Eugen C. Minca

ALK Status Testing in Non–Small Cell Lung Carcinoma: Correlation Between Ultrasensitive IHC and FISH

ALK gene rearrangements in advanced non-small cell lung carcinomas (NSCLC) are an indication for targeted therapy with crizotinib. Fluorescence in situ hybridization (FISH) using a recently approved companion in vitro diagnostic class FISH system commonly assesses ALK status. More accessible IHC is challenged by low expression of ALK-fusion transcripts in NSCLC. We compared ultrasensitive automated IHC with FISH for detecting ALK status on 318 FFPE and 40 matched ThinPrep specimens from 296 patients with advanced NSCLC. IHC was concordant with FFPE-FISH on 229 of 231 dual-informative samples (31 positive and 198 negative) and with ThinPrep-FISH on 34 of 34 samples (5 positive and 29 negative). Two cases with negative IHC and borderline-positive FFPE-FISH (15% and 18%, respectively) were reclassified as concordant based on negative matched ThinPrep-FISH and clinical data consistent with ALK-negative status. Overall, after including ThinPrep-FISH and amending the false-positive FFPE-FISH results, IHC demonstrated 100% sensitivity and specificity (95% CI, 0.86 to 1.00 and 0.97 to 1.00, respectively) for ALK detection on 249 dual-informative NSCLC samples. IHC was informative on significantly more samples than FFPE-FISH, revealing additional ALK-positive cases. The high concordance with FISH warrants IHCs routine use as the initial component of an algorithmic approach to clinical ALK testing in NSCLC, followed by reflex FISH confirmation of IHC-positive cases.

ALK Status Testing in Non–Small-Cell Lung Carcinoma by FISH on ThinPrep Slides with Cytology Material

Introduction: Oncogenic anaplastic lymphoma kinase (ALK) gene rearrangements in non–small-cell lung carcinomas (NSCLC) provide the basis for targeted therapy with crizotinib and other specific ALK inhibitors. Treatment eligibility is conventionally determined by the Food and Drug Administration–approved companion diagnostic fluorescence in situ hybridization (FISH) assay on paraffin-embedded tissue (PET). On limited samples such as fine needle aspiration–derived cytoblocks, FISH for ALK is often uninformative. FISH performed on liquid-based ThinPrep slides (ThinPrep-FISH) may represent a robust alternative. Methods: Two hundred thirty cytology samples from 217 patients with advanced NSCLC, including a consecutive series of 179 specimens, were used to generate matched ThinPrep slides and paraffin cytoblocks. The same ThinPrep slides used for cytologic diagnosis were assessed by standard ALK break-apart two-color probe FISH, after etching of tumor areas. Ultrasensitive ALK immunohistochemistry (IHC) on corresponding cytoblocks [D5F3 antibody, OptiView signal amplification] served as the reference data set. Results: ThinPrep-FISH ALK signals were robust in 228 of 230 cases and not compromised by nuclear truncation inherent in paraffin-embedded tissue–FISH; only two samples displayed no signals. Nine of 178 informative cases (5%) in the consecutive series and 18 of 228 informative cases (7.8%) overall were ALK rearranged by ThinPrep-FISH. In 154 informative matched ThinPrep-FISH and cytoblock-IHC samples, 152 were concordant (10, 6.5% ALK status positive; 142, 92.2% ALK status negative), and two (1.3%) were ThinPrep-FISH positive but IHC negative (sensitivity 100%, specificity 98.6%, overall agreement 98.7%). Conclusion: Detection of ALK gene rearrangements in liquid cytology ThinPrep slides derived from patients with NSCLC can be confidently used for clinical ALK molecular testing.

Cytokeratin 20-negative Merkel cell carcinoma is infrequently associated with the Merkel cell polyomavirus

Merkel cell carcinoma is a rare, highly aggressive cutaneous neuroendocrine carcinoma most commonly seen in sun-damaged skin. Histologically, the tumor consists of primitive round cells with fine chromatin and numerous mitoses. Immunohistochemical stains demonstrate expression of neuroendocrine markers. In addition, cytokeratin 20 (CK20) is expressed in ∼95% of cases. In 2008, Merkel cell carcinoma was shown to be associated with a virus now known as Merkel cell polyomavirus in ∼80% of cases. Prognostic and mechanistic differences between Merkel cell polyomavirus-positive and Merkel cell polyomavirus-negative Merkel cell carcinoma may exist. There has been the suggestion that CK20-negative Merkel cell carcinomas less frequently harbor Merkel cell polyomavirus, but a systematic investigation for Merkel cell polyomavirus incidence in CK20-negative Merkel cell carcinoma has not been done. To test the hypothesis that Merkel cell polyomavirus is less frequently associated with CK20-negative Merkel cell carcinoma, we investigated 13 CK20-negative Merkel cell carcinomas from the files of the Cleveland Clinic and the University of Michigan for the virus. The presence or absence of Merkel cell polyomavirus was determined by quantitative PCR performed for Large T and small T antigens, with sequencing of PCR products to confirm the presence of Merkel cell polyomavirus. Ten of these (77%) were negative for Merkel cell polyomavirus and three (23%) were positive for Merkel cell polyomavirus. Merkel cell polyomavirus is less common in CK20-negative Merkel cell carcinoma. Larger series and clinical follow-up may help to determine whether CK20-negative Merkel cell carcinoma is mechanistically and prognostically unique.

Ultrasensitive RNA In Situ Hybridization for Detection of Restricted Clonal Expression of Low-Abundance Immunoglobulin Light Chain mRNA in B-Cell Lymphoproliferative Disorders

OBJECTIVES To assess the feasibility of using a novel ultrasensitive bright-field in situ hybridization approach (BRISH) to evaluate κ and λ immunoglobulin messenger RNA (mRNA) expression in situ in B-cell non-Hodgkin lymphoma (NHL). METHODS A series of 110 semiconsecutive clinical cases evaluated for lymphoma with historic flow cytometric (FCM) results were assessed with BRISH. RESULTS BRISH light chain restriction (LCR) results were concordant with FCM in 108 (99%) of 109 evaluable cases. Additional small B-cell lymphoma cohorts were successfully evaluated. CONCLUSIONS BRISH analysis of κ and λ immunoglobulin mRNA expression is a sensitive tool for establishing LCR in B-cell NHL when FCM results are not available.

EGFR mutational genotyping of liquid based cytology samples obtained via fine needle aspiration (FNA) at endobronchial ultrasound of non-small cell lung cancer (NSCLC)

OBJECTIVES Epidermal growth factor receptor (EGFR) gene mutation status should be determined in all patients with advanced, non-squamous non-small cell lung carcinoma (NSCLC) to guide targeted therapy with EGFR tyrosine kinase inhibitors. EGFR mutations are commonly tested by Sanger sequencing or allele specific polymerase chain reaction (ASPCR) on formalin-fixed paraffin-embedded (FFPE) samples including cell blocks (CB) that may fail due to absence of tumor cells. The cell pellet from cytology specimens obtained at the time of endobronchial guided ultrasound fine needle aspiration (EBUS FNA) (EBUS-TBNA, transbronchial needle aspiration) represents an alternative resource for additional tissue. Here we demonstrate the utility of using the FNA cell pellet versus for the detection of EGFR mutations in NSCLC. MATERIALS AND METHODS For internal validation, 39 cytology samples from patients with NSCLC referred for EGFR testing were analyzed using the EGFR rotor-gene Q (RGQ) PCR assay (Qiagen). Thereafter, a consecutive series of 228 EBUS FNA samples were tested. RESULTS The ASPCR assay demonstrated acceptable intra-assay, inter-assay and inter-lot reproducibility, sensitivity, and specificity. For the consecutive series, only 6/228 (2.6%) failed analysis (5 due to insufficient DNA yield). Of 228 EBUS FNA cell pellets tested 32 (14.0%) demonstrated clinically relevant mutations. RESULTS AND CONCLUSION ASPCR can reliably detect EGFR gene mutations in FNA preparations from patients with NSCLC obtained at EBUS.

High MET receptor expression but not gene amplification in ALK 2p23 rearrangement positive non-small-cell lung cancer.

Introduction: Overexpression of MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) and MET gene amplification have been well-documented in non–small-cell lung cancer (NSCLC). Activated MET signaling plays an important role in human cancer tumorigenesis, metastasis, and drug resistance. However, the deregulation of MET/HGF pathway in NSCLC harboring ALK gene rearrangement (ALK[+]), which is sensitive to dual ALK and MET inhibitor Crizotinib, has not been reported. Methods: We performed systematic analysis of MET/HGF expression by immunohistochemistry (IHC) and MET gene amplification by dual color, dual hapten bright field in situ hybridization in 19 ALK(+) and 73 ALK(−) NSCLC tumor tissues from those who had clinical ALK rearrangement test done at the Cleveland Clinic from August 2010 to January 2013. IHC scoring was interpreted on a standard four-tier system. Results: The percentage of MET IHC score 0, 1+, 2+, and 3+ were 5.5%, 27.8%, 50.0%, and 16.7% in ALK(+) group, compared with 28.8%, 33.9%, 23.7%, and 13.6% in ALK(−) group, respectively. The MET high expression (IHC score 2 or 3) was significantly higher in ALK(+) group statistically (66.7% versus 37.3%, p = 0.03). HGF-high expression (IHC score 2 or 3) was 33.3% in ALK(+) and 15.8% in ALK(−) (p = 0.17). We identified eight cases in ALK(−) and one case in ALK(+) tumor who had MET gene amplification (18.4% versus 7.1%, p = 0.43) by dual color, dual hapten bright field in situ hybridization. No significant correlation between MET protein receptor expression and gene amplification was identified. Conclusions: Our study demonstrated for the first time that MET receptor expression, but not MET gene amplification, is significantly increased in ALK(+) NSCLC. MET gene amplification is a relatively rare event in this unique population compared with ALK(−) NSCLC.

Comparison between melanoma gene expression score and fluorescence in situ hybridization for the classification of melanocytic lesions

Melanoma accounts for most skin cancer-related deaths and has an increasing incidence. Accurate diagnosis and distinction from atypical nevi can be at times difficult using light microscopy alone. Fluorescence in situ hybridization (FISH) and melanoma gene expression score (myPath, Myriad Genetics) have emerged as ancillary tools to further aid in this differential diagnosis. Our aim in this study was to correlate FISH results, gene expression score, consensus histopathologic impression and clinical outcome on a series of 117 challenging melanocytic lesions collected from three separate institutions. The lesions were separated into two groups: 39 histopathologically unequivocal lesions (15 malignant, 24 benign) and 78 challenging lesions interpreted by expert consensus (27 favor malignant, 30 favor benign, and 21 ambiguous). Melanoma-FISH was performed using probes for 6p25, 11q13, 8q24, and 9p21/CEP9 and scored according to established criteria. Analysis by myPath gene expression score was performed and interpreted by the manufacturer as ‘benign’, ‘indeterminate,’ or ‘malignant’. In the unequivocal group, melanoma-FISH and myPath score showed 97 and 83% agreement with the histopathologic diagnosis, respectively, with 93 and 62% sensitivity, 100 and 95% specificity, and 80% inter-test agreement. In the challenging group, FISH and the myPath score showed 70 and 64% agreement with the histopathologic interpretation, respectively, with 70% inter-test agreement and similar sensitivities and specificities. The inter-test agreement was 73% overall, excluding indeterminate results. Discordant test results occurred in 27/117 cases from both unequivocal and challenging groups. Melanoma-FISH and gene expression score are valuable ancillary tools, though both have limitations and return discordant results in a subset of cases. Follow-up studies with more extensive clinical outcome data are warranted to establish the accuracy of these tests for the classification of melanocytic lesions.

Superficial malignant peripheral nerve sheath tumor with overlying intradermal melanocytic nevus mimicking spindle cell melanoma

Malignant peripheral nerve sheath tumors are rare soft tissue sarcomas with histological and immunohistochemical similarities to spindle cell melanoma. Although spindle cell melanoma is significantly more common, both tumors may express S100 and lack staining for HMB‐45, Melan‐A or MITF. Here we present a case of superficial malignant peripheral nerve sheath tumor with diffuse S100 positivity arising in a subtle neurofibroma in close proximity to an intradermal melanocytic nevus. This configuration had led to prior misdiagnosis as a desmoplastic melanoma arising in the nevus and to sentinel lymph node biopsy. Identification of the background neurofibroma, as well as CD34 positivity raised consideration of a low grade malignant peripheral nerve sheath tumor, which was confirmed via observation of Schwannian differentiation on electron microscopy. The importance of distinguishing these two tumors is stressed owing to the difference in management.

Genomic microarray analysis on formalin-fixed paraffin-embedded material for uveal melanoma prognostication.

Cytogenetic alterations are strong outcome prognosticators in uveal melanoma (UVM). Monosomy 3 (-3) and MYC amplification at 8q24 are commonly tested by fluorescence in situ hybridization (FISH). Alternatively, microarray analysis provides whole genome data, detecting partial chromosome loss, loss of heterozygosity (LOH), or abnormalities unrepresented by FISH probes. Nonfixed frozen tissue is conventionally used for microarray analysis but may not always be available. We assessed the feasibility of genomic microarray analysis for high resolution interrogation of UVM using formalin-fixed paraffin-embedded tissue (FFPET) as an alternative to frozen tissue (FZT). Enucleations from 44 patients (clinical trial NCT00952939) yielded sufficient DNA from FFPET (n = 34) and/or frozen tissue (n = 41) for comparative genomic hybridization and select single nucleotide polymorphism analysis (CGH/SNP) on Roche-NimbleGen OncoChip arrays. CEP3 FISH analysis was performed on matched cytology ThinPrep material. CGH/SNP analysis was successful in 30 of 34 FFPET and 41 of 41 FZT samples. Of 27 paired FFPET/FZT samples, 26 (96.3%) were concordant for at least four of six major recurrent abnormalities (-3, +8q, -1p, +6p, -6q, -8p), and 25 of 27 (92.6%) were concordant for -3. Results of CGH/SNP were concordant with the CEP3 FISH results in 27 of 30 (90%) FFPET and 38 of 41 (92.6%) FZT cases; partial -3q was detected in two CEP3 FISH-negative cases and whole chromosome 3, 4, and 6 SNP-LOH in one case. CGH detection of -3, +8q, -8p on FFPET and FZT showed significant correlation with the clinical outcome measures (metastasis development, time to progression, survival). Results of the UVM genotyping by CGH/SNP on FFPET are highly concordant with those of the FZT analysis and with those of the CEP3 FISH analysis, and therefore CGH/SNP is a practical method for UVM prognostication. Genome-wide coverage provides additional data with potential relevance to UVM biology, diagnosis, and prognosis.

Stenotrophomonas maltophila cellulitis in an immunocompromised patient presenting with purpura, diagnosed on skin biopsy

Stenotrophomas maltophilia is an opportunistic Gram‐negative bacillus and an important cause of nosocomial infections, particularly in immunosuppressed individuals. Although infections with this organism are most often in the form of pneumonia, bacteremia and endocarditis, awareness of the impact of S. maltophilia skin infections has been increasing. Here we describe a case of S. maltophilia cellulitis in a 65‐year‐old man with severe neutropenia and purpuric skin lesions to highlight the critical histopathological findings and correlate them with the clinical manifestations of the skin infection with this organism. Because identification of S. maltophilia can be challenging and infections are difficult to manage, this case illustrates essential considerations regarding the multifaceted histopathological, dermatological, clinical and microbiological aspects of the diagnosis and treatment of S. maltophilia cellulitis in a severely immunocompromised patient. Cognizance of the increasing incidence of nosocomial infections with uncommon microorganisms such as S. maltophilia is necessary when presented with atypical cutaneous manifestations, particularly in immunocompromised patients.