Molecular profiling of primary uveal melanomas with tumor-infiltrating lymphocytes.

In contrast to other cancers, the presence of tumor-infiltrating lymphocytes (TILs) in uveal melanoma is associated with a poor prognosis. However, how TILs may promote disease progression and what regulates their infiltration has not yet been established. To address these clinically relevant outstanding questions, T cell, immune regulatory, and chemokine gene expression profiles of 57 enucleated uveal melanoma tumors were compared, encompassing 27 with TILs and 30 without,. Tumors with infiltrating lymphocytes expressed more CD8A mRNA, as well as IFNG, TGFB1, and FOXP3 transcripts. Other T helper associated cytokines and T helper transcription factors were not differentially expressed, nor were mediators of lymphocyte cytotoxicity. The immune inhibitors INDO, PDCA1, CTLA4, and LAG3, and the non-classical MHC Class I target of CD8+ T regulatory cells, HLA­E, were significantly higher in tumors with TILs. FAS was also significantly higher. The C-C chemokine ligands CCL4, CCL5, and CCL20 were higher in tumors with TILs. Levels of CCL5 were most strongly correlated with levels of CD8A. Chemokine receptors were not differentially expressed. Molecular profiling of uveal melanoma tumors with TILs supports the existence of an immunosuppressive tumor microenvironment and suggests roles for CD8+ regulatory T cells, as well as specific chemokines, in fostering uveal melanoma disease progression.

Automated Bright-Field Dual-Color In Situ Hybridization for MDM2: Interobserver Reproducibility and Correlation With Fluorescence In Situ Hybridization in a Series of Soft Tissue Consults.

CONTEXT: -Atypical lipomatous tumors/well-differentiated liposarcomas contain alterations in the 12q13-15 region resulting in amplification of MDM2 and nearby genes. Identifying MDM2 amplification is a useful ancillary test, as the histologic mimics of atypical lipomatous tumors/well-differentiated liposarcomas have consistently shown a lack of MDM2 amplification. OBJECTIVE: -To assess the interobserver reproducibility of a bright-field assay for MDM2 amplification (dual-color, dual-hapten in situ hybridization [DDISH]) among reviewers with varying degrees of experience with the assay and to assess the concordance of MDM2 DDISH with MDM2 fluorescence in situ hybridization (FISH). DESIGN: -In total, 102 cases were assessed in parallel for MDM2 by FISH and DDISH. MDM2 amplification was defined as an MDM2 to chromosome 12 ratio of 2.0 or greater, whereas an MDM2 to chromosome 12 ratio of less than 2 was nonamplified. Fluorescence in situ hybridization was scored in the routine clinical laboratory and DDISH was evaluated by 3 different pathologists blinded to the final diagnosis and FISH results. RESULTS: -Fluorescence in situ hybridization categorized 27 cases (26%) as MDM2 amplified and 75 cases (74%) as nonamplified; the consensus DDISH diagnosis was 98% concordant with FISH. Agreement between MDM2 DDISH by each reviewer and MDM2 FISH was highly concordant (99%, 98%, and 98%, respectively, for reviewers 1, 2 and 3). The κ agreement of the 3 reviewers scoring DDISH was excellent (κ = 0.949, 0.95, and 0.95, respectively, for reviewers 1, 2, and 3). CONCLUSIONS: -This study highlights excellent concordance between DDISH and FISH in MDM2 copy number assessment. Moreover, excellent interobserver reproducibility of the DDISH assay was found among reviewers with varying levels of experience evaluating bright-field assays.

MET and PTEN gene copy numbers and Ki-67 protein expression associate with pathologic complete response in ERBB2-positive breast carcinoma patients treated with neoadjuvant trastuzumab-based therapy.

BACKGROUND: Pathologic complete response (pCR) after neoadjuvant chemotherapy for breast cancer is associated with improved prognosis in aggressive tumor subtypes, including ERBB2- positive tumors. Recent adoption of pCR as a surrogate endpoint for clinical trials in early stage breast cancer in the neoadjuvant setting highlights the need for biomarkers that, alone or in combination, help predict the likelihood of response to treatment. METHODS: Biopsy specimens from 29 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color bright field in situ hybridization (dual ISH) using probes for MET, TOP2A, PTEN, and PIK3CA genes, each paired with centromeric probes to their respective chromosomes (chromosomes 7, 17, 10, and 3). Ki-67 expression was assessed by immunohistochemistry (IHC). Various parameters describing copy number alterations were evaluated for each gene and centromere probe to identify the optimal parameters for clinical relevance. Combinations of ISH parameters and IHC expression for Ki-67 were also evaluated. RESULTS: Of the four genes and their respective chromosomes evaluated by ISH, two gene copy number parameters provided statistically significant associations with pCR: MET gain or loss relative to chromosome 7 (AUC = 0.791, sensitivity = 92 % and specificity = 67 % at optimal cutoff, p = 0.0032) and gain of PTEN (AUC = 0.674, sensitivity = 38 % and specificity = 100 % at optimal cutoff, p = 0.039). Ki-67 expression was also found to associate significantly with pCR (AUC = 0.726, sensitivity = 100 % and specificity = 42 % at optimal cutoff, p = 0.0098). Combining gain or loss of MET relative to chromosome 7 with Ki-67 expression further improved the association with pCR (AUC = 0.847, sensitivity = 92 % and specificity = 83 % at optimal cutoffs, p = 0.0006). CONCLUSIONS: An immunogenotypic signature of low complexity comprising MET relative copy number and Ki-67 expression generated by dual ISH and IHC may help predict pCR in ERBB2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab. These findings require validation in additional patient cohorts.

iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors.

BACKGROUND: Uveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis. METHODS: Eight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch's membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors. RESULTS: Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens. CONCLUSIONS: The present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.

Immunohistochemistry for BRAF V600E in the Differential Diagnosis of Hairy Cell Leukemia vs Other Splenic B-Cell Lymphomas.

OBJECTIVES: Recent reports have used immunohistochemistry (IHC) with a mutation-specific antibody to detect the BRAF V600E mutation, which is found in nearly all cases of hairy cell leukemia (HCL). To date, however, only a small number of non-HCL, splenic B-cell lymphomas have been examined by IHC. METHODS: We analyzed 121 cases, including 26 HCLs, 52 non-HCL splenic lymphomas, 22 chronic lymphocytic leukemias/small lymphocytic lymphomas (CLLs/SLLs), and 21 plasma cell neoplasms (PCNs) for BRAF V600E expression by IHC. Molecular testing for BRAF V600E was performed in a subset of cases, using allele-specific polymerase chain reaction and/or Sanger sequencing. RESULTS: Twenty-six (100%) of 26 HCL cases were positive by IHC vs one (1%) of 95 non-HCL cases. Positive staining was identified in one (2%) of 44 splenic marginal zone lymphomas (SMZLs), while each of 22 CLLs/SLLs, 21 PCNs, six unclassifiable splenic lymphomas, and two HCL variants were negative. IHC and molecular results were concordant in all cases examined (21 HCLs and 21 non-HCLs, including the BRAF+ SMZLs). CONCLUSIONS: The detection of BRAF V600E by IHC is useful in the distinction of HCLs from other splenic-based lymphomas, although the identification of at least rare SMZLs containing this abnormality illustrates the continuing need for a multiparameter approach to diagnosis.

Detection of MGMT promoter methylation in glioblastoma using pyrosequencing.

Recent clinical trials on patients with glioblastoma revealed that O6-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient's response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.

Detection of MGMT promoter methylation in glioblastoma using pyrosequencing.

Recent clinical trials on patients with glioblastoma revealed that O(6)-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient's response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.

Lymphoma of prosthetic aortic graft presenting as recurrent embolization.

We describe the case of a patient who presented with transient ischemic attack 4 years after bicuspid aortic valve repair, ascending aorta, and hemiarch replacement. Workup included cross-sectional imaging consistent with thrombus in the ascending aorta graft. Warfarin was initiated, but another episode of cerebral ischemia occurred despite therapeutic anticoagulation. Surgery was performed to avoid further embolization as re-replacement with a homograft aorta. Histologic analysis of the material found within the graft demonstrated large B cell lymphoma. At 39 months' follow-up, there have been no additional episodes of embolization and no evidence of recurrent cancer.

Detection of immunoglobulin light-chain restriction in cutaneous B-cell lymphomas by ultrasensitive bright-field mRNA in situ hybridization.

BACKGROUND: Detection of immunoglobulin light-chain restriction is important in the diagnosis of B-cell non-Hodgkin lymphoma (B-NHL). Flow-cytometry, commonly used to evaluate light-chain restriction, is impractical to be used in cutaneous specimens. Immunohistochemical and conventional chromogenic in situ hybridization (CISH) methods on formalin-fixed-paraffin-embedded (FFPE) tissue lack sufficient sensitivity to detect low-level light-chain expression in B-NHL without plasmacytic differentiation. Ultrasensitive bright-field mRNA-ISH (BRISH) for in situ light-chain detection in cutaneous B-NHL has been assessed. DESIGN: Kappa/lambda mRNA was detected using two-color BRISH (RNAscope 2xPlex, Advanced Cell Diagnostics) on 27 FFPE skin biopsies and excisions from patients with available B-cell PCR clonality studies: 16 clonal B-cell lesions (6 follicle center lymphoma, 5 marginal zone lymphoma, 3 large B-cell lymphoma, and 2 other) and 11 non-clonal B-cell proliferations. RESULTS: BRISH was successful in 15/16 clonal B-cell lesions and 11/11 non-clonal proliferations. Light-chain restriction was detected in 15/15 clonal lesions and in 1/11 non-clonal proliferations (96.1% overall concordance with clonality PCR). In 4/5 marginal zone lymphomas, light-chain restriction was detected as strong monotypic mRNA expression in a B-cell subset, consistent with plasmacytic differentiation. CONCLUSION: Ultrasensitive BRISH can successfully detect light-chain restriction in B-NHL from FFPE skin specimens and may be a useful adjunct ancillary tool in cases not resolved by CISH or immunohistochemical methods.

Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma.

We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)-DLBCL compared with activated B-cell (ABC)-DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the "gold standard" GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL.

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.

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

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.

Frequency, interobserver reproducibility and clinical significance of equivocal peaks in PCR clonality testing using Euroclonality/BIOMED-2 primers.

AIMS: PCR studies for lymphoid clonality are now widely employed, especially using Euroclonality/BIOMED-2 primers. Criteria for interpretation as a clonal result, however, have proven controversial. This study examines the frequency and clinical significance of equivocal amplification patterns and measures the interobserver reproducibility of clonality interpretations. METHODS: At our institution, results of each primer set are first classified as clonal, non-clonal or abnormal (equivocal peak on polyclonal background). Final results for all primer sets are then collectively reported as positive (≥1 clonal result), negative (non-clonal results) or indeterminate (≥1 abnormal result) for a clonal population. Results of 274 consecutive clonality cases were reviewed, and the interobserver reproducibility of individual primer set reactions and final results was determined in a subset of 30 cases. RESULTS: 44/161 (27%) B-cell and 50/163 (31%) T-cell cases contained at least one abnormal peak. Of these, 29 (64%) and 31 (62%), respectively, showed clonal results in another primer set. Interobserver reproducibility was excellent for most primer sets and for final interpretations, but only fair to good for IGK V-J and TCRB D-J1+2 primer sets. A definitive diagnosis of lymphoma was rendered in 93%, 20% and 6% of B-cell cases and 90%, 42%, and 14% of T-cell cases positive, indeterminate or negative for a clonal population, respectively. CONCLUSIONS: Using a subjective approach, abnormal (equivocal) peaks are frequently observed in routine practice. However, most cases with abnormal peaks contain clonal rearrangements in other primer sets, facilitating overall interpretation of final results with excellent interobserver reproducibility.

BCL2 antibodies targeted at different epitopes detect varying levels of protein expression and correlate with frequent gene amplification in diffuse large B-cell lymphoma.

Patients with aggressive, BCL2 protein-positive (+) diffuse large B-cell lymphoma (DLBCL) often experience rapid disease progression that is refractory to standard therapy. However, there is potential for false-negative staining of BCL2 using the standard monoclonal mouse 124 antibody that hinders the identification of these high-risk DLBCL patients. Herein, we compare 2 alternative rabbit monoclonal antibodies (E17 and SP66) to the 124 clone in staining for BCL2 in formalin-fixed, paraffin-embedded DLBCL tissues. Overall, in 2 independent DLBCL cohorts, E17 and SP66 detected BCL2 expression more frequently than 124. In the context of MYC expression, cases identified as BCL2 (+) with SP66 demonstrated the strongest correlation with worse overall survival. The 124 clone failed to detect BCL2 expression in the majority of translocation (+), amplification (+), and activated B-cell DLBCL cases in which high levels of BCL2 protein are expected. Using dual in situ hybridization as a new tool to detect BCL2 translocation and amplification, we observed similar results as previously reported for fluorescence in situ hybridization for translocation but a higher amplification frequency, indicating that BCL2 amplification may be underreported in DLBCL. Among the discrepant cases, phosphorylation of BCL2 at T69 and/or S70 was more common than in the concordant cases and may contribute to the 124 false negatives, in addition to previously associated mutations within the epitope region. The accurate detection of BCL2 expression is important in the prognosis and treatment of DLBCL particularly with new anti-BCL2 therapies.

Concomitant detection of HER2 protein and gene alterations by immunohistochemistry (IHC) and silver enhanced in situ hybridization (SISH) identifies HER2 positive breast cancer with and without gene amplification.

INTRODUCTION: HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number. METHODS: Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis. RESULTS: Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification. CONCLUSIONS: The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.

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.

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.

An international interpretation study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators.

INTRODUCTION: The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non-small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay. METHODS: Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made. RESULTS: Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively. CONCLUSIONS: On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader's score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.