Comparative Analysis of Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer According to 2007 and 2013 American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations.

Purpose To study the effect of the 2013 updates to the 2007 American Society of Clinical Oncology/College of American Pathologists recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer on testing patterns and interpretation in a large regional reference laboratory. Patients and Methods Patient cases with HER2 testing scores for breast biomarker evaluation were selected from our laboratory information system during two 12-month periods (2012 and 2014). The number of tests performed, type of specimens, proportion of HER2-positive and equivocal patient cases, and number of repeat tests on subsequent excisional specimens were examined and compared. Results Although the number of samples tested increased between 2012 and 2014 (2,201 v 2,558 patient cases; 2,278 v 2,659 tumors), HER2 positivity remained constant (15.7% v 15.5%, respectively). The number of repeat tests performed within 6 months more than doubled (122 [5.5%) of 2,201 v 302 [11.8%] of 2,558; P < .001), and the proportion of immunohistochemistry (IHC) 2+ tumors was significantly lower in 2014 than in 2012 (20.3% v 25.3%; P < .001). However, the proportion of patient cases with unresolved HER2 statuses (equivocal by IHC and in situ hybridization) was significantly higher in 2014 (four of 2,278 v 90 of 2,660; P < .001). Conclusion Our findings indicate that the 2013 updates to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer did not affect the overall HER2-positivity rate or the proportion of patients eligible for HER2-targeted therapy. The proportion of tests and repeat tests performed increased, as did the number of patient cases categorized as ISH equivocal. The benefit of targeted therapy in the equivocal group is not proven, so targeted therapy should not be considered for patients in this category which should be redefined in future iterations of the recommendations.

Immunophenotypic Analysis in Early Müllerian Serous Carcinogenesis.

Studies on the immunophenotypes of early forms of serous carcinoma arising from female genital tract are limited. We aimed to examine p53, p16(Ink4a), estrogen receptor (ER), progesterone receptor (PR), ERBB2, WT1, and Ki-67 protein expression in endometrial intraepithelial carcinoma (n=29), serous tubal intraepithelial lesion (n=4) and carcinoma (STIC, n=10), and the putative precursor p53 signature (n=11). Among endometrial intraepithelial carcinoma, 80% demonstrated p53 overexpression and 10% were consistent with a null phenotype. p16(Ink4a) immunostaining were observed in all endometrial intraepithelial carcinoma cases. ER, PR, ERBB2, and WT1 were positive in 54%, 25%, 11%, and 18% of cases, respectively. STIC cases demonstrated p53 overexpression and null phenotype in 90% and 10%, respectively. All STIC cases were p16(Ink4a) and WT1 positive, whereas ER and PR were positive in 70% and 20%, respectively. All STICs were negative for ERBB2. Among serous tubal intraepithelial lesion cases, 75% demonstrated p53 overexpression and 25% a null phenotype. p53 was positive in all 11 p53 signature cases, whereas p16(Ink4a) was universally negative. Finally, ER and PR were positive in 100% and 73% of p53 signature cases, respectively. These results suggest that p16(Ink4a) has a role in early Müllerian serous carcinogenesis but is absent in the earliest noncommitted lesion. p16(Ink4a) immunohistochemistry can be used as an adjunct confirmatory tool in p53-null cases with limited surface area.

Metastatic ovarian carcinoma to the brain: an approach to identification and classification for neuropathologists.

Brain metastasis is an uncommon but increasing manifestation of ovarian epithelial carcinoma and neuropathologists' collective experience with these tumors is limited. We present clinicopathological characteristics of 13 cases of brain metastases from ovarian epithelial carcinoma diagnosed at two academic institutions. The mean ages at diagnosis of the ovarian carcinoma and their subsequent brain metastases were 58.7 and 62.8 years, respectively. At the time of initial diagnosis of ovarian carcinoma the majority of patients had an advanced stage and none had brain metastases as their first manifestation of malignancy. Brain metastases tended to be multiple with ring-enhancing features on neuroimaging. Primary tumors and their brain metastases were all high-grade histologically and the histologic subtypes were: nine high-grade serous carcinoma (HGSC) cases, two clear cell carcinoma (CCC) cases and a single case each of carcinosarcoma and high-grade adenocarcinoma. A recommended histo- and immunopathological approach to these tumours are provided to aid neuropathologists in the recognition and classification of metastatic ovarian carcinoma to the brain.

TNFAIP8: a new effector for Galpha(i) coupling to reduce cell death and induce cell transformation.

Galpha(i)-coupled receptors comprise a diverse family of receptors that induce transformation by largely unknown mechanisms. We previously found that the Galpha(i)-coupled dopamine-D2short (D2S) receptor transforms Balb-D2S cells via Gαi3. To identify new Gαi effectors, a yeast two-hybrid screen was done using constitutively active Gαi3-Q204L as bait, and tumor necrosis factor-alpha (TNFα)-induced protein 8 (TNFAIP8, SCC-S2/NDED/GG2-1) was identified. In contrast, TNFAIP8-related TIPE1 and TIPE2 showed a very weak interaction with Gαi3. In yeast mating, in vitro pull-down, co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays, TNFAIP8 preferentially interacted with activated Gαi proteins, consistent with direct Gαi-TNFAIP8 coupling. Over-expression or depletion of TNFAIP8 using antisense constructs in Balb-D2S cells did not affect D2S-induced signaling to Gαi-dependent inhibition of cAMP. In contrast, antisense depletion of TNFAIP8 completely inhibited spontaneous and D2S-induced foci formation, consistent with a role for TNFAIP8 in Gαi-dependent transformation. To address possible mechanisms, the effect of D2S signaling via TNFAIP8 on TNFα action was examined. D2S receptor activation inhibited TNFα-induced cell death in Balb-D2S cells, but not in cells depleted of TNFAIP8. However, depletion of TNFAIP8 did not prevent D2S-induced inhibition of TNFα-mediated caspase activation, suggesting that D2S/TNFAIP8-induced protection from TNFα-induced cell death is caspase-independent. The data suggest that Gαi-TNFAIP8-mediated rescue of pre-oncogenic cells enhances progression to oncogenic transformation, providing a selective target to inhibit cellular transformation.

GAP1(IP4BP)/RASA3 mediates Galphai-induced inhibition of mitogen-activated protein kinase.

The dopamine D2S receptor (short isoform) couples to inhibitory Galphai/o proteins to inhibit thyrotropin-releasing hormone (TRH)-stimulated p42/p44 mitogen-activated protein kinase (ERK1/2) phosphorylation in GH4ZR7 rat pituitary cells, consistent with its actions to inhibit prolactin gene transcription and cell proliferation. However, the underlying mechanism is unclear. To identify novel Galphai effectors, yeast two-hybrid screening of a GH4ZR7 cDNA library was done using constitutively active Galphai3-Q204L, and multiple clones of the RasGAP cDNA GAP1(IP4BP)/RASA3 were identified. In yeast mating assay, RASA3 preferentially interacted with activated forms of Galphai/o/z proteins, but not with Galphas. A direct interaction was indicated by in vitro pull-down assay, in which S-His-RASA3 preferentially bound guanosine 5'-O-(gamma-thio)triphosphate-activated Galphai3 and Galphai2 compared with guanosine 5'-O-(beta-thio)diphosphate-inactivated proteins. Similarly, in co-immunoprecipitation studies in HEK-293 cells, FLAG-tagged RASA3 preferentially interacted with activated mutants of Galphai3 and Galphai2 compared with wild type proteins. In GH4ZR7 cells, co-immunoprecipitation studies of endogenous proteins demonstrated a Galphai3-RASA3 complex that was induced upon TRH/D2S receptor co-activation. To address RASA3 function in dopamine D2S receptor-induced inhibition of ERK1/2 activity, endogenous RASA3 protein expression was suppressed (70% knockdown) in GH4ZR7 cells stably transfected with full-length antisense cDNA of RASA3. The selected antisense clones had similar levels of dopamine D2S receptor binding and D2S-induced inhibition of cAMP formation compared with parental GH4ZR7 cells. In these clones, D2S-mediated inhibition of TRH-induced phospho-ERK1/2 was reversed by 70-80% compared with parental GH4ZR7 cells. Our results provide a novel mechanism for dopamine D2S-induced inhibition of ERK1/2 and indicate that RASA3 links Galphai proteins to inhibit Gq-induced Ras/ERK1/2 activation.

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants.

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.