Wiktor, Anne E.

Purpose In 1998, the US Food and Drug Administration (FDA) approved human epidermal growth factor receptor 2 (HER2) testing guidelines to determine eligibility for HER2-directed therapy (HDT) in breast cancer. ASCO and the College of American Pathologists published immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) HER2 testing guidelines in 2007 (AC2007) and updated these guidelines in 2013 (AC2013). We compared the HER2 FISH amplification frequency using these three guidelines. Methods Patient samples that were sent to the Mayo Clinic cytogenetics laboratory for FISH testing (n = 2,851; from November 2013 to October 2014) were analyzed. Frequency of HER2 FISH amplification was examined and impact of AC2013 assessed. Results IHC results were available for 1,922 patient samples (67.4%), 137 of which were from Mayo Clinic. Distribution was 2.4% IHC 0, 7.9% IHC 1+, 84.8% IHC 2+, and 2.5% IHC 3+. Among IHC 2+ patients, HER2 FISH positivity was 11.8% (FDA), 9.4% (AC2007), and 24.1% (AC2013). Overall, 11.8% (n = 339) were positive with a FISH ratio ≥ 2.0, 1.3% (n = 35) with a FISH ratio ≥ 2.0 despite a HER2 signal < 4.0, and 3.0% (n = 86) with HER2 signal ≥ 6.0 despite FISH ratio < 2.0. Among 405 patients (14.2%) who were initially considered FISH-equivocal (ratio < 2.0 with HER2 signal ≥ 4.0, but < 6.0; AC2013), use of an alternative chromosome 17 probe reassigned 212 (7.4% overall) patients to FISH-positive and 36 (1.3% overall) patients to FISH-negative, whereas 157 (5.5% overall) patients remained equivocal. Final HER2 positivity with AC2013 (23.6%) was increased (P < .001) compared with FDA (13.1%) and AC2007 (11%) guidelines. Conclusion In a reference laboratory cohort that was highly enriched for IHC 2+ patient samples, AC2013 guidelines led to a larger number of FISH-equivocal patients. Approximately one half of these FISH-equivocal patients (7.4% overall) became HER2-positive upon alternative FISH probe testing. However, these patients would not have participated in the pivotal HDT trials. Clinical utility data on HDT benefit in these patients and other special subsets are needed.

Although HER2‐positive breast cancers demonstrate a propensity for central nervous system (CNS) metastasis, it is unknown whether other HER2‐positive tumors, including adenocarcinomas of the esophagus/gastroesophageal junction (EAC), share this characteristic. Insight into this association may inform the development of HER2‐targeted therapies that penetrate the blood‐brain barrier. We examined HER2 overexpression and gene amplification in 708 patients with EAC who underwent curative‐intent surgery during a time period (1980–1997) when no patient received HER2‐targeted therapy. We identified patients whose site of first cancer recurrence was CNS and those who had a CNS relapse at any time. After a median follow‐up of 61.2 months, 3.4% (24/708) of patients developed CNS relapse (all involved the brain). Patients with HER2‐positive (vs ‐negative) primary tumors showed a higher 5‐year cumulative incidence of CNS relapse as first recurrence (5.8% vs. 1.2%; p = 0.0058) and at any time (8.3% vs. 2.4%; p = 0.0062). In a multivariable model that included covariates previously associated with HER2 or with CNS relapse in breast cancer, HER2 positivity was the only variable that was statistically significantly associated with shorter time to CNS relapse as first recurrence (p = 0.0026) or at any time (hazard ratio 4.3 [95% confidence interval 1.8 to 10.3]; p = 0.001). These are the first data in a non‐breast cancer to demonstrate an association between HER2 positivity and higher CNS relapse risk after surgery, and suggest that HER2‐positive EACs have a predilection for CNS metastases.

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 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.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Abstract Pathologic examination of products of conception (POC) is used to differentiate hydropic abortus (HA), partial hydatidiform mole (PM), and complete hydatidiform mole (CM). Histologic classification of POC specimens can be difficult, and ancillary testing is often required for a definitive diagnosis. This study evaluated 66 POC specimens by flow cytometry, digital image analysis, p57 immunohistochemical analysis, and fluorescence in situ hybridization (FISH). The final diagnosis, based on the combined analysis of all test results, included 33 HAs, 24 PMs, and 9 CMs. The p57 immunostain identified 9 CMs that were evaluated as nontriploid by all other techniques. FISH seems to have the best accuracy (100%) for determining whether a specimen contains a triploid chromosome complement. These data suggest that the combination of p57 and FISH seems to be the best ancillary testing strategy to aid pathologists in the appropriate identification of CM, PM, and HA in POC specimens.