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Possible use and role of molecular techniques in fine-needle aspiration cytology (FNAC) practice
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Possible use and role of molecular techniques in fine-needle aspiration cytology (FNAC) practice
exon sequences to flank rupture points of translocations. PCR applications are centered in diagnosis of solid tumour detecting gene mutations or detecting clone gene rearrangement in lymphoproliferative disorders. PCR analysis can be performed directly with fresh collected material from FNAC, in liquid-based cytology samples or even with cells scraped from FNAC slides. In the first condition, the needle should be washed up in ethanol, methanol or culture mediums like RPMI. The amount and quality of DNA obtained by FNAC for PCR assay does not seem to be a problem and 50e100 cells are adequate to obtain good PCR results. FNAC obtained tumour cells provide excellent representative samples, with poor contamination of stroma or local structures. In situ hybridization methods (ISH) can also be applied to FNAC, permitting, with either fluorescent or chromogenic markers, to detect numerical or structural aberrations of chromosomes. This technique is a reliable technique, particularly useful in cytology as it can be applied directly in smears. Monolayer smears are ideal for ISH techniques and slides with ethanol or air-dried fixed preparations are equally suitable. These techniques are used to detect deletions, insertions or translocations, but can also be used in routine to detect gene amplifications like HER2 in breast carcinoma or NMYC in neuroblastomas. There are two main fields for application of molecular techniques to FNAC: diagnosis and prognosis/therapeutic selection.
Fernando Schmitt Helena Barroca
Abstract Fine-needle aspiration cytology (FNAC) is a recognized ancillary tool in the diagnosis of tumours and infectious diseases. Throughout decades cytopathologists did tremendous marvels with pure morphologic criteria, diagnosing in shrinking amounts of material. The recent understanding of complex cancer biology reshapes the practice of FNAC. The scientific advance of image techniques gave cytopathologists the possibility to go to deep locations and collecting material from previous unlikely locations. This conjunction of knowledge’s and techniques has increased the role of FNAC in the diagnosis, prognosis and in the establishment of new therapeutic targets. Lately, core needle biopsy is gaining popularity and advantage regarding FNAC. The sample limitation of FNAC regarding immunocytochemical staining with proper controls, as well as the limitation in appreciating invasiveness has lead, in some fields, like soft tissue tumours, breast, lymph node, and prostate, to diminish the use of FNAC. However FNAC still is a less invasive, effective, and cost efficient technique. In this review we demonstrate the application of basic molecular techniques to FNAC as a crucial tool in the diagnosis, prognosis and planning of alternative molecular therapeutics of tumours.
Diagnosis
Fernando Schmitt MD PhD FIAC is at the Instituto de Patologia e Imunologia Molecular da Universidade do Porto e IPATIMUP, Faculdade de Medicina da Universidade do Porto, Portugal. Conflicts of interest: none declared.
The recent perception that some tumours carry specific genetic changes in a background of a simple karyotype has lead to the appearance of new commercial available probes of great utility in clinical practice of cytological diagnosis. Soft tissue tumours represent the archetype of this application. A new molecular classification of soft tissue tumours into two main groups has been proposed: (1) tumours with specific genetic alterations and (2) tumours with no specific genetic alterations on a complex background of numerous chromosomal changes. Approximately, 15e20% of mesenchymal tumours belongs to the first group and is associated, in a nearly exclusive and pathognomonic way, to characteristic chromosomal translocations (Table 1). The resulting fusion protein of the specific translocation can act either as a transcription factor, upregulating genes that will promote tumour growth or act in the activation of oncogene promoters with consequent tumour initiation.3 This specific genetic changes revealed such an impact in soft and bone tumour classification that in the recent WHO classification of these tumours, histogenesis is no longer the major determinant in subtyping. In this new classification a combination of diagnostic parameters that include morphology and genotype is advanced. Other tumours like GIST, fibrous dysplasia, and rhabdoid tumours carry specific somatic mutations that act amplifying the response to external stimulus and promote cell cycle progression and growth. These mutations represent a great aid to the cytopathologist assisting them in differential diagnosis of problematic cases or in some cases predicting outcome and selecting patients for targeted therapeutic strategies.
gica do Helena Barroca MD is at the Servic‚o de Anatomia Patolo ~o Joa ~o, Porto, Portugal. Conflicts of interest: Hospital de Sa none declared.
Solid tumours Soft tissue sarcomas: a particular group of sarcomas affecting mainly children and young adults is the small blue round cell
Keywords FNAC; immunocytochemistry; molecular diagnostics
Introduction The molecular techniques most commonly used in FNAC include polymerase chain reaction (PCR) and in situ hybridization (FISH). Other techniques such as in situ PCR, direct sequencing, microarrays and proteomic methodologies are just now entering in the clinical practice in some centres but are still not disseminated in the clinical practice.1,2 PCR methods are ideal for FNAC material and some application are detection of gross chromosomal alterations as deletions and translocations or even point mutations in individual genes. RT-PCR uses cDNA as a template and primers
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identification of EWS/ETS fusion in problematic cases is a helpful tool for cytological differential diagnosis. Rhabdoid tumours show a cytological picture of multiple single polygonal cells with distinct cell borders and clear vesicular nuclei, with occasional single prominent nucleolus. Cytoplasm is fragile but occasionally, intracytoplasmic hyaline-like eosinophilic material is found. Except for the immunoexpression of INI1 marker, all other antibodies expressed by this tumour (vimentin, cytokeratins, EMA, synaptophysin) are also shared by PNET and Meduloblastoma (MB). Cytogenetic and molecular studies are a useful adjunct in the differential diagnosis with these entities. An isochromosome 17q is present in approximately 30e40% of MBs and has not, to date, been observed in AT/RT. Conversely, monosomy or deletion of chromosome 22 is seen in the majority of rhabdoid tumours in all anatomical sites. Karyotypes can be prepared directly from samples obtained from FNAC, or DNA can be isolated and analyzed by PCR-based microsatellite analysis for loss of heterozygoty using probes that map to 22q. HSNF5/INI1 gene seems to be implicated somehow, in an ATP-dependent remodeling chromatin way, thus allowing transcription factor binding to DNA. Some authors hypothesized that the deletion or mutation of INI1 might be associated with the aggressive and usually fatal clinical course in children with AT/RT. This behaviour could also account for some misdiagnosed PNET or MBs, with unpredicted quick outcome. Alveolar rhabdomyosarcoma (ARMS) is another member of this SBRCT group. ARMS accounts for 30% of all rhabdomyosarcomas and represent the most frequent soft tissue tumour seen in childhood. Cytological smears are characterized by loosely cohesive aggregates of uniform, small round blue cells, with little or no specific differentiation. In more differentiated cases, multinucleated neoplastic giant cells with eosinophilic cytoplasm can be present as well as racquet or tadpole cells, helping in differential diagnosis. The importance of recognizing this entity is essential for an adequate treatment. Two forms of translocation have been associated to ARMS: t(2; 13) (q35; q14) in 70% of the cases, and t(1; 13) (p36; q14) in 10e20% of the cases. Recently ARMS with t(1; 13) were found to have better prognosis than those with t(2; 13) (q35; q14). Rhabdomyosarcoma can sometimes exhibit “mixed” embryonal and alveolar phenotypes, and it is not unlikely that in cytological smears, with typical embryonal morphology, the presence of dispersed small round cells are missed or misinterpreted as nude nuclei. This “mixed” morphology gives these tumours a worse prognosis than that of pure embryonal rhabdomyosarcoma, and a clinical outcome similar to that of pure ARMS. FNAC allows the collection of a sample from different parts of the tumour, increasing the possibility of making a correct diagnosis even in heterogeneous neoplasia (Figure 1). Synovial sarcoma (SS) represents 5e10% of soft tissue malignancies in the first two decades of life. Undifferentiated tumours with primitive small round cells occur in 15% of the cases and represent a challenge in the differential cytological diagnosis with all the other small round cell tumours. These tumours fail to immunoexpress most epithelial markers, excepting EMA, and are immunoreactive to CD99. This profile turns differential diagnosis with EFTs imperative. The detection through RT-PCR of a reciprocal translocation t(X; 18) (p11.2;
Chromosomal translocations in soft tissue sarcomas Tumour type
Cytogenetics
Ewing sarcoma/PNET
t(11;22)(q24;q12) t(21;22)(q22;q21) t(7;22)(p22;q12) t(2;22)(q33;q12) t(17;22)(q12;q12) t(11;22)(p13;q12) t(9;22)(q22;q12) t(9;17)(q22;q11) t(9;15)(q22;q21) t(2;13)(q35;q14) t(1;13)(p36;q14) t(12;16)(q13;p11) t(X;18)(p11;q11) t(12;22)(q13;q12) t(X,17)(p11.2;q25) t(17;22)(q22;q13) t(12;15)(p13;q25)
Desmoplastic small round cell tumour Extraskeletal myxoid chondrosarcoma
Alveolar rhabdomyosarcoma Myxoid liposarcoma Synovial sarcoma Clear cell sarcoma Alveolar soft-part sarcoma Dermatofibrosarcoma protuberans Congenital fibrosarcoma/Mesoblastic nephroma Table 1
tumours (SBRCT). This group encompasses a heterogeneous and morphologically similar group of tumours, composed by undifferentiated small round cells. Tumours such as neuroblastoma, PNET/Ewing sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma (undifferentiated type), small cell osteosarcoma, mesenchymal chondrosarcoma and desmoplastic small round cell tumour are included in this group. In most instances, immunocytochemistry is essential to perform the correct differential diagnosis. Some of these tumours, share antigenic determinants and immunostainning is not always concluding. Molecular diagnosis is especially important in detecting specific translocations in Ewing sarcoma, rhabdomyosarcoma, synovial sarcoma or desmoplastic small round cell tumour. Ewing tumour and PNET belongs to a now recognized subgroup and named as the Ewing’s family tumours (EFTs). These tumours account for less than 1% of all soft tissue tumours and affect essentially children and young adults. Cytological smears of EFTs are characterized by a discohesive population of small round blue cells with few distinguishing characteristics. Some cytological minor details, such as the presence of a tigroid background or the presence of dispersed picnotic cells can be suggestive for a trained cytopathologist. CD99 is the most useful immunostain in the differential diagnosis with other malignant round cell tumours. Nevertheless, other tumours, like alveolar rhabdomyosarcoma, undifferentiated synovial sarcoma, and lymphoblastic lymphoma, displaying a similar cytological pattern and CD99 positive, must be individualized. In 85% of EFTs there is a specific tumour-associated translocation t(11; 22) (q24; q12), juxtaposing the EWS gene on chromosome 22 with the FLI1 gene on chromosome 11. This translocation seems to appear very early in tumourogenesis being deeply involved in tumour initiation. Other less frequent variants of EFT translocations have been described, all involving the EWS gene. The
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Figure 1 Two examples of molecular technique application in FNAC material. On the top an example of a rhabdomyosarcoma smear in Giemsa stain (left top) and on the right top a three interphase nuclei studied by FISH with the FKHR Break Apart Rearrangement probe. The upper and lower nuclei exhibit a typical pattern of FKHR rearrangement, with a separation of the probes centromeric (green) and telomeric (red) to the FKHR locus. On the bottom we have the cytological representation of a lung adenocarcinoma (left bottom) and at its right the representation of the respective sequencing EGFR exon 19 showing the presence of mutation Glu 746_Ala750 del.
q11.2) seen in 90% of the cases is highly specific of SS and is essential to achieve a correct diagnosis in problematic cases. This translocation creates a fusion between SS18 (SYT ) gene on chromosome 18 and one of two copies SSX1 or SSX2, of gene SSX on X chromosome. SYT-SSX1 fusions seem to be implicated in epithelial differentiation of SS whether SYT-SSX2 are the most linked to monophasic SS. These fusions codify an abnormal nuclear transcription factor that affects chromatin remodulation. A further effort to establish a relationship between the type of translocation and prognostic has failed till now. Other less frequent malignant round cell tumours also bear specific translocations such as desmoplastic small round cell tumour (DSRCT), mesenchymal chondrosarcoma and myxoid liposarcoma (see Table 1). Desmoplastic small round cell tumour is a highly malignant mesenchymal neoplasm which affects mainly young adults, and has a predilection to abdominal location. Cytological smears resemble those of all other malignant small round cell tumours.
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Occasionally, stromal fragments dispersed in the slide are typically seen. This tumour has a distinctive immune profile, coexpressing vimentin, epithelial, muscle, and neural markers. Characteristically, the expression of desmin has typically a dotlike, paranuclear pattern. Muscle specific markers, like MyoD1 or myogenin are negative. CD99 can also be expressed in 35% of the cases, diffusely staining the cytoplasm. In EFTs membranous unequivocal cytoplasmic membrane staining pattern of CD99 is required for diagnosis, but this requisite is not always easy to assess as there is frequently some degree of cytoplasmic staining. Cytogenetically DSRCT is laureated with a reciprocal translocation, t(11; 22) (p13; q12). Spindle cell tumours also share similar morphology, many of these tumours are undistinguishable in their morphological characteristics and if histological classification is frequently a challenge to most pathologists’ FNAC diagnosis is even more defying. Clear cell sarcoma and monophasic synovial sarcoma are two examples of spindle cell tumours where diagnosis can
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sometimes be dubious. Both these tumours encompass discriminative translocations (Table 1). The variability in cytological features in gastrointestinal stromal tumour (GIST) implies that a diagnosis of GIST should be rendered on cytology in conjunction with immunocytochemistry result of CD117. CD117 antibody recognizes a transmembrane protein, c-Kit, involved in tumour proliferation and shared by other tumours like seminoma, and melanoma, beyond others. C-kit is detected in most GIST’s and represents a hallmark that helps in the differential diagnosis with other mesenchymal tumours. In some GIST were CD117 is negative, the assessment of c-kit mutation by molecular techniques in FNAC sample can be helpful for diagnosis.
the TFE3 gene on chromosome X. Both entities, ASPS and RCC tXp11.2, seem to be associated to the overexpresion of the gene MET receptor tyrosine kinase with increased levels of the MET protein. In the future further studies must be done, exploring the possible response in these tumours with selective inhibitors of the MET tyrosine kinase. Thyroid tumours In thyroid the benefit of using molecular detection of oncogenes on cytology smears is progressing fastly. Oncogenes like BRAF, RAS, RET/PTC have been implicated in thyroid carcinogenesis. The detection of mutation in one of these genes is a strong indicator of malignancy in thyroid. BRAF is the most frequent genetic alteration found and is almost exclusive to papillary thyroid carcinoma (PTC). Apart from BRAF, these oncogenes although sensible concerning malignancy, whenever detected, are not so sensitive discriminating papillary from follicular carcinoma or even adenoma. Still a significant proportion of carcinomas in thyroid lack these genetic markers. Moreover, positivity for RET/PTC has been described on Hashimoto thyroiditis. BRAF mutation is the most common genetic alteration identified in PTC. In about 80% of follicular carcinomas, mutation of RAS or PAX8/PPARg translocation is also found. Recently, Nikiforov et al. suggested that using a panel of markers to detect alterations in BRAF, RAS, PAX8/PPARg and RET/PTC can increase the sensitivity of thyroid FNA in problematic or inconclusive cases.4
Renal cell tumours Apart from sarcomas some renal tumours are also assessed by FNAC, for tumour initial diagnosis and therapeutic planning. Chiefly in European countries, following SIOP protocols, large pediatric renal tumours are initially treated with chemotherapy, for tumour reduction and surgical facility. It is so not uncommon to perform an initial cytological diagnosis by FNAC. A few renal tumours affecting essentially children are also characterized by specific genetic alterations. Mesoblastic nephroma, rhabdoid tumour and more recently described, a special type of renal carcinoma (RCC) Xp11.2 associated, is characterized by specific translocations. Mesoblastic nephroma and some RCC Xp11.2 associated share a gene expression profile with two soft tissue entities respectively: congenital fibrosarcoma and alveolar soft-part sarcoma, respectively (Table 1). This fact should not constitute a surprise, since the kidney is a mesoderm derived organ. Mesoblastic nephroma (MN) is a benign renal tumour, diagnosed essentially in the first 3 months of life. Cytological smears show spindle cells singly or in cohesive fragments with fibroblastic/myofibroblastic bland nuclei. This pattern can simulate the mesenchymal component of a nephroblastoma. In MN it is not uncommon that entrapped local renal tubules of the periphery of the tumour, with embryonal metaplasia, are picked together in FNAC sample, simulating the epithelial component of a Wilm’s tumour. FNAC obtained sample is generally enough and of good quality to perform PCR-based molecular techniques, in order to detect a t(12;15), characteristic of this entity, allowing the correct diagnosis. Recently, it has been described a subset of RCC, in young patients, associated to Xp11.2 genetic alterations. These tumours have distinctive immunohistochemical features. Unlike adulttype RCC’s these tumours do not show intensive immunoreactions to vimentin, or epithelial markers. They are immunoreactive to CD10 and to TFE3 protein. In cytological smears they reproduce what is has been observed in histology: numerous sheets and papillary groups of polyhedric cells with central round nuclei with prominent nucleoli and a clear or granular cytoplasm with ill defined borders. The presence of psammoma bodies is also referred, and should give a clue to cytopathologists. There are two main forms of RCC Xp11.2 associated: RCC t(X; 17) (p11.2; q21) and t(X; 1) (p11.2; p34). Clinically, despite its usual advanced stage presentation, these neoplasms’s course indolently. RCC Xp11.2 associated share with alveolar soft-part sarcoma (ASPS), a similar translocation point
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Pancreatic cysts/tumours Preoperative assessment of pancreatic cystic lesions poses a major diagnostic challenge. Differentiation of benign and potentially malignant pancreatic cysts is difficult even using a multimodal approach. The relatively low sensitivity of cytologic examination has prompted search for other testing with potentially diagnostic yield. A recent review showed that the presence of K-ras mutation that can be done with only 200 mL of the cyst content is quite specific to mucinous cysts.5 In negative K-ras cases, CEA level is able to capture almost 70% of the cases. The drawback of this test is that it needs 1 ml of cyst fluid to perform the assay. Concerning malignancy, cytomorphological findings is the most specific test for diagnosis. However, in the absence of a solid component, pancreatic cysts aspirates frequently have poor cellularity. The specific sequence of K-ras mutation followed by allelic loss is most specific for malignancy. Recently, it was demonstrated that the expression of K homology domain containing protein (KOC), an oncofetal RNA binding protein that regulates insulin-like growth factor and cell proliferation during embryogenesis, is a new marker of malignancy for pancreatic adenocarcinoma and that can be studied in cytological specimens obtained by fine-needle aspiration cytology. KOC expression is limited to invasive pancreatic ductal adenocarcinoma and may thus serve as an adjunct to cytologic cases where atypical features preclude a definitive diagnosis of malignancy.
Hematolymphoid disorders In many institutions FNAC is used as a first line approach to evaluate lymphadenopathies. FNAC yields a high rate of conclusive cytological diagnoses in the assessment of Hodgkin
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disease or large cell non-Hodgkin lymphomas (79e90%). Association of cytology and flow cytometry is used in some centres with success.6 Nevertheless difficulties appear in some situations where despite the best efforts of cytology and flow cytometry some lymphomas remain impossible to diagnose. The combined use of morphology, flow cytometry for immunophenotyping and genetic analysis has increased the accuracy of diagnosis and correct categorization of lymphomas on FNAC samples. The most frequently used molecular techniques in this field are PCR and FISH. Molecular techniques in lymphomas aim two main objectives: 1 e detection of clonality and 2 e detection of molecular characteristics linked to specific subtype of lymphomas. Detection of clonality represents the essential step in the differential diagnosis of reactive lymphadenopathies and lymphoma. Lymphomas develop from a single clone of B or T cells, and as so, all neoplastic lymphocyte population present an identical Immunoglobulin (B cells) or TCR (T cells) gene rearrangement. Clonality can be demonstrated indirectly through immunocytochemistry or flow cytometry, establishing the presence of a light chain restriction in B-cell lymphomas or aberrant expression of T cell antigen in a predominant cell population, or in a direct way, by PCR.7 The presence, in a PCR test, of a monoclonal band is however not necessary an undisputed synonymous of malignancy. In some specific clinical settings it is not infrequent to find minor monoclonal reactive lymphocyte proliferations. Consequently it is advisable that PCR results are interpreted together with clinical and cytological features as well as other ancillary techniques. Another approach is the detection of molecular characteristics linked to specific subtype of lymphomas. There are several lymphomas associated to balanced translocations (Table 2). Follicular lymphoma, mantle cell lymphoma, Burkitt’s lymphoma and anaplastic large cell lymphoma are, in high percentage of cases, associated to oncogenic markers whose identification helps the cytopathologist in the correct diagnosis. However failure to identify these markers does not by itself excludes diagnosis. Cytogenetic and molecular breakpoints are heterogeneous and complex and some molecular techniques are best suitable for some entities than others. For example the detection of t(14:18) in follicular lymphomas by karyotype occurs in 75% of the cases, by PCR in 50e70% of the cases, whether the detection of aberrant expression of the BCL2 protein is found in 90% of the cases. In Burkitt lymphoma, FISH
analysis is considered the most rapid and appropriate method for detection of t(8:14) and in anaplastic large cell lymphoma immunophenotypic analysis is the more appropriated method for diagnosis, leaving PCR for confirmation in inconclusive cases or for follow-up. Identification of these chromosomal translocations helps cytopathologists in the correct classification of lymphomas, but also represents an advance in prognostic assessment and staging. For example, the BCL2 rearrangement and the presence of a translocation of BCL6 in diffuse large B-cell lymphomas are mutually exclusive and this last one is linked to a worse prognosis.
Prognosis and therapeutic selection The knowledge of genetic, epigenetic or metabolic features that contribute to cancer development and growth, represent good targets for cancer therapy. This fact has lead scientist into a run seeking out for better cancer therapies with good efficacy and low toxicity. Loss of cell cycle control, uncontrolled proliferation, inappropriate survival, decreased apoptosis, immortalization, invasion, angiogenesis and metastasis, are all deregulated mechanisms that characterize cancer and that represent excellent targets to work out for novel therapies. Till now emphasis has been given essentially on cell cycle, proliferation and angiogenic involved targets, but other mechanisms-based therapies are been investigated, especially those inducing apoptosis, telomerase inhibitors, invasiveness and metastasis inhibitors. Here we will mention some examples where molecular therapeutic targets/ prognostic indicators can be studied in FNAC material.8 GIST has recently gained the interest of researchers, in the sense that it embodies the model of an outstanding metabolic response to a targeted therapeutic drug, the imatinib. In GIST the mutation of c-kit gene, a transmembrane receptor connected with tyrosine kinase receptor, leads to an autonomous enhance stimulation of the tyrosine kinase cascade with consequent tumour proliferation and development. These tumours are also associated to plateletderived growth factor receptor (PDGFRA) gene mutation also a member of the type III tyrosine kinase receptor family. Mutation c-kit or PDGFRA are mutually exclusive and represent two different alternative oncogenic events leading to similar biological consequences. As important as classifying GIST tumours in low or high grade, it is essential to realize whose tumours will be the best responders to therapy. Tumour clinical response to Imatinib is determined by the affected exon in c-kit mutation. In GIST four different regions of KIT have been found to be involved, exon 11 (juxtamembrane domain), exon 9 (extracellular domain), exon 13 and exon 17 (tyrosine kinase domain). Also PDGFRA mutations vary according to the exon involved (exon 18 e tyrosine kinase domain or exon 12 e juxtamembrane domain). The highest overall response rates have been reported in patients with GIST harbouring c-kit exon 11 mutations. The clinical response rate decreases to 45% in patients with c-kit exon 9 mutations. PDGFRA exon 18 point mutations involving amino acids 841e843 are clinically resistant to imatinib. However, the single exon 18 mutation D842Y is associated with good response to Imatinib. Our group demonstrated the feasibility of performing molecular analysis of c-kit and PDGFRA genes in cytologic material obtained by endoscopic ultrasound-guided FNA from GISTs.9 PCR amplification and DNA sequencing revealed c-Kit mutations in 61% of
Chromosomal translocations and affected genes in lymphomas Lymphoma
Chromosomal translocation
Affected genes
Follicular lymphoma Burkitt lymphoma Diffuse large B-cell lymphoma Mantle cell lymphoma Anaplastic large cell lymphoma Malt lymphoma
t(14:18)(q32:q21) t(8:14)(q24:q32) t(3:14)(q27:32)
BCL2 C-MYC BCL6
t(11:14)(q13:q32) t(2:5)(p23:q35)
BCL1 NPM, ALK
t(11:18)(p21:q21)
Table 2
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GIST cases, in accordance with previously published ranges (30e90%). Nearly 95% (19/20) of c-kit-mutant tumors carried exon 11 mutations. In this study, we have shown that GISTs could be diagnosed pre-operatively on EUS-FNA specimens. In addition to immunocytochemistry, molecular analysis can be done in formalin-fixed and paraffin-embedded cell block material obtained from these aspirates, avoiding more invasive diagnostic procedures to obtain a tissue diagnosis. In nowadays, with two RTK inhibitors available, there is an imperative need in refining GIST diagnosis and including a molecular analysis of both c-Kit and PDGFRA in the current diagnostic protocol. Neuroblastoma exhibits a variety of clinical behaviours. In neuroblastoma cytology and genetics work together defining the biological behaviour. In half of all the patients with neuroblastoma spread and metastasis have already occurred by the time they are diagnosed. These tumours behave in an aggressive way, leading to the patient death and require proper treatment. Neuroblastoma treatment settles in pre-established risk groups, defining different biologic behaviour groups and ensuing different therapeutic strategies. These groups combine age at diagnosis, clinical stage (INSS-International Neuroblastoma Staging System), histology (INPC-International Neuroblastoma Pathology Classification), MYCN oncogene status, deletion of the short arm of chromosome 1 and DNA index. DNA content and mainly, N-myc amplification have been established as independent prognostic markers in neuroblastomas. At the time of diagnosis, material should be kept to evaluate DNA content, and also to perform FISH or Southern blotting analysis in order to detect N-myc amplification. Several reports, including from our group indicate that FNAC of neuroblastic tumours allow the collection of enough material to evaluate the DNA content of the tumour and detection N-myc amplification.10 The other example that we will discuss is the assessment of EGFR mutations in non-small cell lung cancer (NSCLC) (Figure 1). The most common oncogenic mutations in the tyrosine kinase (TK) domain of EGFR are small in-frame deletions in exon 19 and a point mutation (L858R) in exon 21. These mutations likely cause constitutive activation of the kinase and confer dramatic sensitivity to TK inhibitors gefitinib and erlotinib. Unfortunately the effect of these drugs is limited in time because of the emergence of drug resistance. A second mutation, a substitution T790M in exon 20, appears in about half of all patients with acquired resistance to TKIs. Screening of EGFR mutations, both for selecting patients for treatment and for detecting the resistance mutation, is thus extremely important. However, the most common method of mutation detectioninvolving DNA purification from the whole tumour sample, PCR-based amplification, and sequencing e has limitations, the most important of which is the need of large-sized samples. Cytologic samples are frequently used to diagnose NSCLC and in many times are the only available sample. Recently, it was developed new methods to detect EGFR mutations in samples with a limited number of tumour cells and in cytologic specimens. The method involves three steps: direct microdissection of tumour cells into PCR buffer; a first round of PCR for each EGFR exon and determination of EGFR status by length analysis or TaqMan Assay using the first PCR product as a template. This method is complemented by further analysis using nested PCR and sequencing. With this methodology was demonstrated that four cells are sufficient to determine EGFR mutation status. The
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determination of EGFR mutations in adenocarcinomas of the lung can be performed directly on archival cytological material, scraping the cells from the smears. Whole genomic amplification (WGA) is a technique that allowed high-fidelity in vitro reproduction of quality template DNA opening the door to further array-based analysis of multiple genes. However, this technology requires a great amount of starting DNA material, been a drawback for using cytological specimens. Recently it was demonstrated the feasibility of RNA amplification of low-volume lung specimens for detection of mutations in multiple genes that have prognostic value in NSCLC. These authors showed that in contrast with EGFR and P53 mutations, KRAS and CMET mutations have a deleterious effect on overall survival in advanced-stage NSCLC. Furthermore, the increasingly more coherent data on the genetics of cancers and the advent of next generation sequencing has led to the identification of additional somatic rearrangements (e.g. translocations) in epithelial malignancies, some of which are predictive of response to targeted agents (e.g. ALK4 rearrangements in lung cancer). It is, therefore, possible that the classification of epithelial malignancies will require the identification of specific somatic rearrangements in the not so distant future.11 FNAC offers a suitable alternative to biopsy in a variety of clinical settings, in which it may be useful to obtain material to study prognostic and predictive markers. There are many studies showing the possibility of using FNA to the study response to therapy and evaluation of morphological changes of tumour cells with minimal discomfort for the patients. This is particularly relevant to obtain material from metastatic sites. Although not so frequent, is well document variability in HER2 amplification in primary and metastatic breast cancer and in the K-RAS mutational status in primary and metastatic colon cancer. The possibility of use cytological material for high-throughput technology may allow retrospectively identification of a molecular profile or surrogate marker characteristic of responding tumours, even when the demonstration of activity is limited to a small group of patients. In turn, this profile or marker could be used prospectively for patient enrolment into subsequent studies with selected patients. At the time of tumour progression, one could also consider assessment of newly acquired genetic alterations to select the next line of therapy. A
REFERENCES 1 Hwang TS. Molecular biologic techniques in cytopathologic diagnosis. Korean J Pathol 2009; 43: 387e92. 2 Schmitt FC, Longato-filho A, Valent A, Vielh P. Molecular techniques in cytopathology practice. J Clin Pathol 2008; 61: 258e65. 3 Bovee J, Hogendoorn PCW. Molecular pathology of sarcomas: concepts and clinical implications. Virchows Arch 2010; 456: 193e9. 4 Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab 2009; 94: 2092e8. 5 Shen J, Brugge WR, Dimaio CJ, Pitman MB. Molecular analysis of pancreatic cyst fluid: a comparative analysis with current practice of diagnosis. Cancer Cytopathol 2009; 117: 217e27. 6 Barroca H, Marques C, Candeias J. Comparative study of fine needle aspiration cytology diagnosis, flow cytometry immunophenotyping and histology in lesions clinically suspected of lymphoproliferative disorder: a study of 113 patients. Acta Cytol 2008; 52: 124e32.
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7 Venkatraman L, Catherwood MA, Patterson A, Lioe TF, McCluggage WG, Anderson NH. Role of polymerase chain reaction and immunocytochemistry in the cytological assessment of lymphoid proliferations. J Clin Pathol 2006; 59: 1160e5. 8 Schmitt FC. Cells carry the clue for targeted treatment: a new horizon for cytopathology. Cytopathology 2007; 18: 275e7. 9 Gomes AL, Bardales RH, Milanezi F, Reis RM, Schmitt F. Molecular analysis of c-Kit and PDGFRA in GISTs diagnosed by EUS. Am J Clin Pathol 2007; 127: 1e8. 10 Barroca H, Carvalho JL, Gil da Costa MJ, Cirnes L, Seruca R, Schmitt FC. Detection of N-myc amplification in neuroblastomas using southern blotting on fine needle aspirates. Acta Cytol 2001; 45: 169e72. 11 Ding L, Gad Getz G, Wheeler DA, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008; 455: 1069e75.
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Practice points C
C
C
C
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PCR and FISH are the most commonly used molecular methods in FNAC material Molecular methods help as well as in the diagnosis of tumors and in determining therapeutical targets in some neoplastic diseases The ‘small blue round cell tumours’ (SBRCT) are the main target group in using molecular diagnostics on cytology material Using a panel of markers detecting BRAF, RAS, PAX8/PPARg and RET/PTC may increase the sensitivity of thyroid FNAC
Ó 2011 Elsevier Ltd. All rights reserved.
Possible use and role of molecular techniques in fine-needle aspiration cytology (FNAC) practice
exon sequences to flank rupture points of translocations. PCR applications are centered in diagnosis of solid tumour detecting gene mutations or detecting clone gene rearrangement in lymphoproliferative disorders. PCR analysis can be performed directly with fresh collected material from FNAC, in liquid-based cytology samples or even with cells scraped from FNAC slides. In the first condition, the needle should be washed up in ethanol, methanol or culture mediums like RPMI. The amount and quality of DNA obtained by FNAC for PCR assay does not seem to be a problem and 50e100 cells are adequate to obtain good PCR results. FNAC obtained tumour cells provide excellent representative samples, with poor contamination of stroma or local structures. In situ hybridization methods (ISH) can also be applied to FNAC, permitting, with either fluorescent or chromogenic markers, to detect numerical or structural aberrations of chromosomes. This technique is a reliable technique, particularly useful in cytology as it can be applied directly in smears. Monolayer smears are ideal for ISH techniques and slides with ethanol or air-dried fixed preparations are equally suitable. These techniques are used to detect deletions, insertions or translocations, but can also be used in routine to detect gene amplifications like HER2 in breast carcinoma or NMYC in neuroblastomas. There are two main fields for application of molecular techniques to FNAC: diagnosis and prognosis/therapeutic selection.
Fernando Schmitt Helena Barroca
Abstract Fine-needle aspiration cytology (FNAC) is a recognized ancillary tool in the diagnosis of tumours and infectious diseases. Throughout decades cytopathologists did tremendous marvels with pure morphologic criteria, diagnosing in shrinking amounts of material. The recent understanding of complex cancer biology reshapes the practice of FNAC. The scientific advance of image techniques gave cytopathologists the possibility to go to deep locations and collecting material from previous unlikely locations. This conjunction of knowledge’s and techniques has increased the role of FNAC in the diagnosis, prognosis and in the establishment of new therapeutic targets. Lately, core needle biopsy is gaining popularity and advantage regarding FNAC. The sample limitation of FNAC regarding immunocytochemical staining with proper controls, as well as the limitation in appreciating invasiveness has lead, in some fields, like soft tissue tumours, breast, lymph node, and prostate, to diminish the use of FNAC. However FNAC still is a less invasive, effective, and cost efficient technique. In this review we demonstrate the application of basic molecular techniques to FNAC as a crucial tool in the diagnosis, prognosis and planning of alternative molecular therapeutics of tumours.
Diagnosis
Fernando Schmitt MD PhD FIAC is at the Instituto de Patologia e Imunologia Molecular da Universidade do Porto e IPATIMUP, Faculdade de Medicina da Universidade do Porto, Portugal. Conflicts of interest: none declared.
The recent perception that some tumours carry specific genetic changes in a background of a simple karyotype has lead to the appearance of new commercial available probes of great utility in clinical practice of cytological diagnosis. Soft tissue tumours represent the archetype of this application. A new molecular classification of soft tissue tumours into two main groups has been proposed: (1) tumours with specific genetic alterations and (2) tumours with no specific genetic alterations on a complex background of numerous chromosomal changes. Approximately, 15e20% of mesenchymal tumours belongs to the first group and is associated, in a nearly exclusive and pathognomonic way, to characteristic chromosomal translocations (Table 1). The resulting fusion protein of the specific translocation can act either as a transcription factor, upregulating genes that will promote tumour growth or act in the activation of oncogene promoters with consequent tumour initiation.3 This specific genetic changes revealed such an impact in soft and bone tumour classification that in the recent WHO classification of these tumours, histogenesis is no longer the major determinant in subtyping. In this new classification a combination of diagnostic parameters that include morphology and genotype is advanced. Other tumours like GIST, fibrous dysplasia, and rhabdoid tumours carry specific somatic mutations that act amplifying the response to external stimulus and promote cell cycle progression and growth. These mutations represent a great aid to the cytopathologist assisting them in differential diagnosis of problematic cases or in some cases predicting outcome and selecting patients for targeted therapeutic strategies.
gica do Helena Barroca MD is at the Servic‚o de Anatomia Patolo ~o Joa ~o, Porto, Portugal. Conflicts of interest: Hospital de Sa none declared.
Solid tumours Soft tissue sarcomas: a particular group of sarcomas affecting mainly children and young adults is the small blue round cell
Keywords FNAC; immunocytochemistry; molecular diagnostics
Introduction The molecular techniques most commonly used in FNAC include polymerase chain reaction (PCR) and in situ hybridization (FISH). Other techniques such as in situ PCR, direct sequencing, microarrays and proteomic methodologies are just now entering in the clinical practice in some centres but are still not disseminated in the clinical practice.1,2 PCR methods are ideal for FNAC material and some application are detection of gross chromosomal alterations as deletions and translocations or even point mutations in individual genes. RT-PCR uses cDNA as a template and primers
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identification of EWS/ETS fusion in problematic cases is a helpful tool for cytological differential diagnosis. Rhabdoid tumours show a cytological picture of multiple single polygonal cells with distinct cell borders and clear vesicular nuclei, with occasional single prominent nucleolus. Cytoplasm is fragile but occasionally, intracytoplasmic hyaline-like eosinophilic material is found. Except for the immunoexpression of INI1 marker, all other antibodies expressed by this tumour (vimentin, cytokeratins, EMA, synaptophysin) are also shared by PNET and Meduloblastoma (MB). Cytogenetic and molecular studies are a useful adjunct in the differential diagnosis with these entities. An isochromosome 17q is present in approximately 30e40% of MBs and has not, to date, been observed in AT/RT. Conversely, monosomy or deletion of chromosome 22 is seen in the majority of rhabdoid tumours in all anatomical sites. Karyotypes can be prepared directly from samples obtained from FNAC, or DNA can be isolated and analyzed by PCR-based microsatellite analysis for loss of heterozygoty using probes that map to 22q. HSNF5/INI1 gene seems to be implicated somehow, in an ATP-dependent remodeling chromatin way, thus allowing transcription factor binding to DNA. Some authors hypothesized that the deletion or mutation of INI1 might be associated with the aggressive and usually fatal clinical course in children with AT/RT. This behaviour could also account for some misdiagnosed PNET or MBs, with unpredicted quick outcome. Alveolar rhabdomyosarcoma (ARMS) is another member of this SBRCT group. ARMS accounts for 30% of all rhabdomyosarcomas and represent the most frequent soft tissue tumour seen in childhood. Cytological smears are characterized by loosely cohesive aggregates of uniform, small round blue cells, with little or no specific differentiation. In more differentiated cases, multinucleated neoplastic giant cells with eosinophilic cytoplasm can be present as well as racquet or tadpole cells, helping in differential diagnosis. The importance of recognizing this entity is essential for an adequate treatment. Two forms of translocation have been associated to ARMS: t(2; 13) (q35; q14) in 70% of the cases, and t(1; 13) (p36; q14) in 10e20% of the cases. Recently ARMS with t(1; 13) were found to have better prognosis than those with t(2; 13) (q35; q14). Rhabdomyosarcoma can sometimes exhibit “mixed” embryonal and alveolar phenotypes, and it is not unlikely that in cytological smears, with typical embryonal morphology, the presence of dispersed small round cells are missed or misinterpreted as nude nuclei. This “mixed” morphology gives these tumours a worse prognosis than that of pure embryonal rhabdomyosarcoma, and a clinical outcome similar to that of pure ARMS. FNAC allows the collection of a sample from different parts of the tumour, increasing the possibility of making a correct diagnosis even in heterogeneous neoplasia (Figure 1). Synovial sarcoma (SS) represents 5e10% of soft tissue malignancies in the first two decades of life. Undifferentiated tumours with primitive small round cells occur in 15% of the cases and represent a challenge in the differential cytological diagnosis with all the other small round cell tumours. These tumours fail to immunoexpress most epithelial markers, excepting EMA, and are immunoreactive to CD99. This profile turns differential diagnosis with EFTs imperative. The detection through RT-PCR of a reciprocal translocation t(X; 18) (p11.2;
Chromosomal translocations in soft tissue sarcomas Tumour type
Cytogenetics
Ewing sarcoma/PNET
t(11;22)(q24;q12) t(21;22)(q22;q21) t(7;22)(p22;q12) t(2;22)(q33;q12) t(17;22)(q12;q12) t(11;22)(p13;q12) t(9;22)(q22;q12) t(9;17)(q22;q11) t(9;15)(q22;q21) t(2;13)(q35;q14) t(1;13)(p36;q14) t(12;16)(q13;p11) t(X;18)(p11;q11) t(12;22)(q13;q12) t(X,17)(p11.2;q25) t(17;22)(q22;q13) t(12;15)(p13;q25)
Desmoplastic small round cell tumour Extraskeletal myxoid chondrosarcoma
Alveolar rhabdomyosarcoma Myxoid liposarcoma Synovial sarcoma Clear cell sarcoma Alveolar soft-part sarcoma Dermatofibrosarcoma protuberans Congenital fibrosarcoma/Mesoblastic nephroma Table 1
tumours (SBRCT). This group encompasses a heterogeneous and morphologically similar group of tumours, composed by undifferentiated small round cells. Tumours such as neuroblastoma, PNET/Ewing sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma (undifferentiated type), small cell osteosarcoma, mesenchymal chondrosarcoma and desmoplastic small round cell tumour are included in this group. In most instances, immunocytochemistry is essential to perform the correct differential diagnosis. Some of these tumours, share antigenic determinants and immunostainning is not always concluding. Molecular diagnosis is especially important in detecting specific translocations in Ewing sarcoma, rhabdomyosarcoma, synovial sarcoma or desmoplastic small round cell tumour. Ewing tumour and PNET belongs to a now recognized subgroup and named as the Ewing’s family tumours (EFTs). These tumours account for less than 1% of all soft tissue tumours and affect essentially children and young adults. Cytological smears of EFTs are characterized by a discohesive population of small round blue cells with few distinguishing characteristics. Some cytological minor details, such as the presence of a tigroid background or the presence of dispersed picnotic cells can be suggestive for a trained cytopathologist. CD99 is the most useful immunostain in the differential diagnosis with other malignant round cell tumours. Nevertheless, other tumours, like alveolar rhabdomyosarcoma, undifferentiated synovial sarcoma, and lymphoblastic lymphoma, displaying a similar cytological pattern and CD99 positive, must be individualized. In 85% of EFTs there is a specific tumour-associated translocation t(11; 22) (q24; q12), juxtaposing the EWS gene on chromosome 22 with the FLI1 gene on chromosome 11. This translocation seems to appear very early in tumourogenesis being deeply involved in tumour initiation. Other less frequent variants of EFT translocations have been described, all involving the EWS gene. The
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Figure 1 Two examples of molecular technique application in FNAC material. On the top an example of a rhabdomyosarcoma smear in Giemsa stain (left top) and on the right top a three interphase nuclei studied by FISH with the FKHR Break Apart Rearrangement probe. The upper and lower nuclei exhibit a typical pattern of FKHR rearrangement, with a separation of the probes centromeric (green) and telomeric (red) to the FKHR locus. On the bottom we have the cytological representation of a lung adenocarcinoma (left bottom) and at its right the representation of the respective sequencing EGFR exon 19 showing the presence of mutation Glu 746_Ala750 del.
q11.2) seen in 90% of the cases is highly specific of SS and is essential to achieve a correct diagnosis in problematic cases. This translocation creates a fusion between SS18 (SYT ) gene on chromosome 18 and one of two copies SSX1 or SSX2, of gene SSX on X chromosome. SYT-SSX1 fusions seem to be implicated in epithelial differentiation of SS whether SYT-SSX2 are the most linked to monophasic SS. These fusions codify an abnormal nuclear transcription factor that affects chromatin remodulation. A further effort to establish a relationship between the type of translocation and prognostic has failed till now. Other less frequent malignant round cell tumours also bear specific translocations such as desmoplastic small round cell tumour (DSRCT), mesenchymal chondrosarcoma and myxoid liposarcoma (see Table 1). Desmoplastic small round cell tumour is a highly malignant mesenchymal neoplasm which affects mainly young adults, and has a predilection to abdominal location. Cytological smears resemble those of all other malignant small round cell tumours.
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Occasionally, stromal fragments dispersed in the slide are typically seen. This tumour has a distinctive immune profile, coexpressing vimentin, epithelial, muscle, and neural markers. Characteristically, the expression of desmin has typically a dotlike, paranuclear pattern. Muscle specific markers, like MyoD1 or myogenin are negative. CD99 can also be expressed in 35% of the cases, diffusely staining the cytoplasm. In EFTs membranous unequivocal cytoplasmic membrane staining pattern of CD99 is required for diagnosis, but this requisite is not always easy to assess as there is frequently some degree of cytoplasmic staining. Cytogenetically DSRCT is laureated with a reciprocal translocation, t(11; 22) (p13; q12). Spindle cell tumours also share similar morphology, many of these tumours are undistinguishable in their morphological characteristics and if histological classification is frequently a challenge to most pathologists’ FNAC diagnosis is even more defying. Clear cell sarcoma and monophasic synovial sarcoma are two examples of spindle cell tumours where diagnosis can
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sometimes be dubious. Both these tumours encompass discriminative translocations (Table 1). The variability in cytological features in gastrointestinal stromal tumour (GIST) implies that a diagnosis of GIST should be rendered on cytology in conjunction with immunocytochemistry result of CD117. CD117 antibody recognizes a transmembrane protein, c-Kit, involved in tumour proliferation and shared by other tumours like seminoma, and melanoma, beyond others. C-kit is detected in most GIST’s and represents a hallmark that helps in the differential diagnosis with other mesenchymal tumours. In some GIST were CD117 is negative, the assessment of c-kit mutation by molecular techniques in FNAC sample can be helpful for diagnosis.
the TFE3 gene on chromosome X. Both entities, ASPS and RCC tXp11.2, seem to be associated to the overexpresion of the gene MET receptor tyrosine kinase with increased levels of the MET protein. In the future further studies must be done, exploring the possible response in these tumours with selective inhibitors of the MET tyrosine kinase. Thyroid tumours In thyroid the benefit of using molecular detection of oncogenes on cytology smears is progressing fastly. Oncogenes like BRAF, RAS, RET/PTC have been implicated in thyroid carcinogenesis. The detection of mutation in one of these genes is a strong indicator of malignancy in thyroid. BRAF is the most frequent genetic alteration found and is almost exclusive to papillary thyroid carcinoma (PTC). Apart from BRAF, these oncogenes although sensible concerning malignancy, whenever detected, are not so sensitive discriminating papillary from follicular carcinoma or even adenoma. Still a significant proportion of carcinomas in thyroid lack these genetic markers. Moreover, positivity for RET/PTC has been described on Hashimoto thyroiditis. BRAF mutation is the most common genetic alteration identified in PTC. In about 80% of follicular carcinomas, mutation of RAS or PAX8/PPARg translocation is also found. Recently, Nikiforov et al. suggested that using a panel of markers to detect alterations in BRAF, RAS, PAX8/PPARg and RET/PTC can increase the sensitivity of thyroid FNA in problematic or inconclusive cases.4
Renal cell tumours Apart from sarcomas some renal tumours are also assessed by FNAC, for tumour initial diagnosis and therapeutic planning. Chiefly in European countries, following SIOP protocols, large pediatric renal tumours are initially treated with chemotherapy, for tumour reduction and surgical facility. It is so not uncommon to perform an initial cytological diagnosis by FNAC. A few renal tumours affecting essentially children are also characterized by specific genetic alterations. Mesoblastic nephroma, rhabdoid tumour and more recently described, a special type of renal carcinoma (RCC) Xp11.2 associated, is characterized by specific translocations. Mesoblastic nephroma and some RCC Xp11.2 associated share a gene expression profile with two soft tissue entities respectively: congenital fibrosarcoma and alveolar soft-part sarcoma, respectively (Table 1). This fact should not constitute a surprise, since the kidney is a mesoderm derived organ. Mesoblastic nephroma (MN) is a benign renal tumour, diagnosed essentially in the first 3 months of life. Cytological smears show spindle cells singly or in cohesive fragments with fibroblastic/myofibroblastic bland nuclei. This pattern can simulate the mesenchymal component of a nephroblastoma. In MN it is not uncommon that entrapped local renal tubules of the periphery of the tumour, with embryonal metaplasia, are picked together in FNAC sample, simulating the epithelial component of a Wilm’s tumour. FNAC obtained sample is generally enough and of good quality to perform PCR-based molecular techniques, in order to detect a t(12;15), characteristic of this entity, allowing the correct diagnosis. Recently, it has been described a subset of RCC, in young patients, associated to Xp11.2 genetic alterations. These tumours have distinctive immunohistochemical features. Unlike adulttype RCC’s these tumours do not show intensive immunoreactions to vimentin, or epithelial markers. They are immunoreactive to CD10 and to TFE3 protein. In cytological smears they reproduce what is has been observed in histology: numerous sheets and papillary groups of polyhedric cells with central round nuclei with prominent nucleoli and a clear or granular cytoplasm with ill defined borders. The presence of psammoma bodies is also referred, and should give a clue to cytopathologists. There are two main forms of RCC Xp11.2 associated: RCC t(X; 17) (p11.2; q21) and t(X; 1) (p11.2; p34). Clinically, despite its usual advanced stage presentation, these neoplasms’s course indolently. RCC Xp11.2 associated share with alveolar soft-part sarcoma (ASPS), a similar translocation point
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Pancreatic cysts/tumours Preoperative assessment of pancreatic cystic lesions poses a major diagnostic challenge. Differentiation of benign and potentially malignant pancreatic cysts is difficult even using a multimodal approach. The relatively low sensitivity of cytologic examination has prompted search for other testing with potentially diagnostic yield. A recent review showed that the presence of K-ras mutation that can be done with only 200 mL of the cyst content is quite specific to mucinous cysts.5 In negative K-ras cases, CEA level is able to capture almost 70% of the cases. The drawback of this test is that it needs 1 ml of cyst fluid to perform the assay. Concerning malignancy, cytomorphological findings is the most specific test for diagnosis. However, in the absence of a solid component, pancreatic cysts aspirates frequently have poor cellularity. The specific sequence of K-ras mutation followed by allelic loss is most specific for malignancy. Recently, it was demonstrated that the expression of K homology domain containing protein (KOC), an oncofetal RNA binding protein that regulates insulin-like growth factor and cell proliferation during embryogenesis, is a new marker of malignancy for pancreatic adenocarcinoma and that can be studied in cytological specimens obtained by fine-needle aspiration cytology. KOC expression is limited to invasive pancreatic ductal adenocarcinoma and may thus serve as an adjunct to cytologic cases where atypical features preclude a definitive diagnosis of malignancy.
Hematolymphoid disorders In many institutions FNAC is used as a first line approach to evaluate lymphadenopathies. FNAC yields a high rate of conclusive cytological diagnoses in the assessment of Hodgkin
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disease or large cell non-Hodgkin lymphomas (79e90%). Association of cytology and flow cytometry is used in some centres with success.6 Nevertheless difficulties appear in some situations where despite the best efforts of cytology and flow cytometry some lymphomas remain impossible to diagnose. The combined use of morphology, flow cytometry for immunophenotyping and genetic analysis has increased the accuracy of diagnosis and correct categorization of lymphomas on FNAC samples. The most frequently used molecular techniques in this field are PCR and FISH. Molecular techniques in lymphomas aim two main objectives: 1 e detection of clonality and 2 e detection of molecular characteristics linked to specific subtype of lymphomas. Detection of clonality represents the essential step in the differential diagnosis of reactive lymphadenopathies and lymphoma. Lymphomas develop from a single clone of B or T cells, and as so, all neoplastic lymphocyte population present an identical Immunoglobulin (B cells) or TCR (T cells) gene rearrangement. Clonality can be demonstrated indirectly through immunocytochemistry or flow cytometry, establishing the presence of a light chain restriction in B-cell lymphomas or aberrant expression of T cell antigen in a predominant cell population, or in a direct way, by PCR.7 The presence, in a PCR test, of a monoclonal band is however not necessary an undisputed synonymous of malignancy. In some specific clinical settings it is not infrequent to find minor monoclonal reactive lymphocyte proliferations. Consequently it is advisable that PCR results are interpreted together with clinical and cytological features as well as other ancillary techniques. Another approach is the detection of molecular characteristics linked to specific subtype of lymphomas. There are several lymphomas associated to balanced translocations (Table 2). Follicular lymphoma, mantle cell lymphoma, Burkitt’s lymphoma and anaplastic large cell lymphoma are, in high percentage of cases, associated to oncogenic markers whose identification helps the cytopathologist in the correct diagnosis. However failure to identify these markers does not by itself excludes diagnosis. Cytogenetic and molecular breakpoints are heterogeneous and complex and some molecular techniques are best suitable for some entities than others. For example the detection of t(14:18) in follicular lymphomas by karyotype occurs in 75% of the cases, by PCR in 50e70% of the cases, whether the detection of aberrant expression of the BCL2 protein is found in 90% of the cases. In Burkitt lymphoma, FISH
analysis is considered the most rapid and appropriate method for detection of t(8:14) and in anaplastic large cell lymphoma immunophenotypic analysis is the more appropriated method for diagnosis, leaving PCR for confirmation in inconclusive cases or for follow-up. Identification of these chromosomal translocations helps cytopathologists in the correct classification of lymphomas, but also represents an advance in prognostic assessment and staging. For example, the BCL2 rearrangement and the presence of a translocation of BCL6 in diffuse large B-cell lymphomas are mutually exclusive and this last one is linked to a worse prognosis.
Prognosis and therapeutic selection The knowledge of genetic, epigenetic or metabolic features that contribute to cancer development and growth, represent good targets for cancer therapy. This fact has lead scientist into a run seeking out for better cancer therapies with good efficacy and low toxicity. Loss of cell cycle control, uncontrolled proliferation, inappropriate survival, decreased apoptosis, immortalization, invasion, angiogenesis and metastasis, are all deregulated mechanisms that characterize cancer and that represent excellent targets to work out for novel therapies. Till now emphasis has been given essentially on cell cycle, proliferation and angiogenic involved targets, but other mechanisms-based therapies are been investigated, especially those inducing apoptosis, telomerase inhibitors, invasiveness and metastasis inhibitors. Here we will mention some examples where molecular therapeutic targets/ prognostic indicators can be studied in FNAC material.8 GIST has recently gained the interest of researchers, in the sense that it embodies the model of an outstanding metabolic response to a targeted therapeutic drug, the imatinib. In GIST the mutation of c-kit gene, a transmembrane receptor connected with tyrosine kinase receptor, leads to an autonomous enhance stimulation of the tyrosine kinase cascade with consequent tumour proliferation and development. These tumours are also associated to plateletderived growth factor receptor (PDGFRA) gene mutation also a member of the type III tyrosine kinase receptor family. Mutation c-kit or PDGFRA are mutually exclusive and represent two different alternative oncogenic events leading to similar biological consequences. As important as classifying GIST tumours in low or high grade, it is essential to realize whose tumours will be the best responders to therapy. Tumour clinical response to Imatinib is determined by the affected exon in c-kit mutation. In GIST four different regions of KIT have been found to be involved, exon 11 (juxtamembrane domain), exon 9 (extracellular domain), exon 13 and exon 17 (tyrosine kinase domain). Also PDGFRA mutations vary according to the exon involved (exon 18 e tyrosine kinase domain or exon 12 e juxtamembrane domain). The highest overall response rates have been reported in patients with GIST harbouring c-kit exon 11 mutations. The clinical response rate decreases to 45% in patients with c-kit exon 9 mutations. PDGFRA exon 18 point mutations involving amino acids 841e843 are clinically resistant to imatinib. However, the single exon 18 mutation D842Y is associated with good response to Imatinib. Our group demonstrated the feasibility of performing molecular analysis of c-kit and PDGFRA genes in cytologic material obtained by endoscopic ultrasound-guided FNA from GISTs.9 PCR amplification and DNA sequencing revealed c-Kit mutations in 61% of
Chromosomal translocations and affected genes in lymphomas Lymphoma
Chromosomal translocation
Affected genes
Follicular lymphoma Burkitt lymphoma Diffuse large B-cell lymphoma Mantle cell lymphoma Anaplastic large cell lymphoma Malt lymphoma
t(14:18)(q32:q21) t(8:14)(q24:q32) t(3:14)(q27:32)
BCL2 C-MYC BCL6
t(11:14)(q13:q32) t(2:5)(p23:q35)
BCL1 NPM, ALK
t(11:18)(p21:q21)
Table 2
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GIST cases, in accordance with previously published ranges (30e90%). Nearly 95% (19/20) of c-kit-mutant tumors carried exon 11 mutations. In this study, we have shown that GISTs could be diagnosed pre-operatively on EUS-FNA specimens. In addition to immunocytochemistry, molecular analysis can be done in formalin-fixed and paraffin-embedded cell block material obtained from these aspirates, avoiding more invasive diagnostic procedures to obtain a tissue diagnosis. In nowadays, with two RTK inhibitors available, there is an imperative need in refining GIST diagnosis and including a molecular analysis of both c-Kit and PDGFRA in the current diagnostic protocol. Neuroblastoma exhibits a variety of clinical behaviours. In neuroblastoma cytology and genetics work together defining the biological behaviour. In half of all the patients with neuroblastoma spread and metastasis have already occurred by the time they are diagnosed. These tumours behave in an aggressive way, leading to the patient death and require proper treatment. Neuroblastoma treatment settles in pre-established risk groups, defining different biologic behaviour groups and ensuing different therapeutic strategies. These groups combine age at diagnosis, clinical stage (INSS-International Neuroblastoma Staging System), histology (INPC-International Neuroblastoma Pathology Classification), MYCN oncogene status, deletion of the short arm of chromosome 1 and DNA index. DNA content and mainly, N-myc amplification have been established as independent prognostic markers in neuroblastomas. At the time of diagnosis, material should be kept to evaluate DNA content, and also to perform FISH or Southern blotting analysis in order to detect N-myc amplification. Several reports, including from our group indicate that FNAC of neuroblastic tumours allow the collection of enough material to evaluate the DNA content of the tumour and detection N-myc amplification.10 The other example that we will discuss is the assessment of EGFR mutations in non-small cell lung cancer (NSCLC) (Figure 1). The most common oncogenic mutations in the tyrosine kinase (TK) domain of EGFR are small in-frame deletions in exon 19 and a point mutation (L858R) in exon 21. These mutations likely cause constitutive activation of the kinase and confer dramatic sensitivity to TK inhibitors gefitinib and erlotinib. Unfortunately the effect of these drugs is limited in time because of the emergence of drug resistance. A second mutation, a substitution T790M in exon 20, appears in about half of all patients with acquired resistance to TKIs. Screening of EGFR mutations, both for selecting patients for treatment and for detecting the resistance mutation, is thus extremely important. However, the most common method of mutation detectioninvolving DNA purification from the whole tumour sample, PCR-based amplification, and sequencing e has limitations, the most important of which is the need of large-sized samples. Cytologic samples are frequently used to diagnose NSCLC and in many times are the only available sample. Recently, it was developed new methods to detect EGFR mutations in samples with a limited number of tumour cells and in cytologic specimens. The method involves three steps: direct microdissection of tumour cells into PCR buffer; a first round of PCR for each EGFR exon and determination of EGFR status by length analysis or TaqMan Assay using the first PCR product as a template. This method is complemented by further analysis using nested PCR and sequencing. With this methodology was demonstrated that four cells are sufficient to determine EGFR mutation status. The
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determination of EGFR mutations in adenocarcinomas of the lung can be performed directly on archival cytological material, scraping the cells from the smears. Whole genomic amplification (WGA) is a technique that allowed high-fidelity in vitro reproduction of quality template DNA opening the door to further array-based analysis of multiple genes. However, this technology requires a great amount of starting DNA material, been a drawback for using cytological specimens. Recently it was demonstrated the feasibility of RNA amplification of low-volume lung specimens for detection of mutations in multiple genes that have prognostic value in NSCLC. These authors showed that in contrast with EGFR and P53 mutations, KRAS and CMET mutations have a deleterious effect on overall survival in advanced-stage NSCLC. Furthermore, the increasingly more coherent data on the genetics of cancers and the advent of next generation sequencing has led to the identification of additional somatic rearrangements (e.g. translocations) in epithelial malignancies, some of which are predictive of response to targeted agents (e.g. ALK4 rearrangements in lung cancer). It is, therefore, possible that the classification of epithelial malignancies will require the identification of specific somatic rearrangements in the not so distant future.11 FNAC offers a suitable alternative to biopsy in a variety of clinical settings, in which it may be useful to obtain material to study prognostic and predictive markers. There are many studies showing the possibility of using FNA to the study response to therapy and evaluation of morphological changes of tumour cells with minimal discomfort for the patients. This is particularly relevant to obtain material from metastatic sites. Although not so frequent, is well document variability in HER2 amplification in primary and metastatic breast cancer and in the K-RAS mutational status in primary and metastatic colon cancer. The possibility of use cytological material for high-throughput technology may allow retrospectively identification of a molecular profile or surrogate marker characteristic of responding tumours, even when the demonstration of activity is limited to a small group of patients. In turn, this profile or marker could be used prospectively for patient enrolment into subsequent studies with selected patients. At the time of tumour progression, one could also consider assessment of newly acquired genetic alterations to select the next line of therapy. A
REFERENCES 1 Hwang TS. Molecular biologic techniques in cytopathologic diagnosis. Korean J Pathol 2009; 43: 387e92. 2 Schmitt FC, Longato-filho A, Valent A, Vielh P. Molecular techniques in cytopathology practice. J Clin Pathol 2008; 61: 258e65. 3 Bovee J, Hogendoorn PCW. Molecular pathology of sarcomas: concepts and clinical implications. Virchows Arch 2010; 456: 193e9. 4 Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab 2009; 94: 2092e8. 5 Shen J, Brugge WR, Dimaio CJ, Pitman MB. Molecular analysis of pancreatic cyst fluid: a comparative analysis with current practice of diagnosis. Cancer Cytopathol 2009; 117: 217e27. 6 Barroca H, Marques C, Candeias J. Comparative study of fine needle aspiration cytology diagnosis, flow cytometry immunophenotyping and histology in lesions clinically suspected of lymphoproliferative disorder: a study of 113 patients. Acta Cytol 2008; 52: 124e32.
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7 Venkatraman L, Catherwood MA, Patterson A, Lioe TF, McCluggage WG, Anderson NH. Role of polymerase chain reaction and immunocytochemistry in the cytological assessment of lymphoid proliferations. J Clin Pathol 2006; 59: 1160e5. 8 Schmitt FC. Cells carry the clue for targeted treatment: a new horizon for cytopathology. Cytopathology 2007; 18: 275e7. 9 Gomes AL, Bardales RH, Milanezi F, Reis RM, Schmitt F. Molecular analysis of c-Kit and PDGFRA in GISTs diagnosed by EUS. Am J Clin Pathol 2007; 127: 1e8. 10 Barroca H, Carvalho JL, Gil da Costa MJ, Cirnes L, Seruca R, Schmitt FC. Detection of N-myc amplification in neuroblastomas using southern blotting on fine needle aspirates. Acta Cytol 2001; 45: 169e72. 11 Ding L, Gad Getz G, Wheeler DA, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 2008; 455: 1069e75.
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Practice points C
C
C
C
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PCR and FISH are the most commonly used molecular methods in FNAC material Molecular methods help as well as in the diagnosis of tumors and in determining therapeutical targets in some neoplastic diseases The ‘small blue round cell tumours’ (SBRCT) are the main target group in using molecular diagnostics on cytology material Using a panel of markers detecting BRAF, RAS, PAX8/PPARg and RET/PTC may increase the sensitivity of thyroid FNAC
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