Tenner, K.

Abstract Background: While previous study showed that the enrichment of immune-related gene expression was associated with outcome in HER2+ patients receiving sequential or concurrent trastuzumab (H), stromal tumor infiltrating lymphocytes (sTIL) have not been consistently shown to associate with outcome in this group of patients. Given that TIL scoring may be subjective, we analyzed molecular signatures of different subsets of tumor infiltrating immune cell populations, using NanoStringTM gene expression data to assess molecular TIL (mTIL) signature enrichment and intrinsic subtype as a function of relapse-free survival (RFS). Methods: NanoStringTM technology was used to quantify mRNA in samples from 1,280 patients in N9831, 168 patients in FinHer, and 170 patients in FinXX. In N9831, patients in arm A were treated with chemotherapy alone (AC-T), arm B received chemotherapy followed by sequential H (AC-T-H), and arm C received H concurrently with chemotherapy (AC-TH). In the FinHer trial, H was given concurrently for 9 weeks and either 1 year or 9 weeks in FinXX trial. Cox proportional hazard ratio (HR) was used to determine the association of each gene signature with RFS. Different immune subset signatures, including CD45, B-cells, CD8 T-cells, cytotoxic-cells, and T-cells were analyzed using algorithms developed by NanoString. Results: In N9831, CD45, cytotoxic-cell, and T-cell signatures were significantly associated with improved RFS in patients receiving chemotherapy alone and AC-T-H. However, none of the mTIL signatures were significantly associated with outcome in patients receiving AC-TH. Patients lacking CD45 enrichment had better outcome when H was given concurrently with chemotherapy. The 10-year Kaplan-Meier estimates for RFS in arm B patients with CD45 enrichment or no enrichment were 81.3% and 72.6%, respectively (HR 0.63 [95% CI, 0.42-0.93]; p = 0.02), and in arm C were 83.6% and 79.8%, respectively (HR 0.79, 95%CI 0.49-1.28; p = 0.34). Among patients with HER2-enriched subtype, all of the mTIL signatures were associated with improved RFS in arm A (AC-T) and B (AC-T-H) but remained non-significant in arm C (AC-TH). In patients with luminal subtypes, mTIL signatures were not significantly associated with outcome in patients treated with chemotherapy alone. Similar findings were observed in the FinHer and FinXX trials, in which, none of mTIL signatures were significantly associated with outcome among patients who received H. Conclusion: This analysis sheds light on previous discrepancy between immune-related gene signature and sTIL findings. Our data also suggests that the poor prognosis associated with lack of infiltrating immune cells can be partly overcome by the concomitant administration of H with chemotherapy. mTIL signatures, specifically CD45, cytoxic, and T cells, were prognostically associated with improved outcome in patients receiving chemotherapy without concurrent trastuzumab. Understanding the role of the immune system in response to H will require a higher degree of granularity than can be achieved by histological quantification of TILs. Further studies are needed to validate the significance of mTIL signatures as predictive or prognostic biomarker in HER+ patients. Citation Format: Chumsri S, Serie DJ, Mashadi-Hossein A, Tenner KS, Lauttia SL, Moreno-Aspitia A, McLaughlin SA, Nassar A, Warren S, Danaher P, Colon-Otero G, Lindman H, Joensuu H, Perez EA, Thompson EA. Prognostic value of molecular tumor infiltrating lymphocyte (mTIL) signatures in HER2-positive breast cancer patients in N9831 and FinHer/FinXX trials [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-06.

Abstract Importance: 20-25% of patients with early stage HER2-positive breast cancer develop tumor relapse after adjuvant trastuzumab. Identification of such patients is a key goal for clinical management decisions. Objective: To assess molecular heterogeneity among early stage HER2-positive patients using the Prosigna™ algorithm, to define intrinsic subtypes, and to determine the clinical significance of such heterogeneity. Design: The NanoString® platform was used to measure the abundance of the PAM50 subtype signature transcripts. Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna™ algorithm to define intrinsic subtype and risk scores. Subtypes were evaluated for recurrence-free survival following chemotherapy with or without trastuzumab. Setting: Samples were obtained from a multi-center randomized phase III trial of chemotherapy versus chemotherapy plus trastuzumab. Participants: All tumors were centrally evaluated for HER2 positivity, defined as IHC 3+ and/or FISH >2.0; 1392 patients were evaluated for molecular subtype. Intervention(s): Patients received adjuvant chemotherapy (doxorubicin plus cyclophosphamide followed by paclitaxel) (n=484) or chemotherapy plus trastuzumab (n=908). Main Outcome Measure(s): The primary outcome was recurrence-free survival as a function of subtype and treatment. Results: Patients with HER2-positive tumors with HER2-enriched features comprised about 70% of the sample cohort, and these individuals received significant benefit from adjuvant trastuzumab (HR=0.68, 95%CI: 0.52, 0.89, p=0.005), as did the relatively fewer patients (291/1392) with Luminal-type tumors (HR=0.52, 95%CI: 0.32, 0.85, p=0.01). The sample cohort contained a small number of patients with tumors having Basal-like features (97/1392), and the data suggest that these individuals may have received less benefit from trastuzumab, beyond that received from chemotherapy alone (HR=1.06, 95%CI:0.53,2.13, p=0.87). Conclusions: The majority of HER2-positive tumors are classified as HER2-enriched or Luminal using the Prosigna algorithm, and patients with such tumors benefit from adjuvant trastuzumab. About 10% of HER2-positive tumors exhibit Basal-like genomic features, and such tumors appear to recur at fairly similar frequency irrespective of treatment with chemotherapy or chemotherapy plus trastuzumab. Patients with HER2-positive/Basal-like tumors may represent a cohort that should be considered for enrollment in trials to evaluate emerging novel HER2-targeted agents, other targeted therapies, or combinations of both approaches. Support provided in part by CA129949 and CA15083. Citation Format: Perez EA, Ballman KV, Mashadi-Hossein A, Tenner KS, Kachergus JM, Norton N, Necela BM, Carr JM, Ferree S, Perou CM, Cheang MCU, Thompson EA. Intrinsic subtype and therapeutic response among early stage HER2-positive breast tumors from the North Central cancer treatment group (Alliance) N9831 trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-04.

Abstract Background: Prediction of benefit from trastuzumab in patients (pts) with HER2+ breast cancer remains an important goal. We sought to investigate the predictive value of quantitative measurement of HER2, HER3, HER4, EGFR, ER and PTEN protein expression on the benefit of trastuzumab in the phase III HER2+ adjuvant N9831 study for pts randomized to chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C). Methods: For each marker, we evaluated quantitative expression, relationship with demographic data, and association with disease-free survival (DFS) of pts. Freshly cut tissue microarray slides with up to three-fold redundancy per specimen from the N9831 cohort were treated identically using the AQUA (Camp, et al; Nat Med 2002, JCO 2008) method of quantitative immunofluorescence for each marker. HER2 was tested with CB11 (mouse monoclonal, Biocare, Inc.) and preliminary results were available for 698 of nearly 1400 pt specimens to be tested. The minimum value per pt was used in statistical analysis. Specimens were classified with high versus low expression based on a median value cutpoint for each marker. Median follow-up was 7.0 yrs. Results: Quantitative HER2 was compared with centrally performed HER2 testing by IHC and FISH. Median quantitative HER2 via AQUA was 10,017 units for the HER2 IHC 3+ group (n=607) versus 1058, 831, and 970 for the HER2 IHC 2+ (n=68), 1+ (n=11), and 0 (n=11) groups, respectively. The Spearman correlation between quantitative HER2 and FISH HER2/CEP17 ratio was 0.32 (p<0.001). High quantitative HER2 was associated with lower percentage of hormone receptor positivity (48% vs 59%, chi-sq p=0.003) but not associated with age, race, nodal positivity, tumor histology, grade, or size. High HER2 did not impact DFS in any arm of the study (See Table). Data for additional HER2 testing, HER3, HER4, EGFR, ER and PTEN are in process and will be ready by September, 2011. Conclusions: Similar to results based on standard HER2 testing by IHC and FISH in N9831, quantitative HER2 did not impact benefit from adjuvant trastuzumab. Results for additional markers will be presented. Our complete quantitative results for a second epitope on HER2, HER3, HER4, ER and EGFR will be the first report of these markers in a large patient cohort in the adjuvant setting. Disease Free Survival Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-03.

Abstract Abstract #6149 Background: Internal Review Boards and Data and Safety Monitoring Boards often require protocol-specified adverse event (AE) stopping rules for safety monitoring. However, availability of safety data for investigational agent(s) may be limited at the time of protocol development in the phase II setting. This meta-analysis was undertaken to quantify the variability of AE rates in North Central Cancer Treatment Group (NCCTG) phase II metastatic breast cancer (MBC) clinical trials and to investigate whether study factors are associated with AE rates for the development of AE stopping rules in future phase II clinical trials.
 Methods: All closed NCCTG phase II MBC clinical trials using CTC v2.0 or CTCAE v3.0 for AE monitoring were selected. Rates of G3/4 AEs (overall, hematologic [H], and non-hematologic [NH]) and of study discontinuation [SD] due to AEs were calculated for each trial. Associations between study factors [number of agents (single vs combination); line of therapy (first-line only vs other); and type of therapy (chemotherapy vs other)] and AE rates were assessed via Wilcoxon rank sum tests.
 Results: 15 trials met inclusion criteria. 7 used CTCAE v3.0; 6 investigated single agents; 5 investigated first-line therapy; and 11 investigated chemotherapy regimens. 694 pts were evaluable for AE analysis. The G3/4 AE rate across trials was 68% (16-98%) overall, 45% (0-96%) H, and 51% (16-78%) NH. The overall rate of SD due to AEs was 13% (0-40%). The overall rate of G3/4 AEs was significantly lower in single agents vs combination regimens (30% vs 86%, p=0.004). This association held for H (2% vs 66%, p<0.0001) and NH (27% vs 57%, p=0.03) Aes. The rate of SD due to AEs was also significantly lower in single agents vs combination regimens (0.5% vs 15%, p=0.04). The rate of G3/4 AEs was significantly higher in first-line only trials vs other trials (overall: 90% vs 47%, p=0.04; H: 73% vs 5%, p=0.02; NH: 66% vs 41%, p=0.08). The rate of SD due to AEs was also significantly higher in first-line only trials (18% vs 4%, p=0.008). The only significant association with chemotherapy vs other therapy was G3/4 H AE rates (51% vs 2%, p=0.02).
 Discussion: High variability in G3/4 AE rates overall and in H and NH AEs was noted in these 15 trials. There is also high variability in the rates of SD due to AEs. The data suggest that clinical trials with single agent regimens have lower AE rates than combination regimens and, surprisingly, trials of first-line therapy only have higher AE rates than other trials. This may be due to more aggressive therapy being tested in the first-line setting. Although data from previous trials with the investigational agent(s) should be used for developing AE stopping rules when available, this study suggests that study factors such as number of agents and line of therapy can be useful when previous data are limited. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6149.