This test is a TaqMelt™-based PCR assay based on the detection of target DNA using complementary primer pairs and two oligonucleotide probes labeled with different fluorescent dyes.106 However, testing laboratories are currently exploiting for BRAF mutational analysis in melanoma the equipment and the methodology already validated for other mutational assays. Several studies showed that BRAF mutational analysis on archival and fresh direct smears or on CBs from FNAs of metastatic melanomas is feasible and accurate (Fig. 6).105

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Limitations of mutation testing in cytopathology The translation of the mutational tests from dedicated research labs to cytopathologic diagnostic settings is problematic. As detailed earlier, the performance of any given molecular method is highly dependent not only on the analytical sensitivity of the technique but also on several pre-analytical issues. To increase the robustness of mutational testing in cytology, there is a need for an increased standardization of cytopreparation protocols. All mutational assays on cytology are “home brew.” Although a rigorous validation process is required, the combinations among preparation features (source, type, fixation, and staining), tumor cell enrichment modality (manual dissection or LCM) and the subsequent molecular analytical method (SS vs. non-sequencing) are numberless. Thus, it is impossible that a single testing laboratory can evaluate the effects of any possible combination. This calls for a change of cytotechnologists' and cytopathologists' mentality to collect and process the cytological samples for both microscopy and molecular tests. Preserving the informativeness of the aspirated material for both traditional and molecular approaches may be crucial. For example, in thyroid and pancreatic cytology, DNA

Fig. 6 – Lymph nodal FNA from a melanoma metastasis. The Diff-Quik slide showed dyshesive plasmacitoid neoplastic cells (A) whose nuclei were round to oval with an evident nucleolus better appreciated in Papanicolaou preparation (B); in the background, several “tangles” represent the residual lymphocytes hosts. (C) Electropherogram of exon 11 BRAF gene PCR products showing the V600E mutation (arrow).

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mutational testing can refine indeterminate microscopic diagnosis only when cytological specimens are properly handled to provide morphological and molecular information. To this end, each of the steps of traditional cytology, such as preparation of FNAB material, search for morphological criteria, and assignment to the correct diagnosis, needs to be preserved, and collection of additional material for molecular testing by further aspiration of nodules, needle washout with nucleic acids preserving solution, or storage of FNABs samples at low temperature should not interfere with routine practice. Then the relevance of the additional diagnostic information is fulfilled when interpreted in the context of cytology by pathologists. The relevance of the correct application of positive and negative controls when applying PCR techniques to smears cannot be overemphasized.11 Formalin fixation and cell block (CB) embedding, the standard procedures when additional cellular material is harvested for immunocytochemical studies, may yield false-positive results. By using DNA extraction kits specifically developed for paraffin samples, amplifying small PCR fragments (150–200 bp) and performing all assays in duplicate, the reliability of DNA testing on CBs increases,12 minimizing the risk of artifacts and false-positive mutation detection.12 The potential sources of false negatives must also be considered. A major limitation of some testing methods, and in particular of Sanger sequencing, is the low analytical sensitivity of the test. The cytopathologist should provide an estimate of the percentage of neoplastic cells in the smear areas that will be used for DNA extraction, and that percentage should exceed the established limit of detection of the assay. When this requirement is not met, a laboratory may carry out the testing anyway, but if mutations are not detected, the laboratory will have to interpret the result as “undeterminated” (Fig. 3). In conclusion, one of the basic requirements to favor the implementation of cytology-based DNA mutational tests is educational. As pointed out clearly in a very recent and comprehensive review by Clark,2 there is necessity for “pathologists to be significantly engaged with the clinical and therapeutic aspects of cancer care to be knowledgeable partners in this process.” The pathologist should not only appreciate the reasons for testing but should also understand the underlined methodology of the various polymerase chain reaction (PCR)-based assays. Based only on such knowledge, the pathologist can refer specimens for testing in an appropriate and timely manner. This urges for more updated training programs that will provide the opportunity to the future cytopathologists to understand techniques and to incorporate new technologies into cytological practice.107

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