Diagnosis of anaplastic lymphoma kinase rearrangement in ...

Review Article

Diagnosis of Anaplastic Lymphoma Kinase Rearrangement in Cytological Samples Through a Fluorescence In Situ Hybridization–Based Assay: Cytological Smears Versus Cell Blocks Federica Zito Marino, BD1,2; Giulio Rossi, MD3; Matteo Brunelli, MD, PhD4; Maria Gabriella Malzone, BD1; Giuseppina Liguori, MD1; Giuseppe Bogina, BD5; Alessandro Morabito, MD6; Gaetano Rocco, MD7; Renato Franco, MD, PhD2; and Gerardo Botti, MD1

Anaplastic lymphoma kinase (ALK) status analysis of lung cytological specimens should be successfully encouraged in routine practice because biopsy specimens are not always available. To date, the US Food and Drug Administration has approved both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) as diagnostic tests for identifying ALK-positive patients eligible for treatment with crizotinib. Although ALK IHC is an optimal diagnostic tool, FISH becomes mandatory in equivocal cases. ALK FISH of paraffin-embedded tissue material is still the gold standard, whereas the cytological specimen assay has not yet been completely standardized. Many controversial data have been reported on the adequacy of cytology cell blocks (CBs) versus conventional smears for FISH testing. This review discusses some critical issues related to ALK FISH of cytological samples, including the triaging of collected specimens to optimize the material, the use of CBs versus conventional smears, and alternative methods for an ALK rearrangement diagnosis. Conventional smears have the advantages of an immediate evaluation, no probe tissue-related artifactual loss, no fixationrelated alterations, and usually sufficient material for an analytic preparation. On the other hand, CBs have several advantages, including the appropriate conservation of the tissue architecture, an absence of problems related to cell overlapC 2017 American ping, and the ability to evaluate neoplastic cells in a dark field. Cancer Cytopathol 2017;125:303-12. V

Cancer Society. KEY WORDS: anaplastic lymphoma kinase (ALK); cell block; cytological smears; fluorescence in situ hybridization (FISH); lung cytology.

INTRODUCTION Approximately 70% of lung cancer diagnoses are based on small biopsy specimens or cytological samples, with most patients being at an advanced stage.1 However, only in 2004 were cytological specimens belatedly included in the World Health Organization lung cancer classification system.2 The International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society have provided a new standardized terminology and diagnostic criteria for small biopsy specimens and cytological samples.3 In recent Corresponding author: Renato Franco, MD, PhD, Pathology Unit, Luigi Vanvitelli University of Campania, Via Luciano Armanni 20, 80100, Naples, Italy; Fax: (011) 39 0817764582; [email protected] or [email protected] 1 Pathology Unit, Istituto Nazionale Tumori Fondazione G. Pascale, Naples, Italy; 2Pathology Unit, Luigi Vanvitelli University of Campania, Naples, Italy; 3Unit of Pathologic Anatomy, Azienda USL Valle d’Aosta, Aosta, Italy; 4Anatomic Pathology Section Department of Pathology, University of Verona, Verona, Italy; 5Section of Pathologic Anatomy, Sacro Cuore Don Calabria Hospital, Negrar, Italy; 6Medical Oncology Unit, Department of Thoracic Surgical and Medical Oncology, Istituto Nazionale Tumori Fondazione G. Pascale, Naples, Italy; 7Division of Thoracic Surgery, Department of Thoracic Surgical and Medical Oncology, Istituto Nazionale Tumori Fondazione G. Pascale, Naples, Italy.

The last 2 authors are cosenior authors. Received: October 11, 2016; Revised: December 15, 2016; Accepted: December 19, 2016 Published online February 14, 2017 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21835, wileyonlinelibrary.com

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years, the use of lung cytology in molecular diagnosis has progressively increased, especially for unresectable lung cancer.4 To date, the identification of new therapeutic targets, including epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase rearrangement (ALK-R), has greatly changed the treatment of patients with non–small cell lung cancer (NSCLC). In their molecular testing guidelines for the selection of lung cancer patients who are potentially suitable for EGFR- and anaplastic lymphoma kinase (ALK)–targeted therapy, the International Association for the Study of Lung Cancer, the College of American Pathologists, and the Association for Molecular Pathology recommend EGFR and ALK testing for all patients with an adenocarcinoma component, regardless of other clinical and pathological features.5 The ALK gene encodes for a tyrosine kinase receptor that may transduce various different pathways, including Extracellular signal-Regulated Kinase (ERK), Janus Kinase 3-Signal Trasducer and Activator of Trascription (JAKSTAT3) and Phosphoinositide 3-Kinase (PI3K)-AKT pathways that are crucial to cell proliferation and survival. The ALK gene may be involved in several mutations, including rearrangements leading to a constitutively active chimeric protein kinase with oncogenic properties.6 ALKR is detected in approximately 2% to 7% of NSCLC cases, and the patients harboring this aberration have a good clinical response to treatment with crizotinib, a specific inhibitor.6 Crizotinib acts as a protein kinase inhibitor through competitive binding within the receptor adenosine triphosphate–binding pocket; initially developed as a c-MET inhibitor, it has shown significant efficacy in advanced ALK-R NSCLC.7 In this context, the integration of an ALK-R diagnosis into routine cytological practice is mandatory in the treatment decision tree for advanced NSCLC. Lung cytological specimens represent optimal materials for a molecular diagnosis using polymerase chain reaction (PCR)–based assays; they have demonstrated high sensitivity and specificity for identifying EGFR and KRAS mutations.4,8 Conversely, ALK fluorescence in situ hybridization (FISH) testing of cytological biomaterials has not yet been completely standardized9; indeed, many controversial data are reported in the literature. At first, the US Food and Drug Administration (FDA) approved FISH as the only diagnostic tool for crizotinib treatment eligibility, with formalin-fixed, paraffin-embedded tissue, not including 304

cytological samples, being exclusively used.10 The guidelines subsequently proposed by the College of American Pathologists suggested that lung cytological specimens, including both cell blocks (CBs) and cytological conventional smears (CCSs), could also be suitable for ALK FISH.5,11 To date, the only probe approved as a companion diagnostic assay by the FDA for an ALK inhibitor is the Vysis LSI ALK Break Apart FISH Probe Kit (Abbott Molecular). The ALK break-apart probe is designed through the labeling of the 30 (telomeric) part of the fusion breakpoint in Spectrum Orange and the 50 (centromeric) part in Spectrum Green.10 In June 2015, the FDA approved the Ventana ALK (D5F3) CDx immunohistochemistry (IHC) assay for selecting ALK-positive patients eligible for treatment with crizotinib.12 The Italian Medicines Agency has proposed an algorithm for the selection of ALK-R patients; it has suggested prescreening by IHC and confirmation by FISH in equivocal cases as an alternative to FISH testing alone.13 IHC is an optimal diagnostic tool in most cases; however, FISH is a mandatory and discriminating test for equivocal cases. In the literature, some cases have shown discordant results with ALK FISH and IHC, likely because of technical factors.14 ALK IHC false-negatives may be caused by a different variant rearrangement that leads to a structural change in the fusion protein, which results in inaccessibility of the ALK epitope.15 ALK FISH of cytological samples has some open issues, especially with respect to the most adequate samples with which to perform the analysis. To date, many controversial data have been reported on the adequacy of CBs versus CCSs for ALK FISH testing. In this review, we discuss some critical points related to ALK FISH in cytology, including the triaging of collected specimens to optimize the material for the test, the advantages of CBs versus CCSs, and alternative methods for detecting the ALK gene status.

TRIAGE OF FINE-NEEDLE ASPIRATION CYTOLOGY (FNAC) SAMPLES FOR OPTIMIZING MATERIAL FOR ALK FISH Several techniques, such as bronchial brushing/washing, bronchoalveolar lavage, FNAC, malignant pleural effusion (MPE) fluid collection, and the collection of exfoliated cells in the sputum, are currently used to collect pulmonary cytological samples.4 FNAC is a rapid, efficient, and minimally invasive technique finalized for the diagnosis of both superficial and deep pulmonary lesions.16,17 FNAC triage also provides a Cancer Cytopathology

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ALK Rearrangement in Cytological Samples/Marino et al

Figure 1. Alternative FNAC processing workflows for molecular testing in patients with non–small cell lung cancer. (A) Traditional FNAC triage. The material from an FNAC needle pass is used to prepare 2 smears (DQ and Pap); the remaining needle rinse can be used for CB preparation. EGFR and ALK tests can be performed on the CB material. (B) Alternative workflow using UNSs. The material from an FNAC needle pass is used to prepare 2 smears (DQ and Pap); the remaining needle rinse can be used to prepare some UNSs on which EGFR and ALK testing can be performed. Then, possibly remaining needle rinse can be used for CB preparation. (C) Alternative workflow using only 1 slide to perform both EGFR and ALK tests through microdissection. A DQ smear prepared via FNAC is examined to identify 2 areas rich in tumor cells. A manual microdissection is performed in an area of the DQ smear for the EGFR test. Then, the remaining DQ smear is destained, and ALK FISH is performed. Finally, possibly remaining needle rinse can be used for CB preparation. (D) Alternative workflow using only 1 slide to perform ALK FISH first and then EGFR testing. A DQ smear prepared via FNAC is destained, and ALK FISH is performed; cells from the same sample are then scraped off the slides and used for EGFR testing. Finally, the remaining needle rinse can be used for CB preparation. ALK indicates anaplastic lymphoma kinase; CB, cell block; dDQ, destained Diff-Quik; DQ, DiffQuik (air-dried smear); EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; FNAC, fineneedle aspiration cytology; Pap, Papanicolaou (alcohol-fixed smear); UNS, extra unstained smear.

rapid onsite evaluation of the cellularity to reduce falsenegatives due to the absence of tumor material.18,19 Moreover, a rapid onsite evaluation allows the acquisition of tumor cells without contamination of stromal tissue and also ensures that enough material can be gathered for ancillary tests while the patient is still accessible.20 The traditional triage of FNAC permits the preparation of 2 smears from each needle pass, which are used for conventional staining. In addition, depending on the amount of aspirated material, the remaining needle rinse can be used for CB preparation to perform ancillary Cancer Cytopathology

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testing, including immunocytochemistry (ICC), EGFR, and ALK testing, and to preserve archived material20,21 (Fig. 1A). A possible alternative workflow is the preparation of additional unstained smears for ICC and later EGFR and ALK analysis and CB preparation; this would depend on the remaining material. The presence of tumor cells could be assessed either with a previously stained smear or with unstained direct smears by light microscopy20,21 (Fig. 1B). Recent studies have shown the possibility of assessing the EGFR and ALK status with a single slide.22,23 Betz et al22 proposed using the same stained smear first for an EGFR mutation test, with tumor cells obtained through manual microdissection, and then for ALK FISH (Fig. 1C). Similarly, Ulivi et al23 showed the feasibility of performing both molecular analyses with the same slide: they performed ALK FISH first and then an EGFR test because the FISH analysis did not interfere with the PCR-based assay (Fig. 1D). Furthermore, Minca et al24 suggested that ThinPrep slides prepared from FNAC could be valid alternative cytological materials for ALK status determination. The use of ThinPrep could have several advantages, including minimal artifacts due to air drying and smear crushing, homogeneous cell enrichment, and the preservation of the entire nucleus, which would provide more accurate signal counts. An MPE is a frequent complication in advanced lung cancer and occurs in approximately 10% to 50% of NSCLC cases; it is associated generally with the adenocarcinoma histotype.25-28 The CCS procedure is widely used for an MPE diagnosis and has a sensitivity of approximately 40% to 70%, although several difficulties can arise in the differentiation of reactive mesothelial cells from malignant cells. An MPE is easily collected with not too invasive methods and thus provides a useful material also for molecular diagnosis in clinical practice. Numerous studies have demonstrated the usefulness of pleural effusions in various cancers for the identification of several molecular aberrations such as p53, FHIT, and KRAS gene mutations.29-31 In lung cancer, previous studies have shown the clinical feasibility of an EGFR test using an MPE; it has resulted in a predictive value for the responsiveness and resistance to treatment with EGFR tyrosine kinase inhibitors.32-37 In the literature, several studies28-41 have also proven a possible use of MPEs for ALK status analysis, with CB materials preferred to CCSs (Table 1). 305

Review Article TABLE 1. ALK Rearrangement Analysis of MPEs Reported in the Literature ALK FISH, No. Sample Type MPE CB MPE CB MPE CB

ALK ICC, No.

Total No.

Unevaluable

Negative

Positive

Unevaluable

Negative

Positive

Reference

66 63 52

0 5 14

64 52 33

2 6 5

0 0 0

63 55 44

3 8 8

38 40 41

Abbreviations: ALK, anaplastic lymphoma kinase; CB, cell block; FISH, fluorescence in situ hybridization; ICC, immunocytochemistry; MPE, malignant pleural effusion.

In particular, a good concordance in ALK detection has been found between MPE CBs and matched tissue samples; this suggests that MPEs could be a valid diagnostic material when tissue is not available.41 However, ALK FISH of an MPE is not always evaluable because of the scarcity of malignant cells and the difficulty in the identification of tumor cells in a dark field.42 Therefore, ALK ICC could be preferred to FISH testing with MPE specimens.40,41 Finally, ALK status determination with an MPE represents a challenge in advanced NSCLC; therefore, further studies are required to validate its routine use.

CRITICAL ISSUES RELATED TO ALK FISH IN CYTOLOGY In the context of molecular diagnosis, cytological specimens are often more suitable for PCR-based assays than FISH-based assays. DNA is readily extracted from routine cytology slides. Furthermore, DNA extracted from archived cytological samples remains generally stable and intact for a long time (at least 6 months and up to 5 years).43,44 Conversely, FISH-based assays using cytological samples have several limitations. Although ALK FISH of histological specimens has always been considered the gold standard for identifying ALK-R, some critical factors have been reported for cytological materials (Fig. 2). ALK FISH of cytological specimens is influenced by poor cellularity and a low percentage of tumor cells. Several studies have investigated the minimum percentage of tumor cells needed for EGFR and KRAS analyses of NSCLC cytological specimens. Thus, for a PCR-based analysis, an acceptable range varies from 1% to 50% of tumor cells.45-48 However, the proportion of valuable tumor cells must be at least 60% for ALK FISH interpretation, and this sometimes is hardly available in cytological samples.49 Moreover, the evaluation of malignant cells in a dark field is generally complicated, especially with 306

cytological samples, for which there is no referring tissue architecture. In this scenario, an absolute number of malignant cells has to be preserved to make an appropriate interpretation (at least 50 nuclei). The threshold for a positive ALK FISH result has been established for tissue slides, whereas each laboratory needs to determine its own threshold for ALK-negative cytological specimens until a consensus is reached. Recently, a novel automated platform combining cytomorphology and FISH assays has been developed to identify tumor cells more easily in dark-field microscopy. The automated FISH assay allows the enumeration of the signals in selected cells and enhances the precision and reproducibility. However, this platform is not readily available to all pathology laboratories.14 ALK FISH of cytological samples is influenced by the different staining methods and treatments used before the FISH analysis.50,51 In the literature, a FISH assay using NSCLC cytological samples showed high feasibility, which ranged from 79% to 97%, despite several critical technical points.22,52-54 Recently, Proietti et al53 demonstrated that cytological samples (both CCSs and CBs) might be more adequate for ALK FISH testing than small biopsy specimens. However, the optimal cytological sample preparation between CBs and CCSs for ALK FISH has not been yet assessed. There may be concerns about using diagnostic cytological slides for FISH because of a legal requirement that cytology laboratories archive diagnostic slides for several years and the potential need to review slides even years after a diagnosis. It is possible to restain slides after FISH analysis.22 In this context, digital cytology could improve several features, including not only image archiving, proficiency testing, and second opinions but especially also critical legal and administrative issues. Digitized images of the most appropriate fields of view or whole slides could solve many problems; therefore, accurate process validation and a Cancer Cytopathology

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Figure 2. ALK FISH analysis of cytological samples: examples of the major critical issues. (A) ALK FISH of a CB of good quality. (B) ALK FISH of a CCS with overlapping nuclei, which make the interpretation of fluorescent signals difficult. (C) ALK FISH of a CCS with a limited number of neoplastic nuclei. (D) ALK FISH of a CB with artifacts due to a nonstandardized pre-analytic phase. ALK indicates anaplastic lymphoma kinase; CB, cell block; CCS, cytological conventional smear; FISH, fluorescence in situ hybridization.

regulatory environment for digital pathology are needed in the near future.55

USE OF CONVENTIONAL CYTOLOGICAL SMEARS IN THE ALK-R DIAGNOSIS Much evidence has demonstrated that CCSs represent an optimal platform for EGFR and KRAS mutation testing for a lung molecular diagnosis.22,56-58 CCSs are ideal samples for PCR-based assays because their processing does not require the use of formalin for fixation. Generally, an alcohol-based fixation process leads to the extraction of high-quality DNA; this excludes possible formalin-related crosslinking of nucleic acids and proteins.22,59,60 In addition, the microdissection of specific tumor areas on a smear allows the collection of selected tumor cells, and this increases the success rate of the assay, especially in cases with low cellularity.45,59,61,62 In the literature, several studies have investigated the use of CCSs for ALK FISH testing, and controversial data have been reported; it has been concluded that CCSs could be effective materials for ALK Cancer Cytopathology

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FISH assessment in comparison with CBs (Table 2).21,22,52-54 CCSs are especially more appropriate than CBs for those cases with insufficient material and low cellularity because the evaluation of the sample is immediate.53 Moreover, the use of CCSs overcomes some limitations related to FISH in formalin-fixed, paraffin-embedded specimens, including the problems of formalin fixation and the loss of probe signals due to nuclear truncation.22,24,53 In previous studies,53,54 different types of stained CCSs have been used to assess the ALK status by FISH, and they have demonstrated similar results. Betz et al22 demonstrated the feasibility of ALK FISH with Diff-Quik staining; they recommended destaining in acid-alcohol solutions to reduce autofluorescence from residual staining, which could interfere with probe signal visualization. In addition, a comparison of exfoliative and aspiration specimens showed that they were equally satisfactory for ALK FISH testing.53 Despite the aforementioned advantages, some limitations related to ALK FISH of CCSs have also been 307

Review Article TABLE 2. ALK Rearrangement Diagnosis in Cytology: CBs Versus CCSs in the Literature ALK FISH, No. Sample Type (No.) CCS/CB (32/32)* CB (55) CCS (94)/CB (86) CCS (55) CB (24)/CCS (91)

ALK ICC, No.

Total No.

Unevaluable

Negative

Positive

Unevaluable

Negative

Positive

Reference

32 55 180 55 115

0 3 20/26 6 3/19

27/28 49 68/59 39 16/52

5/4 3 6/1 10 5/1

NA 0 NA NA NA

NA 54 NA NA NA

NA 1 NA NA NA

22 52 53 54 63

Abbreviations: ALK, anaplastic lymphoma kinase; CB, cell block; CCS, cytological conventional smear; FISH, fluorescence in situ hybridization; ICC, immunocytochemistry. a The same cases were analyzed on cytological smears and cell blocks.

described. A recent study63 showed that in a series of cytological samples obtained by transbronchial needle aspiration or endobronchial ultrasound, a higher percentage of unsatisfactory samples was found with CCSs (26%) versus CBs (12%). FISH failure with CCSs was mainly due to cell overlapping and fixation immediately after FNAC. Conversely, other studies24 have reported a higher failure rate for the test with CBs versus CCSs.

CB USE IN THE ALK-R DIAGNOSIS The use of CBs again is a technique traditionally used for tests such as ICC and molecular analysis. CB use is currently increasing in diagnostic pathology as an ancillary tool for FNAC.20,64 CBs are processed similarly to histological samples; this includes formalin fixation and paraffin embedding. This procedure leads to appropriate preservation of the tissue architecture, nuclear-cytoplasmic details, cell membrane integrity, and crisp chromatin details. Thus, CBs provide better morphological recognition of histological patterns and improve the diagnostic yield by 15%.42,65,66 Because ALK testing is recommended for adenocarcinomas and mixed lung cancers with an adenocarcinoma component, the subtyping and the diagnostic accuracy ensured by CBs might be relevant to the selection of potential patients sensitive to ALK inhibitors. Moreover, CBs represent valuable archival material and a source for multiple serial sections for several additional analyses.60,67 However, CBs frequently show inadequate cellularity and benign cellular elements in the background, such as inflammatory cells, bronchial epithelial cells, and stromal cells.20 Sometimes, insufficient material from a cytology CB preparation, a low percentage of available tumor cells, a possible loss of tumor cells in deeper sections, and an inability to assess cellularity at the time of the sample collection could significantly affect the success of ALK FISH.20,66 308

CB preparations may show significant differences due to the different methods used, such as agar, plasma/ thrombin, and colloid bag methods.68,69 To date, an automated CB system is also available, and it allows faster preparation and higher cellularity in comparison with traditional CB methods.70,71 According to the literature, several studies have performed ALK FISH on CBs, and they have resulted in discrepant data (Table 2). Bravaccini et al63 reported that the percentage of unevaluable ALK FISH cases with CBs was much lower than the percentage with CCSs. Conversely, other studies have registered a failure rate with CBs of approximately 30% due to insufficient material with cytology CB preparations.24,53 These controversial results suggest that the standardization of CB methods is needed to optimize the handling of cytological samples in daily practice and to improve the quality of the specimens for FISH testing.

ALTERNATIVE ASSAYS FOR DETECTING THE ALK STATUS IN CYTOLOGY ALK IHC represents an option for identifying ALKpositive cases: it is simpler, quicker, and less expensive than a FISH assay. In the last few years, several studies have performed ALK IHC on histological specimens with different monoclonal anti-ALK antibodies such as D5F3, 5A4, and D9E4.72-74 Similarly to histological samples, ICC with the clone ALK-1 monoclonal antibody on cytological specimens showed low specificity and sensitivity.52,75,76 IHC with the anti-ALK antibody clone 5A4 showed a sensitivity of 95% and a specificity of 100% for NSCLC resection specimens and bronchial biopsies.77,78 Similarly, the clone 5A4 demonstrated a high sensitivity in endobronchial Cancer Cytopathology

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ultrasound–derived cytology materials.79,80 The antibody anti-ALK D5F3 on the Ventana platform has been approved to select ALK-positive patients for treatment with specific inhibitors. Although only a few studies have used ALK ICC to assess ALK-R, ICC has shown a high concordance with FISH also in cytological samples24,79 (Table 2). Liu et al81 demonstrated with CBs that ALK ICC with D5F3 has a higher sensitivity and a lower specificity than FISH. However, the authors did not use a binary scoring system (positive vs negative), which is currently approved for anti-ALK D5F3 staining interpretation, but instead used a semiquantitative scoring system (0, absent/ barely perceptible expression; 1, weak/moderate multifocal expression; 2, strong staining in most cells). A recent study has shown that ICC with anti-ALK D5F3 might be an optimal method for assessing ALK-R in MPEs and even more suitable than FISH, particularly for suboptimal specimens with few tumor cells.41 Another assay for detecting ALK-R in both histological and cytological specimens is reverse transcription–polymerase chain reaction (RT-PCR). Much evidence has proven that RT-PCR is an accurate method, with a good concordance between archived cytological slides and matched tissue blocks having been reported.82 Moreover, this test has shown a high sensitivity for identifying ALK-R also in MPE CB samples.81,83,84 In addition, Kanaji et al85 proposed a rapid approach for detecting ALK-R by nested multiplex RT-PCR during a bronchoscopic examination. Although this procedure could be interesting in routine clinical practice, it is not used to select patients for the therapy because confirmation by another assay is required. Finally, RT-PCR could be a good alternative candidate for detecting ALK fusion, especially in archived cytology slides; however, it is not currently approved by the FDA for selecting patients eligible for treatment with crizotinib.

the integrated use of FISH and ICC assays, the possible use of MPEs for molecular diagnosis, and the definition of the most adequate cytological sample preparation (CBs or CCSs). In particular, ALK ICC could be a rapid and easy method for the diagnosis of ALK-R with cytological material; however, validation studies are required. Standardized management of cytological samples for ALK status analysis is needed to optimally select patients with advanced NSCLC who are awaiting treatment when tissue samples are not available. In conclusion, CCSs have the advantages of an immediate evaluation, no probe tissue-related artifactual loss, no fixation-related alterations, and usually sufficient material for an analytic preparation, whereas CBs may enable the appropriate conservation of tissue architecture and ensure an easier evaluation of neoplastic cells in a dark field.

FUNDING SUPPORT This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Project IG 2013 N.14425 FZM is a fellow of AIRC.

CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. REFERENCES 1. 2.

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CONCLUSION Cytological samples represent precious material for an ALK-R diagnosis, especially for patients with inoperable NSCLC. FNAC samples are routinely used for molecular testing; however, a standard FNAC processing workflow to optimize the available material has not yet been defined. As for the diagnosis of ALK-R with cytological samples, several challenges are waiting for a consensus; they include Cancer Cytopathology

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