In breast cancer, overexpression of ErbB2 or aberrant regulation of survivin, a member of the inhibitor of apoptosis family, is associated with resistance to chemo/hormone therapy and predicts for a poor clinical outcome. A functional link between the two predictive factors has not been previously shown. Here, using genetic and pharmacologic approaches to block ErbB2 signaling, we show that ErbB2 regulates survivin protein expression in ErbB2-overexpressing breast cancer cells. Selective knockdown of ErbB2 using small interfering RNA markedly reduced survivin protein, resulting in apoptosis of ErbB2-overexpressing breast cancer cell lines such as BT474. Alternatively, inhibition of ErbB2 signaling using lapatinib (GW572016), a reversible small-molecule inhibitor of ErbB1/ErbB2 tyrosine kinases, at pharmacologically relevant concentrations, leads to marked inhibition of survivin protein with subsequent apoptosis. The effect of lapatinib on survivin seems to be predominantly posttranslational, mediated by ubiquitin-proteosome degradation as lactacystin, a proteosome inhibitor, reverses these effects. Furthermore, lapatinib down-regulated the expression of Histagged survivin, which was under the transcriptional control of a heterologous promoter, providing additional evidence supporting a posttranslational mechanism of regulation. In contrast, trastuzumab and gefitinib failed to down-regulate survivin in ErbB2-overexpressing breast cancer cells. Importantly, the clinical relevance of these findings was illustrated in patients with ErbB2-overexpressing breast cancer whose clinical response to lapatinib was associated with marked inhibition of survivin in their tumors. These findings shed new light on the mechanism by which ErbB2 overexpression protects against apoptotic stimuli in breast cancer and identifies therapeutic interventions to improve clinical outcomes in these aggressive tumors. (Cancer Res 2006; 66(3): 1640-7)

Context.-Recurrent epidermal growth factor receptor (EGFR) mutations are seen in a subset of pulmonary adenocarcinomas. These mutations are targeted by EGFR inhibitors and are a biomarker for response to EGFR inhibitor therapies. Initial data have indicated an increased frequency of activating EGFR mutations in nonsmoking Asian females. However, there are very few studies of global scope that address the question of mutation distribution across the population of lung cancer.Objective.-To determine the frequency of EGFR mutations in exons 18 through 21 detected in clinical laboratories participating in the College of American Pathologists proficiency testing program for EGFR in calendar year 2013.Design.-We reviewed the surveys from 170 clinical laboratories from 20 countries that participated in the College of American Pathologists EGFR proficiency testing program. The proficiency testing includes questions regarding the total numbers of tests performed at each common mutation site, including both activating and resistance mutations, and their frequency. Countries were grouped into regional groups in order to assess frequency of mutation by type, and to indirectly assess ethnic differences in mutation frequencies.Results.-Among the treatment-sensitive activating mutations, the most common are exon 19 mutations (n ¼ 10 802 of 136 533 cases; 7.9% of total cases tested) and the exon 21 L858R mutation (n ¼ 10 351 of 136 533 cases; 7.6% of total cases tested) and the least common are exon 20 mutations (n ¼ 466 of 136 533 cases; 0.3% of total cases tested). The T790M mutation in exon 20 is the more common resistance mutation (n ¼ 1010 of 136 533 cases; 0.7% of all cases tested). The highest activating mutation frequency is seen in southern Asia (n ¼ 4260 of 9337 cases; 46%) and the lowest activating mutation frequencies are in South and North America (n ¼ 113 of 1439 cases and 7926 of 86 654 cases; 8% and 9%, respectively).Conclusions.-Our data confirm that activating EGFR mutations are more common in southern Asia and that the distribution of activating EGFR mutations varies significantly across the regions. Similarly, the frequency and distribution of resistance mutations also show significant variation when comparing southern Asia with other regions.

Background Diff-Quik stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. We describe the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and report DNA quality and success rates of FNA smears compared with other specimens from one year of clinical use. Methods A validation set of 10 slides from 9 patients with prior clinical EGFR Sanger sequencing and KRAS pyrosequencing (5 KRAS +/EGFR−, 4 KRAS/EGFR−) underwent DNA extraction, quality assessment, and targeted NGS. Subseqently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in one calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. Results All 10 validation samples met the DNA quality threshold (ΔCT threshold <8, range −2.2 to 4.9) and yielded 0.5 to 22 μg of DNA. KRAS and EGFR mutation status from FNA smears by NGS were concordant with previous clinical testing for all 10 samples. In the one year review, FNA smears were 100% successful, suggesting performance equivalent to or better than established specimen types, including FNA cell blocks. DNA quality by ΔCT was significantly better from FNA smears than from biopsies, resections, and FNA cell blocks. Conclusions We conclude that FNA smears of lung adenocarcinomas are a high quality alternative specimen for a targeted NGS panel with a high success rate in clinical practice.

BACKGROUND BRAF mutation status, and therefore eligibility for BRAF inhibitors, is currently determined by sequencing methods. We assessed the validity of VE1, a monoclonal antibody against the BRAF V600E mutant protein, in the detection of mutant BRAF V600E melanomas as classified by DNA pyrosequencing. METHODS The cases were 76 metastatic melanoma patients with only one known primary melanoma who had had BRAF codon 600 pyrosequencing of either their primary (n=19), metastatic (n=57) melanoma, or both (n=17). All melanomas (n=93) were immunostained with the BRAF VE1 antibody using a red detection system. The staining intensity of these specimens was scored from 0 – 3+ by a dermatopathologist. Scores of 0 and 1+ were considered as negative staining while scores of 2+ and 3+ were considered positive. RESULTS The VE1 antibody demonstrated a sensitivity of 85% and a specificity of 100% as compared to DNA pyrosequencing results. There was 100% concordance between VE1 immunostaining of primary and metastatic melanomas from the same patient. V600K, V600Q, and V600R BRAF melanomas did not positively stain with VE1. CONCLUSIONS This hospital-based study finds high sensitivity and specificity for the BRAF VE1 immunostain in comparison to pyrosequencing in detection of BRAF V600E in melanomas.

Objective MicroRNA expression in formalin fixed paraffin embedded tissue (FFPE) or plasma may add value for cancer management. Methods The GastroGenus miR Panel was developed to measure 55 cancer-specific human microRNAs, Epstein-Barr virus (EBV)-encoded microRNAs, and controls. This Q-rtPCR panel was applied to 100 FFPEs enriched for adenocarcinoma or adjacent non-malignant mucosa, and to plasma of 31 patients. Results In FFPE, microRNAs upregulated in malignant versus adjacent benign gastric mucosa were hsa-miR-21, -155, -196a, -196b, -185, and -let-7i. Hsa-miR-18a, 34a, 187, -200a, -423-3p, -484 and -744 were downregulated. Plasma of cancer versus non-cancer controls had upregulated hsa-miR-23a, -103 and -221 and downregulated hsa-miR-378, -346, -486-5p, -200b, -196a, -141 and -484. EBV infected versus uninfected cancers expressed multiple EBV-encoded microRNAs, and concomitant dysregulation of four human microRNAs suggests that viral infection may alter cellular biochemical pathways. Conclusion Human microRNAs were dysregulated between malignant and benign gastric mucosa and between plasma of cancer patients and non-cancer controls. Strong association of EBV microRNA expression with known EBV status underscores the ability of microRNA technology to reflect disease biology. Expression of viral microRNAs in concert with unique human microRNAs provides novel insights into viral oncogenesis and reinforces the potential for microRNA profiles to aid in classifying gastric cancer subtypes. Pilot studies of plasma suggest the potential for a non-invasive addition to cancer diagnostics.