E-cadherin clone 36 nuclear staining dictates adverse disease outcome in lobular breast cancer patients.

Breast cancer is a heterogeneous disease and additional biomarkers for individually predicting patient outcomes are needed. Aberrant membrane E-cadherin immunoexpression has been demonstrated in lobular breast cancer. Also, E-cadherin nuclear staining has been reported, associating with prognosis in various tumors. Here, we explore whether membrane or nuclear staining of E-cadherin has the potential to dictate prognosis of patients with lobular breast cancer. We selected a cohort of 285 consecutively diagnosed lobular breast cancer patients and performed immunohistochemistry for E-cadherin (clones 36, EP700Y, and NCH38) and P-cadherin (clone 56C1) in representative formalin-fixed paraffin-embedded blocks. All patients were female, HER2-negative and surgically treated in a single institution. Survival curves were computed by Kaplan-Meier analysis. Hazard ratios and respective 95% confidence intervals were estimated using Cox regression models. Statistical significance was set at p < 0.05. Nuclear staining for E-cadherin clone 36 was frequent (35%), contrarily to other antibodies tested. Negative correlation was found between nuclear and membrane E-cadherin clone 36 immunostaining (rs = -0.30, p < 0.001), whereas positive correlation was found between membrane immunoexpression of E-cadherin clone 36 and P-cadherin (rs = 0.31, p < 0.001). Patients with any evidence of E-cadherin clone 36 nuclear immunostaining disclosed significantly worse overall survival, disease-specific-survival and disease/progression-free survival (hazard ratio = 2.059, 95% confidence interval 1.313-3.230; hazard ratio = 1.980, 95% confidence interval 1.121-3.495; and hazard ratio = 2.341, 95% confidence interval 1.403-3.905, respectively). Differences in survival were more remarkable when considering nuclear E-cadherin immunoexpression in ≥50% tumor cells. Poorer survival was maintained in multivariable analysis, after adjusting for age, menopausal and PR status, treatment course, vascular invasion, tumor grade and stage. Our results support the use of antibodies against the cytoplasmic domain of E-cadherin, such as clone 36, which may reveal nuclear immunostaining and indicate more aggressive clinical course in patients with lobular breast cancer. We hypothesize that E-cadherin is cleaved and translocated to nucleus functioning as transcription factor.

Analytical and clinical performance of progesterone receptor antibodies in breast cancer.

OBJECTIVE: Comparison of analytical and immunohistochemical performance of progesterone receptor (PR) antibodies with correlation to recurrence of invasive breast cancer treated with endocrine therapy. METHODS: The binding-affinity kinetics of PR clones 1E2, 1A6, 16 and 636 were compared using synthetic peptides derived from identified epitopes on a Biacore T200. A cohort of 351 cases (Hormone Receptor (HR)+/HER2-) were stained for PR expression with immunohistochemistry (IHC) and scored according to ASCO/CAP criteria. RESULTS: The stability of the antigen/antibody complex was greater for the 1E2 clone compared to 1A6, 16 and 636 clones. PR IHC on archival tissue resulted in 94.3% (299/317) concordance with clones. CONCLUSION: Clones evaluated in this study had a high level of concordance with IHC despite PR (1E2) demonstrating higher analytical binding properties than other clones. In a minority of cases (1.3% for 1E2 and 2.5% for 636) IHC results could convert estrogen receptor (ER)-/PR- to ER-/PR+ tumors, making these patients potentially eligible for endocrine therapy.

Bloom syndrome radials are predominantly non-homologous and are suppressed by phosphorylated BLM.

Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.

Correlation of ROS1 Immunohistochemistry With ROS1 Fusion Status Determined by Fluorescence In Situ Hybridization.

CONTEXT.­: The ability to determine ROS1 status has become mandatory for patients with lung adenocarcinoma, as many global authorities have approved crizotinib for patients with ROS1-positive lung adenocarcinoma. OBJECTIVE.­: To present analytical correlation of the VENTANA ROS1 (SP384) Rabbit Monoclonal Primary Antibody (ROS1 [SP384] antibody) with ROS1 fluorescence in situ hybridization (FISH). DESIGN.­: The immunohistochemistry (IHC) and FISH analytical comparison was assessed by using 122 non-small cell lung cancer samples that had both FISH (46 positive and 76 negative cases) and IHC staining results available. In addition, reverse transcription-polymerase chain reaction (RT-PCR) as well as DNA and RNA next-generation sequencing (NGS) were used to further examine the ROS1 status in cases that were discrepant between FISH and IHC, based on staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive. Here, we define the consensus status as the most frequent result across the 5 different methods (IHC, FISH, RT-PCR, RNA NGS, and DNA NGS) we used to determine ROS1 status in these cases. RESULTS.­: Of the IHC scoring methods examined, staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive had the highest correlation with a FISH-positive status, reaching a positive percentage agreement of 97.8% and negative percentage agreement of 89.5%. A positive percentage agreement (100%) and negative percentage agreement (92.0%) was reached by comparing ROS1 (SP384) using a cutoff for staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells to the consensus status. CONCLUSIONS.­: Herein, we present a standardized staining protocol for ROS1 (SP384) and data that support the high correlation between ROS1 status and ROS1 (SP384) antibody.

Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway.

Formaldehyde is a reactive chemical that is commonly used in the production of industrial, laboratory, household, and cosmetic products. The causal association between formaldehyde exposure and increased incidence of cancer led the International Agency for Research on Cancer to classify formaldehyde as a carcinogen. Formaldehyde-induced DNA-protein crosslinks (DPCs) elicit responses involving nucleotide excision repair (NER) and homologous recombination (HR) repair pathways; however, little is known about the cellular and genetic changes that subsequently lead to formaldehyde-induced genotoxic and cytotoxic effects. Herein, investigations of genes that modulate the cytotoxic effects of formaldehyde exposure revealed that of five NER-deficient Chinese Hamster Ovary (CHO) cell lines tested, XPF- and ERCC1-deficient cells were most sensitive to formaldehyde treatment as compared to wild-type cells. Cell cycle analyses revealed that formaldehyde-treated XPF-deficient cells exhibited an immediate G2/M arrest that was associated with altered cell ploidy and apoptosis. Additionally, an elevated number of DNA double-strand breaks (DSBs), chromosomal breaks and radial formation were also observed in XPF-deficient cells following formaldehyde treatment. Formaldehyde-induced DSBs occurred in a replication-dependent, but an XPF-independent manner. However, delayed DSB repair was observed in the absence of XPF function. Collectively, our findings highlight the role of an XPF-dependent pathway in mitigating the sensitivity to formaldehyde-induced DNA damage as evidenced by the increased genomic instability and reduced cell viability in an XPF-deficient background. In addition, centrosome and microtubule abnormalities, as well as enlarged nuclei, caused by formaldehyde exposure are demonstrated in a repair-proficient cell line.