Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples.

UNLABELLED: The human epidermal growth factor receptor 2 (HER2, also known as erbB2) gene is involved in signal transduction for cell growth and differentiation. It is a cell surface receptor tyrosine kinase and a proto-oncogene. Overexpression of HER2 is of clinical relevance in breast cancer due to its prognostic value correlating elevated expression with worsening clinical outcome. At the same time, HER2 assessment is also of importance because successful anti-tumor treatment with Herceptin® is strongly correlated with HER2 overexpression in the tumor (approximately 30% of all breast tumors overexpress HER2). In a comprehensive national study, Wolff et al. [1] state that "Approximately 20% of current HER2 testing may be inaccurate" which underscores the importance of developing more accurate methods to determine HER2 status. Droplet Digital™ PCR (ddPCR™) has the potential to improve upon HER2 measurements due to its ability to quantitate DNA and RNA targets with high precision and accuracy. Here we present a study which investigates whether ddPCR can be used to assess HER2 transcript levels in formalin-fixed paraffin embedded (FFPE) human breast tumors and whether these ddPCR measurements agree with prior assessments of these same samples by pathologists using immunohistochemistry (IHC) and in some cases fluorescence in situ hybridization (FISH). We also determined the copy number of HER2 in these samples as compared to the CEP17 reference gene. RESULTS: Clinical FFPE samples were successfully studied using ddPCR and compared to results from standard FISH and IHC methodology. The results demonstrate that ddPCR can rank order the samples in complete agreement with the current standard methods and that ddPCR has the added benefit of providing quantitative results, rather than relying on the expert skill of a seasoned pathologist for determination.

Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) testing is routinely performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) analyses for all new cases of invasive breast carcinoma. IHC is easier to perform, but analysis can be subjective and variable. FISH offers better diagnostic accuracy and added confidence, particularly when it is used to supplement weak IHC signals, but it is more labor intensive and costly than IHC. We examined the performance of droplet digital PCR (ddPCR) as a more precise and less subjective alternative for quantifying HER2 DNA amplification. METHODS: Thirty-nine cases of invasive breast carcinoma containing ≥30% tumor were classified as positive or negative for HER2 by IHC, FISH, or both. DNA templates for these cases were prepared from formalin-fixed paraffin-embedded (FFPE) tissues to determine the HER2 copy number by ddPCR. ddPCR involved emulsifying hydrolysis probe-based PCR reaction mixtures containing the ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2] gene and chromosome 17 centromere assays into nanoliter-sized droplets for thermal cycling and analysis. RESULTS: ddPCR distinguished, through differences in the level of HER2 amplification, the 10 HER2-positive samples from the 29 HER2-negative samples with 100% concordance to HER2 status obtained by FISH and IHC analysis. ddPCR results agreed with the FISH results for the 6 cases that were equivocal by IHC analyses, confirming 2 of these samples as positive for HER2 and the other 4 as negative. CONCLUSIONS: ddPCR can be used as a molecular-analysis tool to precisely measure copy number alterations in FFPE samples of heterogeneous breast tumor tissue.

Muir-Torre syndrome.

Muir-Torre syndrome (MTS) is a rare autosomal-dominant genodermatosis characterized by sebaceous neoplasms and one or more visceral malignancies. Sebaceous tumors include sebaceous adenoma and carcinoma, which may be solitary or multiple. Visceral malignancies most often arise in the colorectum and endometrium. Because a subset of patients with phenotypic MTS will have germline mutations in the DNA mismatch repair genes hMSH2 and hMLH1, MTS is considered a phenotypic subtype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer syndrome), in which inherited defects in DNA mismatch repair genes result in microsatellite instability. Pathologists have an important role in the early detection and initial diagnosis of MTS: identification of at-risk individuals allows appropriate screening and surveillance for visceral malignancies, thereby reducing morbidity and mortality. Herein, we describe the clinicopathologic features of MTS.

Novel Integrative Genomics Approach for Associating GWAS Information with Intrinsic Subtypes of Breast Cancer.

Genome-wide association studies (GWAS) have achieved great success in identifying common variants associated with increased risk of developing breast cancer. However, GWAS do not typically provide information about the broader context in which genetic variants operate in different subtypes of breast cancer. The objective of this study was to determine whether genes containing single nucleotide polymorphisms (SNPs, herein called genetic variants) are associated with different subtypes of breast cancer. Additionally, we sought to identify gene regulator networks and biological pathways enriched for these genetic variants. Using supervised analysis, we identified 201 genes that were significantly associated with the six intrinsic subtypes of breast cancer. The results demonstrate that integrative genomics analysis is a powerful approach for linking GWAS information to distinct disease states and provide insights about the broader context in which genetic variants operate in different subtypes of breast cancer.

Russell body gastroenteritis: an aberrant manifestation of chronic inflammation in gastrointestinal mucosa.

First described in 1998, Russell body gastritis is a rare chronic inflammatory condition characterized by abundant intramucosal polyclonal plasma cells, which contain intracytoplasmic eosinophilic globules of immunoglobulins (Russell bodies) that displace the nucleus, with an accompanying chronic inflammatory infiltrate. Russell bodies represent a cellular response to overstimulation of plasma cells, leading to the accumulation of abundant, nondegradable, condensed immunoglobulin in dilated rough endoplasmic reticulum cisternae. Russell body gastritis usually occurs in the gastric antrum, but two cases of Russell body duodenitis have been recently described. Herein, we report an unusual case of Barrett esophagus with prominent lymphoplasmacytic infiltration and Russell bodies, which expands the current spectrum of Russell body gastritis/duodenitis. Given the various anatomic locations in which Russell body gastritis may arise, we suggest that "Russell body gastroenteritis" may be a more appropriate designation for this uncommon reactive condition.

Correlation of Notch1, pAKT and nuclear NF-κB expression in triple negative breast cancer.

Gene expression profiling reveals elevated Notch1 mRNA expression in triple negative breast cancers (TNBC), both basaloid and claudin-low subtypes. Notch ligands, Jagged1 and Jagged2, have been correlated with poor prognosis in TNBC. AKT, an oncogenic protein kinase family that is activated downstream of Notch in breast cancer cell lines, is frequently activated in breast cancer. Recent publications suggest that inhibition of cell growth, migration, invasion, and induction of apoptosis caused by Notch1 or Jagged1 inhibition may be attributed in part to inactivation of the AKT signaling pathway. There is significant evidence that Notch1 activates NF-κB in several models, and that AKT can mediate NF-κB activation. In this study, we evaluated Notch1 protein expression by immunohistochemistry (IHC) and correlated this with expression of pAKT and nuclear NF-κB p65 (RelA) in TNBC. A tissue microarray (TMA) containing 32 formalin-fixed, paraffin-embedded (FFPE) TNBC tumor specimens was constructed from the archival tissue database of the Department of Pathology at UMMC and IHC for Notch1 protein, pAKT 1/2/3 (Ser473), and NF-κB, p65 subunit was performed on the TMA with appropriate positive and negative controls. Of the 32 TNBC in our cohort, 100% expressed Notch1 protein by IHC: 24 (75%) showed cytoplasmic expression, 25 (78%) showed membranous expression, and 17 (53%) showed both cytoplasmic and membranous expression. Overall, 29 (91%) expressed pAKT by IHC: 28 (97%) showed cytoplasmic expression, 14 (48%) showed nuclear expression and 13 (45%) showed both cytoplasmic and nuclear expression. Nuclear staining for NF-κB p65 was detected in all 32 TNBC specimens with variable intensities. On bivariate analysis, cytoplasmic Notch1 was significantly correlated with cytoplasmic pAKT (r = 0.373, P = 0.035) and nuclear NF-κB (r = 0.483, P = 0.005); both cytoplasmic and nuclear pAKT significantly correlated with nuclear NF-κB (r = 0.391, P = 0.027; r = 0.525, P = 0.002, respectively). These results suggest that 1) the cross-talk between Notch1, AKT and NF-κB identified in preclinical models may operate in a significant fraction of human TNBC, and 2) combination therapy with agents targeting these pathways warrants further investigation.

Schwannoma of the oral tongue.

OBJECTIVE: To present a rare, benign lesion of the oral tongue and its treatment. STUDY DESIGN: Case presentation and literature review. METHODS: Case report. RESULTS: A 68 year old male presented with an asymptomatic lateral oral tongue lesion which had been present for several years. Fine needle aspiration was consistent with pleomorphic adenoma versus myoepithelioma. Pathology following complete surgical excision revealed schwannoma. He remains without recurrence following excision. CONCLUSIONS: Oral tongue schwannoma is a rare lesion which is treated with complete surgical excision.

Immunohistochemical analysis of aquaporins in the fetal and adult vocal fold.

OBJECTIVES: The purpose of this study is to identify and compare the presence and location of aquaporins in the fetal and adult larynx using immunohistochemical techniques. METHODS: Immunohistochemical staining was performed on adult and fetal true vocal fold specimens. The presence and location of aquaporins -1, -2 and -3 in the fetal specimens were then compared to the adult specimens. RESULTS: Immunohistochemical analysis revealed a homogenous distribution of aquaporin -3 in the cell cytoplasm of adult lamina propria with a higher concentration in the superficial layer. Aquaporins -1 and -2 were not identified in either the adult specimens. No evidence of aquaporins were identified in the fetal lamina propria. CONCLUSION: Aquaporin -3 was found in the lamina propria of adult true vocal cord specimens with a higher concentration in the superficial layer. No evidence of Aquaporins -1, -2, or -3 was found in fetal specimens.

Histologic changes associated with false-negative sentinel lymph nodes after preoperative chemotherapy in patients with confirmed lymph node-positive breast cancer before treatment.

BACKGROUND: A wide range of false-negative rates has been reported for sentinel lymph node (SLN) biopsy after preoperative chemotherapy. The purpose of this study was to determine whether histologic findings in negative SLNs after preoperative chemotherapy are helpful in assessing the accuracy of SLN biopsy in patients with confirmed lymph node-positive disease before treatment. METHODS: Eighty-six patients with confirmed lymph node-positive disease at presentation underwent successful SLN biopsy and axillary dissection after preoperative chemotherapy at a single institution between 1994 and 2007. Available hematoxylin and eosin-stained sections from patients with negative SLNs were reviewed, and associations between histologic findings in the negative SLNs and SLN status (true negative vs false negative) were evaluated. RESULTS: Forty-seven (55%) patients had at least 1 positive SLN, and 39 (45%) patients had negative SLNs. The false-negative rate was 22%, and the negative predictive value was 67%. The negative SLNs from 17 of 34 patients with available slides had focal areas of fibrosis, some with associated foamy parenchymal histiocytes, fat necrosis, or calcification. These histologic findings occurred in 15 (65%) of 23 patients with true-negative SLNs and in only 2 (18%) of 11 patients with false-negative SLNs (P = .03, Fisher exact test, 2-tailed). The lack of these histologic changes had a sensitivity and specificity for identifying a false-negative SLN of 82% and 65%, respectively. CONCLUSIONS: Absence of treatment effect in SLNs after chemotherapy in patients with lymph node-positive disease at initial presentation has good sensitivity but low specificity for identifying a false-negative SLN.