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Triage of cytologic direct smears for ancillary studies: a case-based illustration and review.

REPORT OF A CASE

A 77-year-old man was initially diagnosed with malignant melanoma on his left earlobe in 2004. This was resected without complications. Eight years later, he developed a recurrence of melanoma at the same location. The re-excision specimen revealed a nodular dermal deposit of melanoma. Subsequent workup included computed tomography scans of his neck, chest,

abdomen, and pelvis. This demonstrated the presence of enlarged and heterogeneous mediastinal and right hilar lymph nodes, concerning for metastatic disease. Specifically, the chest computed tomography scan revealed a 3.1-cm paratracheal lymph node and a 3.1-cm right hilar (station 4R) lymph node. No discrete lung masses were appreciated on imaging. The patient reported no respiratory symptoms but did report a remote cigarette smoking history of approximately 60 pack-years. The patient was referred for endobronchial ultrasound (EBUS)-guided fine-needle aspiration (FNA) for tissue confirmation of his presumed metastatic melanoma.

PATHOLOGIC FINDINGS

A cytopathologist was present on-site during the EBUS-guided FNA procedure of the 4R lymph node. During the on-site assessment, the aspirated material from the first needle pass was expelled onto a slide for the preparation of paired smears, one air-dried and one alcohol fixed. The needle was subsequently rinsed in Roswell Park Memorial Institute (RPMI) medium. The air-dried smear was immediately Diff-Quik (StatLab Medical Products, McKinney, Texas) stained for on-site microscopic examination. The alcohol-fixed smear was set aside for subsequent Papanicolaou staining in the cytopathology laboratory to be completed after the conclusion of the procedure. Microscopic examination of the air-dried, Diff-Quik-stained smear revealed a cellular population of malignant-appearing epithelioid cells arranged predominantly in a discohesive to loosely cohesive pattern (Figure 1). Marked nuclear pleomorphism was appreciated (Figure 2). In isolation, especially in light of the high clinical suspicion of metastatic melanoma, these findings could be consistent with a metastasis from the patient's known malignant melanoma.

In areas, the malignant cells were present as cohesive sheets in which the nuclei demonstrated disordered honeycombing (Figure 3). Three-dimensional spherical clusters of tumor cells, exhibiting community borders, were also appreciated (Figure 4). From these findings, and taking into consideration the anatomic location of the enlarged lymph node being targeted by FNA, the possibility of a metastasis from a second primary malignancy (ie, pulmonary adenocarcinoma) was entertained. Therefore, it was evident that complete workup of this FNA specimen would require immunocytochemical workup in addition to careful cytomorphologic evaluation.

In contrast to the triaging procedure for the first needle pass, the contents from the next needle pass were distributed onto 7 direct smears. Six smears were air-dried and 1 smear was alcohol fixed. One of the air-dried smears was Diff-Quik stained and examined under light microscopy; this needle pass was also cellular and composed of numerous tumor cells. The smears were determined to be of adequate cellularity for cytomorphologic evaluation, confirmatory immunocytochemical workup using a limited panel of antibodies, and anticipated polymerase chain reaction (PCR)-based molecular assays (ie, epidermal growth factor receptor [EGFR] and v-Ki-ras-2 Kirsten rat sarcoma viral oncogene homolog [KRAS] mutation analysis for a final diagnosis of metastatic pulmonary adenocarcinoma, and v-raf murine sarcoma viral oncogene homolog B1 [BRAF] mutation analysis for a final diagnosis of metastatic melanoma). Two additional dedicated passes were performed for the RPMI needle rinse, which would be ultimately used to prepare a cell block. The preliminary diagnosis of malignancy with a differential diagnosis of metastatic melanoma versus metastatic adenocarcinoma was communicated to the bronchoscopist, and EBUS-guided FNA procedure was concluded; the paratracheal lymph node was not sampled.

Four of the 5 extra air-dried direct smears were immediately triaged to the immunoperoxidase laboratory where they were fixed for 30 minutes in formalin at room temperature, rinsed in water, and then processed for immunostaining. Three smears were used for immunocytochemical staining for S100, napsin A, and thyroid transcription factor 1 (TTF-1). The fourth smear was used as a negative control in which the primary antibody was omitted. The fifth air-dried smear was temporarily left unstained.

Shortly after the conclusion of the FNA procedure, Papanicolaou-stained smears were available for microscopic evaluation. Tumor cells arranged in an acinar pattern were readily identified (Figure 5). The nuclei exhibited prominent macronucleoli, and overt cytoplasmic vacuolization was evident in a subset of the malignant cell population (Figures 5 and 6). These features assisted in further bolstering the concern for an adenocarcinoma. Three hours after the air-dried slides were submitted to the immunoperoxidase laboratory, the immunostained slides were available for microscopic examination. The tumor cells exhibited diffuse cytoplasmic, granular immunoreactivity for napsin A and diffuse nuclear immunoreactivity for TTF-1 (Figures 7 and 8, respectively). Background staining was minimal. Immuno-staining for S100 was negative (not shown).

The constellation of cytomorphologic findings and immunophenotype, all ascertained on the same day as the FNA procedure itself, allowed for communication of a diagnosis of metastatic lung adenocarcinoma to the patient's clinical care providers, thereby enabling prompt scheduling of appropriate oncologic referrals. The remaining unstained air-dried smear was Diff-Quik stained. Light microscopic evaluation of this uncoverslipped slide revealed an area on the smear that was enriched with tumor cells. This area was marked on the underside of the slide (Figure 9) and immediately triaged to the molecular diagnostics laboratory for manual microdissection of the tumor cells, DNA isolation using the Pinpoint DNA isolation kit (Zymo Research Corporation, Irvine, California), and subsequent EGFR and KRAS mutation analysis via PCR-based fragment analysis.

On the day following the FNA procedure, the hematoxylin-eosin-stained cell block section was available for review and demonstrated mild to moderate cellularity. The cell block was triaged to the molecular diagnostics laboratory for anaplastic lymphoma kinase gene (ALK) rearrangement analysis. Ultimately, an L858R mutation in EGFR was detected (Figure 10). Neither KRAS mutations nor ALK rearrangements were detected (not shown).

COMMENT

It is well recognized that melanoma can adopt a wide variety of morphologic patterns. In this case, there was a high preprocedural level of clinical suspicion for a metastatic melanoma, and a second primary malignancy was not expected. It is clear that the cytomorphologic features are consistent with malignancy; nonetheless, it could have been tempting to simply dismiss the findings as consistent with metastasis from the patient's known melanoma. Careful cytomorphologic evaluation during the FNA on-site assessment revealed the presence of cohesive sheets of epithelioid cells exhibiting disordered honeycombing, 3-dimensional clusters with community borders, and acinar arrangements. These features should alert the pathologist to entertain the possibility of a pulmonary adenocarcinoma, especially given that the aspirate was obtained from a hilar lymph node. In this case, the TTF-1+/napsin A+/S100- immunophenotype confirmed the diagnosis. A misdiagnosis of metastatic melanoma would have resulted in a lost opportunity to treat this patient's lung cancer and unnecessary melanoma-specific chemotherapy.

Fine-needle aspirates of non-small cell lung carcinoma (NSCLC) are triaged at an increasing frequency for molecular testing including EGFR mutation and ALK gene rearrangement analysis. Approximately 40% of patients with lung cancer have stage IV disease. (1) These patients are not candidates for surgical resection of their primary tumors. Instead, small biopsies can represent the only opportunities to obtain diagnostic tissue. (2) Patients with NSCLCs harboring EGFR mutations are candidates for treatment with tyrosine kinase inhibitors such as erlotinib and gefitinib. (3-5) The L858R point mutation and in-frame deletions in exon 19 represent the two most common clinically relevant mutations in EGFR. (6) The tyrosine kinase inhibitor, crizotinib, has been approved as targeted therapy for NSCLCs harboring ALK rearrangements. (7,8) Rearrangements involving ALK typically result in a fusion protein consisting of echinoderm microtubule-associated protein-like 4 (EML4) and ALK. (9) Fine-needle aspiration also represents a minimally invasive means to establish a tissue diagnosis of metastatic melanoma, allowing for accurate staging and prompt clinical management. Up to 66% of melanomas harbor activating mutations in BRAF. (10) V600E and V600K represent the two most common mutations. (11) Vemurafenib is a BRAF inhibitor that has been approved for the treatment of patients with advanced-stage melanoma whose tumors have been shown to harbor the BRAF V600E mutation. (12) Thus, there has been increased attention to BRAF mutation analysis in FNA samples of metastatic melanoma. Therefore, ensuring adequate cellularity for accurate cytodiagnosis as well as anticipated ancillary testing is paramount.

Cell blocks are traditionally used by many laboratories for ancillary immunocytochemical and molecular studies. This platform is associated with notable strengths and weaknesses. The advantages of this approach include the following: formalin-fixed, paraffin-embedded tissue is treated similarly to that of traditional surgical pathology blocks; multiple serial sections for ancillary studies may be prepared; and a banked archive is maintained for future studies. However, the salient disadvantages of relying solely on this approach include paucicellularity or acellularity in a subset of cases; inability to assess and predict cell block cellularity with certainty at the time of procedure; the pooled nature of the sample; and the possibility that deeper sections from the cell block may not contain adequate tumor cells, initially visualized on the originally prepared hematoxylin-eosin-stained section, for ancillary testing.

Of the disadvantages, variability in cell block cellularity represents the most significant limitation. (2) Compounding this problem, the ability to assure adequate cellularity of the cell block at the time of the FNA procedure is not possible; dedicated passes for the needle rinse do not necessarily ensure adequate cell block cellularity. (2) Furthermore, as the cell block represents a pooled specimen from multiple passes, the tumor cell population from high-yield needle passes may be diluted by background benign elements obtained from off-target needle passes. This is problematic, as the analytic sensitivity of PCR-based molecular diagnostic assays is dependent on a percentage tumor cellularity threshold, below which false-negative results will occur (ie, contaminating benign tissue will be negative for the molecular abnormality being tested).

Alternatively, Diff-Quik-stained cytologic direct smears represent a rich, underused, and "untapped" source of cellular material for ancillary molecular studies. (2,10,13-15) Furthermore, generation of unstained smears allows for the performance of immunocytochemistry, if necessary. (2,14,16,17) Insights gained from these studies provide opportunities to optimize cytologic specimen triage for ancillary immunocytochemical and molecular studies. In our practice, when appropriate, we prepare multiple direct smears for a given FNA needle pass or needle passes (Figure 11) by distributing the cellular material over multiple slides. (2) One smear is air-dried for rapid Diff-Quik staining, the second smear is alcohol fixed for subsequent Papanicolaou staining in the cytopathology laboratory, and the remaining unstained, air-dried smears can be used for ancillary studies. By visualizing representative tumor cells on the Diff-Quik-stained smear at the time of the procedure, the pathologist can infer that tumor cells are present on the unstained smears prepared from the needle pass. A more direct visualization approach, via light microscopic examination of the unstained smear with the condenser flipped, can also be used to verify the presence of tumor cells on the unstained smears. (2)

As illustrated in our case, unstained smears can be immediately submitted for immunocytochemistry and the immunostained slides can be obtained within hours, thereby improving turnaround time in diagnosis. In contrast, cell blocks require processing and preparation of unstained sections before the performance of immunocytochemistry. If molecular testing is necessary, one of the extra unstained, air-dried smears can be Diff-Quik stained, left uncoverslipped, and sent directly to the molecular diagnostics laboratory for tumor cell microdissection and subsequent DNA isolation for molecular analysis. If extra uncoverslipped Diff-Quik-stained smears are not available, previously coverslipped smears can be decoverslipped in xylene and triaged for molecular testing. This overall approach is flexible, forgiving, and avoids the sole reliance on cell blocks for the performance of ancillary studies.

In our practice, we prefer to use Diff-Quik-stained smears over Papanicolaou-stained smears for 2 reasons. First, the cells can be easily visualized on uncoverslipped Diff-Quik-stained smears; this is difficult to do when examining uncoverslipped Papanicolaou-stained slides. Second, Killian and colleagues (15) observed increased DNA fidelity in Diff-Quik-stained cells, compared to Papanicolaou-stained cells. Specifically, increased DNA degradation, as a function of time, was observed for the latter. Of note, whereas BRAF and EGFR mutation analyses are typically performed via PCR-based assays, ALK rearrangement testing is often performed via fluorescence in situ hybridization (FISH). Currently, the prescription of crizotinib requires the use of the Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbott Park, Illinois), which has only been approved for use on formalin-fixed, paraffin-embedded sections. (18) We have recently observed that cytologic direct smears can be effectively used for ALKrearrangement FISH analysis. (19) This approach is advantageous owing to the ability to score entire nuclei, rather than truncated nuclei in paraffin sections.

Overall, the advantages of using direct smears for molecular testing include ease and cost-effectiveness of smear preparation; the ability to directly visualize and select the cellular material to be used for molecular testing; and elimination of the sole reliance on cell blocks for molecular testing. By ensuring that adequate material has been obtained for both cytodiagnosis and anticipated ancillary tests at the time of the FNA procedure while the patient is still accessible, this approach could significantly reduce the frequency of repeated procedures for cases in which insufficient cell block cellularity is encountered. Hence, direct smears, when incorporated into the triage workflow for performing ancillary molecular tests, represent a cost-effective and powerful tool for ancillary testing during the current era of precision medicine.

Caption: Figure 1. Examination of a stained cytologic direct smear, during the on-site assessment, revealed the presence of malignant epithelioid tumor cells arranged in a predominantly discohesive fashion (Diff-Quik, StatLab Medical Products, McKinney, Texas), original magnification x200).

Caption: Figure 2. On higher magnification, marked nuclear pleomorphism is evident in the tumor cells (Diff-Quik, StatLab Medical Products, McKinney, Texas), original magnification x600).

Caption: Figure 3. A cohesive sheet of tumor cells exhibiting disordered honeycombing is present (Diff-Quik, StatLab Medical Products, McKinney, Texas), original magnification x600).

Caption: Figure 4. A cohesive, spherical tumor cell cluster exhibiting a community border is present (Diff-Quik, StatLab Medical Products, McKinney, Texas), original magnification x600).

Caption: Figure 5. Examination of the alcohol-fixed smear, stained after the conclusion of the fine-needle aspiration procedure, revealed the presence of a few cohesive clusters of tumor cells exhibiting acinar arrangements. Prominent nucleoli are appreciable in some of the tumor cell nuclei. Overt cytoplasmic vacuolization is evident in the tumor cell cluster on the lower left aspect of the photomicrograph (Papanicolaou, original magnification x600).

Caption: Figure 6. The tumor cells exhibit a predominantly discohesive pattern. Prominent nucleoli and delicate to overtly vacuolated cytoplasm can be appreciated (Papanicolaou, original magnification x600).

Caption: Figure 7. Immunocytochemistry for napsin A was performed on an unstained direct smear. Coarse, granular cytoplasmic immunoreactivity for napsin A is evident in the tumor cells (original magnification x400).

Caption: Figure 8. Immunocytochemistry for thyroid transcription factor 1 (TTF-1) was performed on an unstained direct smear. Tumor cells exhibited nuclear immunoreactivity for TTF-1 (original magnification x400).

Caption: Figure 9. A Diff-Quik (StatLab Medical Products, McKinney, Texas)-stained direct smear was triaged for molecular testing. An area of the smear enriched with tumor cells is marked on the underside of the slide with a marking pen. The percentage of tumor cellularity (>90%) is indicated on the slide.

Caption: Figure 10. EGFR mutation analysis revealed the presence of an L858R point mutation.

Caption: Figure 11. The contents of given fine-needle aspiration (FNA) passes can be distributed over multiple smears. One smear is Diff-Quik (StatLab Medical Products, McKinney, Texas) stained, another smear is Papanicolaou (Pap) stained, and the remaining extra smears are unstained. Extra smears can be triaged, as indicated, for appropriate ancillary studies. Roswell Park Memorial Institute (RPMI) solution is used for needle rinses and dedicated passes for the cell block.

References

(1.) Azzoli CG, Baker S Jr, Temin S, et al. American Society of Clinical Oncology Clinical Practice Guideline update on chemotherapy for stage IV non-small-cell lung cancer. J Clin Oncol. 2009;27(36):6251-6266.

(2.) Knoepp SM, Roh MH. Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques. Cancer Cytopathol. 2012. In press.

(3.) Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129-2139.

(4.) Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676):1497-1500.

(5.) Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 2004; 101(36):13306-13311.

(6.) Yatabe Y, Mitsudomi T. Epidermal growth factor receptor mutations in lung cancers. Pathol Int. 2007;57(5):233-244.

(7.) Camidge DR, Bang YJ, Kwak EL, et al. Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study. Lancet Oncol. 2012;13(10):1011-1019.

(8.) Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med. 2010;363(18):1693-1703.

(9.) Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448(7153): 561-566.

(10.) Bernacki KD, Betz BL, Weigelin HC, et al. Molecular diagnostics of melanoma fine-needle aspirates: a cytology-histology correlation study. Am J Clin Pathol. 2012;138(5):670-677.

(11.) Long GV, Menzies AM, Nagrial AM, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011;29(10):1239-1246.

(12.) Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364(26):2507-2516.

(13.) Betz BL, Roh MH, Weigelin HC, et al. The application of molecular diagnostic studies interrogating EGFR and KRAS mutations to stained cytologic smears of lung carcinoma. Am J Clin Pathol. 2011;136(4):564-571.

(14.) Hookim K, Roh MH, Willman J, et al. Application of immunocytochemistry and BRAF mutational analysis to direct smears of metastatic melanoma. Cancer Cytopathol. 2012;120(1):52-61.

(15.) Killian JK, Walker RL, Suuriniemi M, et al. Archival fine-needle aspiration cytopathology (FNAC) samples: untapped resource for clinical molecular profiling. J Mol Diagn. 2010;12(6):739-745.

(16.) Knoepp SM, Hookim K, Placido J, et al. The application of immunocytochemistry to cytologic direct smears of metastatic merkel cell carcinoma. Diagn Cytopathol. 2011. In press.

(17.) Roh MH, Schmidt L, Placido J, et al. The application and diagnostic utility of immunocytochemistry on direct smears in the diagnosis of pulmonary adenocarcinoma and squamous cell carcinoma. Diagn Cytopathol. 2012; 40(11):949-955.

(18.) Thunnissen E, Bubendorf L, Dietel M, et al. EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations. Virchows Arch. 2012;461(3):245-257.

(19.) Betz BL, Dixon CA, Weigelin HC, et al. The utilization of stained cytologic direct smears for ALK rearrangement analysis of lung adenocarcinoma. Cancer Cytopathol. 2013. In press.

Michael H. Roh, MD, PhD

Accepted for publication May 16, 2013.

From the Department of Pathology, University of Michigan Health System, Ann Arbor.

The author has no relevant financial interest in the products or companies described in this article.

Presented at New Frontiers in Pathology: An Update for Practicing Pathologists meeting; Homestead Resort; August 3-5, 2012; Glen Arbor, Michigan.

Reprints: Michael H. Roh, MD, PhD, Department of Pathology, University of Michigan Health System, 2G332 UH, 1500 E Medical Center Dr, Ann Arbor, MI 48109-5054 (e-mail: mikro@med.umich. edu).

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Author:Roh, Michael H.
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Case study
Geographic Code:1U7TX
Date:Sep 1, 2013
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