Sukov, William R.

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.

Abstract Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2–ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2–ERG fusion–positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression. Cancer Res; 73(11); 3275–84. ©2013 AACR.

Abstract The determination of HER2 amplification is critical to selecting appropriate patients for HER2 targeted therapy in breast cancer. Dual in situ hybridization (DISH), an alternative to fluorescence in situ hybridization (FISH) and immunohistochemistry, is now available. To compare the FISH and DISH methods, we tested 251 samples enriched for common or difficult-to-assess HER2 anomalies. Seven samples failed DISH testing. There was a 64% (156/244) concordance between FISH and DISH by anomaly (κ = 0.58, 95% confidence interval, 0.51-0.65; P &lt; .0001) and an 83% (203/244) concordance by amplification status (κ = 0.58; 95% confidence interval, 0.47-0.69; P &lt; .0001). DISH resulted in lower estimates of HER2/ centromere 17 ratios than FISH, and many cases that were equivocal with FISH were normal with DISH. DISH did not detect any case with coamplification of HER2 and centromere 17. Using a cohort of difficult specimens, we observed less than 95% concordance between FISH and DISH. DISH may underestimate the HER2/chromosome 17 ratio, or FISH may overestimate this ratio.

Purpose There is increasing recognition of the existence of intratumoral heterogeneity of the human epidermal growth factor receptor (HER2), which affects interpretation of HER2 positivity in clinical practice and may have implications for patient prognosis and treatment. We determined the frequency and prognostic impact of heterogeneous HER2 gene amplification and polysomy 17 in patients with esophageal adenocarcinoma (EAC). Patients and Methods HER2 amplification (by fluorescence in situ hybridization) was examined in surgical EAC specimens (n = 675). HER2 heterogeneity was defined according to consensus guidelines as gene amplification (HER2/CEP17 ratio ≥ 2.0) in more than 5% but less than 50% of cancer cells. No patient received neoadjuvant or HER2-targeted therapy. Cox models were used to assess disease-specific survival (DSS) and overall survival (OS). Results Overall, 117 EACs (17%) demonstrated HER2 amplification, of which 20 (17%) showed HER2 heterogeneity. All HER2-heterogeneous tumors were amplified. Among HER2-amplified tumors, heterogeneous tumors had significantly higher frequency of poor histologic grade and polysomy 17. In multivariable models that included number of metastatic lymph nodes, grade, tumor stage, and polysomy 17, only HER2 heterogeneity and node number were prognostic among HER2-amplified tumors, with heterogeneity showing worse DSS (hazard ratio, 2.04; 95% CI, 1.09 to 3.79; P = .025) and OS (P = .026). Among HER2-nonamplified EACs, polysomy 17 was independently associated with worse DSS (P = .012) and OS (P = .023). Conclusion Among HER2-amplified EACs, 17% show HER2 heterogeneity, which independently predicts for worse cancer-specific death. Among HER2-nonamplified EACs, polysomy 17 is independently associated with worse survival. These novel findings demonstrate aggressive subgroups in HER2-amplified and -nonamplified EACs that have important implications for HER2 analysis and determination of benefit from HER2-targeted therapy.

Abstract Purpose: We examined the frequency, tumor characteristics, and prognostic impact of HER2 protein expression and gene amplification in patients with curatively resected esophageal adenocarcinoma (EAC). Experimental Design: HER2 expression was analyzed by immunohistochemistry (IHC) in surgical EAC specimens (n = 713). Gene amplification was examined by FISH in a large subset (n = 344). Most tumors were T3–4 (66%) or node positive (72%); 95% were located in the esophagus or gastroesophageal junction. No patient received neoadjuvant therapy. Cox models were used. Results: Overall, 17% of EACs were HER2 positive (i.e., IHC3+ or IHC2+ with amplification), with strong agreement between HER2 amplification (HER2/CEP17 ratio ≥2) and expression (κ = 0.83). HER2 positivity was significantly associated with lower tumor grade, less invasiveness, fewer malignant nodes, and the presence of adjacent Barrett's esophagus (BE). EACs with BE had higher odds of HER2 positivity than EACs without BE, independent of pathologic features [OR = 1.8 (95% CI: 1.1–2.8), P = 0.014]. Among all cases, HER2 positivity was significantly associated with disease-specific survival (DSS) in a manner that differed by the presence or absence of BE (Pinteraction = 0.0047). In EACs with BE, HER2 positivity was significantly associated with improved DSS [HR = 0.54 (95% CI: 0.35–0.84), P = 0.0065] and overall survival (P = 0.0022) independent of pathologic features, but was not prognostic among EACs without BE. Conclusions: HER2 positivity was shown in 17% of resected EACs and associated with reduced tumor aggressiveness. EACs with BE had nearly twice the odds of being HER2 positive and, within this subgroup, HER2 positivity was independently associated with improved survival. Clin Cancer Res; 18(2); 546–54. ©2012 AACR.