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We investigated the HE4 variant-specific expression patterns in various normal tissues as well as in normal and malignant endometrial tissues. The relationships between mRNA variants and age, body weight, or survival are analyzed. ICAT-labeled normal and endometrial cancer (EC) tissues were analyzed with multidimensional liquid chromatography followed by tandem mass spectrometry. Levels of HE4 mRNA variants were measured by real-time PCR. Mean mRNA levels were compared among 16 normal endometrial samples, 14 grade 1 and 14 grade 3 endometrioid EC, 15 papillary serous EC, and 14 normal human tissue samples. The relationship between levels of HE4 variants and EC patient characteristics was analyzed with the use of Pearson correlation test. We found that, although all five HE4 mRNA variants are detectable in normal tissue samples, their expression is highly tissue-specific, with epididymis, trachea, breast and endometrium containing the highest levels. HE4-V0, -V1, and -V3 are the most abundant variants in both normal and malignant tissues. All variants are significantly increased in both endometrioid and papillary serous EC, with higher levels observed in grade 3 endometrioid EC. In the EC group, HE4-V1, -V3, and -V4 levels inversely correlate with EC patient survival, whereas HE4-V0 levels positively correlate with age. HE4 variants exhibit tissue-specific expression, suggesting that each variant may exert distinct functions in normal and malignant cells. HE4 levels appear to correlate with EC patient survival in a variant-specific manner. When using HE4 as a biomarker for EC management, the effects of age should be considered.

BACKGROUNDHuman epidermal growth factor receptor 2 (HER2) is a therapeutic target in patients with esophageal adenocarcinoma (EAC), with gene amplification used as a selection criterion for treatment, although to the authors' knowledge the concordance between amplification and HER2 protein expression remains undefined in EAC. Furthermore, the association between HER2 and its interacting partner, human epidermal growth factor receptor 3 (HER3), is unknown yet appears to be of potential therapeutic relevance.METHODSPatients with untreated EACs (N = 673) were analyzed for HER2 amplification and polysomy 17 by fluorescence in situ hybridization in parallel with immunohistochemistry (IHC) (IHC scores of 0‐1+, 2+, and 3+). Amplification was defined as HER2/CEP17 ≥ 2. HER3 expression by IHC was analyzed in randomly selected cases (n = 224). IHC and fluorescence in situ hybridization results were compared using least squares linear regression.RESULTSOverall, 17% of the EACs (116 of 673 EACs) were HER2‐amplified with an amplification frequency that was highest among IHC3+ cases (89%) and declined among IHC2+ cases (13%) and IHC0 to IHC1+ cases (4%). Among HER2‐amplified cases, the level of amplification increased linearly with HER2 membranous expression (HER2/CEP17 ratio: 7.9 in IHC3+ and 5.5 in IHC2+ vs 2.8 in IHC0 to IHC1+ [P < .0001]), with 14% of amplified tumors demonstrating absent/faint expression (IHC0 to IHC1+). Polysomy 17 was not found to be associated with HER2 expression. Cytoplasmic HER3 expression was detected in 87% of tumors (195 of 224 tumors) and was found to be significantly associated with better differentiation (P < .0001). Stepwise increases in HER3 expression were associated with higher HER2 expression levels (P = .0019).CONCLUSIONSLevels of HER2 protein expression and amplification were found to be linearly associated and highly concordant. Among amplified tumors with absent/faint expression, the level of amplification was low. Frequent expression of HER3 suggests its relevance as a therapeutic target, and its significant association with HER2 supports ongoing efforts to inhibit HER2/HER3 in patients with EAC. Cancer 2014;120:415–424. © 2013 American Cancer Society.

Purpose With improved patient care, better diagnosis, and more treatment options after tumor recurrence, outcomes after fluorouracil (FU) -based treatment are expected to have improved over time in early-stage colon cancer. Data from 18,449 patients enrolled onto 21 phase III trials conducted from 1978 to 2002 were evaluated for potential differences in time to recurrence (TTR), time from recurrence to death (TRD), and overall survival (OS) with regard to FU-based adjuvant regimens. Methods Trials were predefined as old versus newer era using initial accrual before or after 1995. Outcomes were compared between patients enrolled onto old- or newer-era trials, stratified by stage. Results Within the first 3 years, recurrence rates were lower in newer- versus old-era trials for patients with stage II disease, with no differences among those with stage III disease. Both TRD and OS were significantly longer in newer-era trials overall and within each stage. The lymph node (LN) ratio (ie, number of positive nodes divided by total nodes harvested) in those with stage III disease declined over time. TTR improved slightly, with larger number of LNs examined in both stages. Conclusion Improved TRD in newer trials supports the premise that more aggressive intervention (oxaliplatin- and irinotecan-based chemotherapy and/or surgery for recurrent disease) improves OS for patients previously treated in the adjuvant setting. Lower recurrence rates with identical treatments in those with stage II disease enrolled onto newer-era trials reflect stage migration over time, calling into question historical data related to the benefit of FU-based adjuvant therapy in such patients.

BACKGROUNDA point mutation (V600E) in the BRAF oncogene is a prognostic biomarker and may predict for nonresponse to anti‐EGFR antibody therapy in patients with colorectal carcinoma. BRAFV600E mutations are frequently detected in tumors with microsatellite instability and indicate a sporadic origin. We used a mutation‐specific antibody to examine mutant BRAFV600E protein expression and its concordance with BRAFV600E mutation data.METHODSPrimary stage III colon carcinomas were analyzed for BRAFV600E mutations in exon 15, and 50 BRAFV600E mutation carriers and 25 wild‐type tumors were selected for analysis of BRAF proteins by immunohistochemistry (IHC). IHC was performed in archival tissue specimens using a pan‐BRAF antibody and a mutation‐specific antibody against BRAFV600E proteins. Staining was scored by 2 pathologists who were blinded to clinical and mutation data.RESULTSUsing a pan‐BRAF antibody, total BRAF protein expression was observed in the tumor cell cytoplasm in 74 of 75 colon carcinomas. A mutation‐specific antibody identified diffuse cytoplasmic staining of mutant BRAFV600E proteins in 49 of 74 cancers. Analysis using a polymerase chain reaction‐based assay revealed that all 49 of these cancers carried BRAFV600E mutations. In contrast, BRAFV600E staining was absent in all 25 tumors that carried wild‐type copies of BRAF.CONCLUSIONSA BRAF mutation‐specific (V600E) antibody detected tumors with BRAFV600E mutations and exhibited complete concordance with a DNA‐based method. These results support the use of IHC as a simplified strategy to screen colorectal cancers for BRAFV600E mutations in clinical practice. Cancer 2013;119:2765–2770. © 2013 American Cancer Society.

AbstractA well‐designed pilot study can advance science by providing essential preliminary data to support or motivate further research, refine study logistics, and demonstrate proof of concept. Often, the outcomes of such studies can be quantified by a success/failure dichotomy. For example, a novel compound may show activation of a neural pathway, or it may not. When an intervention's efficacy is quantified using a dichotomous outcome, probability mass functions can be enumerated to determine the probability that the observed result from a pilot study supports further evaluation of the intervention since there is only a finite, and often small, number of sample configurations possible. The purpose of this research was to determine the probability of an “efficacy signal” for pilot studies using one‐ and two‐sample pilot study designs. Efficacy signal was defined as the probability of observing a more favorable response proportion relative to a historical control (one‐sample setting) or to a concurrent control (two‐sample setting). An enumeration study (exact simulation) was conducted to calculate the efficacy signal probability. One‐sample study designs yielded higher probability of determining an efficacy signal than the two‐sample setting; however, sampling variation must be accounted for. A 68% score confidence interval is recommended for this purpose.

Purpose By using data from North Central Cancer Treatment Group Phase III Trial N0147, a randomized adjuvant trial of patients with stage III colon cancer, we assessed the relationship between smoking and cancer outcomes, disease-free survival (DFS), and time to recurrence (TTR), accounting for heterogeneity by patient and tumor characteristics. Patients and Methods Before random assignment to infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX) or FOLFOX plus cetuximab, 1,968 participants completed a questionnaire on smoking history and other risk factors. Cox models assessed the association between smoking history and the primary trial outcome of DFS (ie, time to recurrence or death), as well as TTR, adjusting for other clinical and patient factors. The median follow-up was 3.5 years among patients who did not experience events. Results Compared with never-smokers, ever smokers experienced significantly shorter DFS (3-year DFS proportion: 70% v 74%; hazard ratio [HR], 1.21; 95% CI, 1.02 to 1.42). This association persisted after multivariate adjustment (HR, 1.23; 95% CI, 1.02 to 1.49). There was significant interaction in this association by BRAF mutation status (P = .03): smoking was associated with shorter DFS in patients with BRAF wild-type (HR, 1.36; 95% CI, 1.11 to 1.66) but not BRAF mutated (HR, 0.80; 95% CI, 0.50 to 1.29) colon cancer. Smoking was more strongly associated with poorer DFS in those with KRAS mutated versus KRAS wild-type colon cancer (HR, 1.50 [95% CI, 1.12 to 2.00] v HR, 1.09 [95% CI, 0.85 to 1.39]), although interaction by KRAS mutation status was not statistically significant (P = .07). Associations were comparable in analyses of TTR. Conclusion Overall, smoking was significantly associated with shorter DFS and TTR in patients with colon cancer. These adverse relationships were most evident in patients with BRAF wild-type or KRAS mutated colon cancer.