Momin T. Siddiqui

Assessment of HER-2/neu status in breast cancer. Automated Cellular Imaging System (ACIS)-assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard.

This retrospective study of formalin-fixed infiltrating breast cancer specimens compared manual immunohistochemical assay with a new image analyzer-assisted immunohistochemical quantitation method, using fluorescence in situ hybridization assay (FISH) as the standard. Following the manual immunohistochemical assay, 189 cases, including most manual immunohistochemically positive and some random negative cases, were analyzed by FISH assay for Her-2/neu gene amplification and by the Automated Cellular Imaging System (ACIS) for immunohistochemical staining. Using the FISH standard, the ACIS immunohistochemical assay attained a higher concordance rate and sensitivity than the manual immunohistochemical assay (91.0% and 88% vs 85.7% and 71%, respectively), with only a slight decrease in specificity (93% vs 96%, respectively). In particular, the ACIS immunohistochemical assay resulted in a higher correlation with the FISH assay in the manual immunohistochemical assay 2+ cases. The ACIS immunohistochemical assay achieved higher accuracy than the manual method according to receiver operating characteristic curve analysis. The ACIS method represents a substantial improvement over the manual method for objective evaluation of the HER-2/neu status.

Aneusomy 17 in Breast Cancer: Its Role in HER-2/neu Protein Expression and Implication for Clinical Assessment of HER-2/neu Status

HER-2/neu protein overexpression in breast cancer is mostly caused by HER-2/neu gene amplification. However, it is unclear whether aneusomy 17 may also play a role. Using immunohistochemistry assay (IHC) with DAKO antibody and manual quantitation, 189 specimens were selected from archival invasive breast cancer specimens, including most IHC-positive and some IHC-negative cases (n = 158 and 31, respectively). They were then analyzed by PathVysion fluorescence in situ hybridization (FISH) assay (Vysis, Inc., Downers Grove, IL) and by an image analyzer (ACIS; ChromaVision Medical Systems, Inc., San Juan Capistrano, CA)–assisted IHC quantitation. Ninety-two cases contained disomy 17 (chromosome 17 centromere, 1.76–2.25 signals per cell) whereas 97 cases had aneusomy 17, including 82 with low polysomy (2.26–3.75 signals per cell), 10 with high polysomy (≥3.76 signals per cell), and 5 with hypodisomy (≤1.75 signals per cell). HER-2/neu protein expression had the highest correlation with HER-2/neu gene dosage (copy number; r = .826), followed by the HER-2/neu gene to chromosome 17 ratio (r = .733). The lowest correlation was with the chromosome 17 copy number (r = .307), on which the 10 cases with high polysomy 17 had a disproportionately high impact. The FISH assay using the PathVysion criterion for HER-2/neu gene amplification (HER-2/neu gene to chromosome 17 ratio, ≥2.00) achieved higher concordance with ACIS IHC than did an alternative FISH criterion (absolute HER-2/neu gene copy number, ≥4.00 signals per cell). Most ACIS IHC-PathVysion FISH–discordant cases contained disomy or low polysomy 17, whereas all 10 cases with high polysomy 17 had no such discordance. However, two cases with monosomy 17 had ACIS IHC-PathVysion FISH discordance, i.e., with gene amplification, but no protein overexpression. Both cases would have had no gene amplification if the alternative FISH criterion had been used. In conclusion, aneusomy 17 is common in breast cancer. Except in a certain subset of cases, aneusomy 17 probably is not a significant factor for HER-2/neu protein expression or for clinical assessment of HER-2/neu status.

Comparison of ThinPrep and Conventional Smears in Detecting Carcinoma in Bile Duct Brushings

Bile duct brushing cytology is a common procedure for the exclusion of adenocarcinoma in the bile duct. The authors evaluated the use of ThinPrep® (TP) to determine whether the information obtained is equivalent to that found with conventional smear cytology (CS).

Stromal PD-L1 Expression Is Associated With Better Disease-Free Survival in Triple-Negative Breast Cancer

OBJECTIVESnTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, and there is no approved targeted therapy. We studied the expression of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) in TNBC.nnnMETHODSnFull-face sections from 136 TNBC cases without neoadjuvant therapy between 2004 and 2013 were stained and evaluated for immune cell PD-1 staining and stromal or tumoral PD-L1 staining using the H-score (staining percentage × intensity). Nottingham histologic grade, lymphovascular invasion (LVI), mitotic count, and tumor-infiltrating lymphocytes (TILs) were evaluated. Tumor size, lymph node status, Ki-67 score, metastasis, overall survival (OS), and disease-free survival (DFS) were retrieved from medical records.nnnRESULTSnOf the 136 TNBC cases, 69 (51%) had any PD-L1 staining and 35 (26%) had PD-L1 staining with an H-score ofu20095 or more; 117 (86%) had any PD-1 staining and 68 (50%) had PD-1 staining with an H-score ofu20095 or more. Tumor size and LVI were significantly associated with worse OS and DFS, and TILs and LVI were significantly associated with metastasis in univariate analysis. Stromal PD-L1 expression was significantly associated with better DFS in multivariate analysis. PD-1 expression was not associated with DFS, OS, or metastasis.nnnCONCLUSIONSnPD-L1 expression is seen in a high proportion of TNBCs and associated with better DFS.

Immunohistochemical Investigation of BRAF p.V600E Mutations in Thyroid Carcinoma Using 2 Separate BRAF Antibodies

Background:Approximately 45% of papillary thyroid carcinomas harbor BRAF p.V600E mutations and current practice algorithms endorse molecular testing for BRAF p.V600E. We assessed the utility of immunohistochemistry to detect BRAF p.V600E mutations in thyroid carcinomas using 2 separate BRAF monoclonal antibodies: one that detects both mutant and wild-type protein (pan-BRAF) and another that detects only the mutant protein (mut-BRAF). Methods:We selected 41 formalin-fixed paraffin-embedded thyroid carcinomas (29 papillary, 1 follicular, 7 medullary, and 4 anaplastic) from 37 thyroidectomies and 4 fine-needle aspirations. Immunohistochemistry was performed using a pan-BRAF (clone EP152Y) or a mut-BRAF (clone VE1) monoclonal antibody. Tumors were considered positive if >10% of neoplastic cells showed moderate (2+) or strong (3+) cytoplasmic staining. BRAF p.V600E mutations were confirmed by molecular pyrosequencing, the gold standard for statistical analysis. Results:pan-BRAF reactivity was observed in 80.5% (n=33) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 46.3% (n=19) were wild type. mut-BRAF reactivity was observed in 46.3% (n=19) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 12.2% (n=5) were wild type. The pan-BRAF antibody detected 14 more false positives (specificity: 29.6%, PPV: 42.4%) compared with the mut-BRAF antibody (specificity: 61.5%, PPV: 73.7%), but both antibodies detected the same 5 false positives. No false negatives were detected with either antibody (sensitivity and NPV 100.0% for both). Conclusions:The suboptimal specificity and PPV limits the diagnostic utility of both antibodies to reliably detect BRAF p.V600E mutations in thyroid carcinoma. However, both antibodies provide excellent sensitivity and NPV and either could be used to exclude BRAF wild-type thyroid carcinomas before molecular testing.

Thyroid Transcription Factor 1 and Napsin A Double Stain: Utilizing Different Vendor Antibodies for Diagnosing Lung Adenocarcinoma

Objective: A combined thyroid transcription factor 1 (TTF-1) and Napsin A double stain has been shown to be useful in the diagnosis of adenocarcinoma (ADC). This study compares differences in double staining patterns among vendor antibodies (Leica, Dako, and Biocare). Study Design: The cohorts included 35 FNA cell blocks of lung ADC and 24 cell blocks of lung squamous cell carcinoma (SqCCA). Double-staining immunohistochemistry was performed with TTF-1 as a brown nuclear stain and Napsin A as a red cytoplasmic stain, using three sets of double stains. Additionally, FISH expression was performed on SqCCAs with aberrant TTF-1 expression. Results: The sensitivity for the double stains ranged from 40 to 74%, while the specificity ranged from 88 to 96%. Two Leica TTF-1-positive SqCCAs also showed low-level amplification by FISH assay not seen in the TTF-1-negative control SqCCAs. Conclusion: The use of Dako TTF-1 antibody paired with Leica Napsin A antibody as a double stain yielded the best results for diagnosing ADC; additionally, the Leica Napsin A-only staining results had the highest positive predictive value at 97%. Both Dako and Biocare antibodies expressed less staining of SqCCAs than Leica staining.

BRAF mutations in metastatic malignant melanoma: comparison of molecular analysis and immunohistochemical expression.

Melanoma is a complex genetic disease, and multiple genetic alterations have been reported to play a role during disease progression. The dysregulation of BRAF signaling has been shown to affect many molecules that promote the continual progression of melanoma. Oncogenic BRAF expression plays a vital role in promoting cell invasion and metastasis in melanoma. It is also associated with poor prognosis in metastatic melanoma. About 40% to 60% of cutaneous melanomas have BRAF mutations, and 90% of the mutations involve a specific substitution at codon 600 (BRAF V600E). In this study, we compared BRAF (V600E) mutation detection by molecular analysis with BRAF expression by immunohistochemical (IHC) analysis using 2 different antibodies. A total of 25 metastatic malignant melanomas were included in this study. 10 of the 25 (40%) cases were positive by molecular analysis using the COBAS 4800 BRAF V600 Real-time PCR assay, and 18 of the 25 (72%) cases were positive by IHC analysis with 2 different antibodies. All positive cases by molecular analysis were positive by both IHC stains (100%). No false negatives were obtained with either IHC stain. Eight of the 25 (32%) were false-positive by both IHC stains. This study demonstrates that IHC analysis is a very sensitive test for evaluation of BRAF mutations in metastatic malignant melanoma and may be useful as an initial screening test.

Expression of SOX10 in Salivary Gland Oncocytic Neoplasms: A Review and a Comparative Analysis with Other Immunohistochemical Markers

Objectives: We evaluated SOX10 (SRY-related HMG-box 10) in differentiating acinic cell carcinoma (AciCC) from other salivary gland neoplasms with oncocytic features on fine-needle aspiration cell blocks (FNA CB) and compared its performance to DOG1 (discovered on gastrointestinal stromal tumor 1). Material and Methods: 35 FNA CB of oncocytic salivary gland neoplasms, i.e. 13 cases of AciCC, 16 of Warthin tumor (WT), 3 of mucoepidermoid carcinoma (MEC) and 3 of oncocytoma (ONC), and 75 salivary gland resections, i.e. 26 AciCC, 7 WT, 36 MEC, 3 ONC, 2 mammary analog secretory carcinomas (MASC) and 1 papillary cystadenoma were stained for SOX10 and DOG1. Results: None of the benign oncocytic neoplasms were immunoreactive for SOX10 on CB or resection, similar to DOG1. On CB, 61.5 and 77% of AciCC were positive for SOX10 and DOG1, respectively. All surgically resected AciCC cases were positive for SOX10 and DOG1; other malignant oncocytic lesions such as MEC and MASC demonstrated variable SOX10 and DOG1 staining. Conclusion: The use of SOX10 may increase the diagnostic accuracy of oncocytic lesions on FNA. In this context, SOX10 is equivalent to DOG1 in ruling out benign lesions such as WT and ONC; however, negative results for SOX10 as well as DOG1 do not favor a benign diagnosis since MEC is often negative for both markers.

Actinomyces associated with persistent vaginal granulation tissue

BACKGROUND: We report a case of symptomatic actinomycosis associated with vaginal suture erosion and granulation tissue refractory to conservative management, in an outpatient setting. CASE: Three months after total vaginal hysterectomy and uterosacral ligament vaginal vault suspension, a woman complained of painless, intermittent vaginal discharge and spotting. Despite cauterization of granulation tissue, vaginal spotting persisted for another month. On re-examination, braided polyester suture that was found underlying the granulation tissue was removed. Recurrent symptoms, together with a biopsy revealing actinomycetes, prompted a trial of oral penicillin VK. With persistent symptoms and discomfort during attempts in the outpatient clinic, the woman eventually required suture removal in the operating room. Her symptoms subsequently resolved without recurrence, and no further antibiotic treatment was required. CONCLUSIONS: Actinomyces may be associated with persistent granulation tissue and vault suspension suture material. In rare circumstances, when tissue debridement and suture removal in the clinic is unsatisfactory, surgical intervention in the operating room may be necessary. Ten days of antibiotic therapy alone did not eradicate the granulation tissue, and symptoms resolved only after complete removal of the underlying permanent suture.

Arginase-1: A Highly Specific Marker Separating Pancreatic Adenocarcinoma from Hepatocellular Carcinoma

Background: Arginase-1 and HepPar-1 are effective immunohistochemical (IHC) markers for hepatocellular carcinoma (HCC). In this study, we explored the possible efficacy of these stains in diagnosing pancreatic adenocarcinoma (PAD). Study Design: Arginase-1 and HepPar-1 IHC was performed on formalin-fixed, paraffin-embedded fine needle aspiration (FNA) cell blocks (CB) of PAD (n = 46), tissue microarray (TMA) of PAD (n = 33), FNA CB of HCC (n = 44) and TMA of HCC (n = 85). Negative controls without carcinoma were also applied (pancreas CB, n = 7; pancreas TMA, n = 3). Results: PAD CB demonstrated arginase-1 positivity in 0 of 46 cases and HepPar-1 positivity in 7 of 46 cases (15%). PAD TMA demonstrated arginase-1 positivity in 0 of 33 cases and HepPar-1 positivity in 4 of 33 cases (12%). HCC CB demonstrated arginase-1 positivity in 37 of 44 cases (84%) and HepPar-1 positivity in 32 of 44 cases (72%). HCC TMA demonstrated arginase-1 positivity in 75 of 85 cases (88%) and HepPar-1 positivity in 80 of 85 cases (94%). Conclusion: Both arginase-1 and HepPar-1 are effective IHC markers of hepatocellular differentiation. Arginase-1 demonstrates superior sensitivity and specificity compared with HepPar-1 in the diagnosis of HCC. However, both arginase-1 and HepPar-1 have a low sensitivity and a very high specificity for PAD.