Chumsri, Saranya

Abstract Background: Blood-based biomarkers (PSA, CEA, CA125) are used to track real-time progression of disease in parallel with imaging. However, while numerous blood biomarkers exist, they are specific to cancer type (i.e., PSA to prostate and CEA to colon) and may not appear in all diseased individuals. Recently, cancer-associated macrophage-like cells (CAMLs), a circulating stromal cell subtype, were identified in various solid cancer types, which were observed increasing in size and in hyperploidy during progressive disease. To assess whether CAML enlargement is a biomarker of progression/response, we tracked CAML growth/shrinkage in a pilot study of patients (n=34). Blood was drawn from patients with lung, prostate, or breast cancers over a 3-month period, baseline through 2 treatment cycles, followed by continued monitoring for 2 years. These data suggest that morphologic assessment of CAMLs (growth/hyperploidy) appear to parallel cancer progression, or response to treatment, in multiple solid tumors. Methods: A prospective multi-institutional study used anonymized peripheral blood samples from 34 cancer patients undergoing therapy [stage I (n=2), II (n=3), III (n=8) & IV (n=21)] with breast (n=10), lung (n=16), & prostate (n=8). Samples were taken prior to therapy (BL), at 1 month (FU1) follow-up and a 3-month (FU2) follow-up, after induction of therapy. Blood was processed by the CellSieve™ microfiltration technique at 4 institutions and stained for cytokeratin 8, 18 and 19, CD14 and CD45. After identification and quantification, CAMLs were measured based on their hyperploidy and cell size. Results: CAMLs were found in 97% of cancer patients at BL, 97% at FU1 and 91% of FU2. Over the 2-year follow-up, 7 patients showed no signs of clinical disease progression, while 27 patients had observable progression. Of the 7 patients who did not progress, only two had CAMLs of ≥50µm at BL and at FU1, but whose CAMLs shrunk to <50 µm by FU2. Of the 27 patients who progressed, 22 patients had ≥50µm CAMLs at all time points, while 5 patients had small <50µm CAMLs at BL. Interestingly, CAMLs in these 5 patients had enlarged to ≥50µm by the FU2 time point. Conclusions: We show that increased polynucleation and CAML enlargement indicate shorter progression-free survival in a number of cancer types after baseline. By monitoring CAML changes over time for the 34 individual patients, we demonstrated correlation of ongoing progression, or response, in tumors to the enlargement or shrinkage in CAMLs at follow-up time points from treatment induction. This pilot study suggests that CAMLs have the potential to monitor the progression/regression of malignancy in real time and suggests the need for larger validation studies. Citation Format: Daniel L. Adams, Raymond Bergan, Martin J. Edelman, Stuart S. Martin, Rena Lapidus, Saranya Chumsri, Cha-Mei Tang, Steven H. Lin. Real-time monitoring of solid tumor progression by circulating stromal cells from early- to late-stage disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1560.

Abstract Background: We previously demonstrated that cancer associated macrophage-like cells (CAMLs) are cancer specific giant polyploid cells circulating in the blood of patients with solid tumors. Building on our initial discovery, others have shown that these hyperploidy cells are an innate immune response that is associated with decreased survival. However to date, no studies have been done to elucidate their clinical significance as in relation to the various stages of malignant disease. We established a 4 year prospective study of 315 patients from a variety of solid tumors (breast, prostate, lung, renal cell, pancreas, and esophageal) comparing the morphological properties of CAMLs in both early and late stage disease as they relate to progression free and overall survival (PFS/OS). These data suggest that CAML size appears have a strong negative correlation with progression and survival of cancer patients, irregardless of stage of disease, indicating their possible use as a non-invasive blood based biomarker in solid tumors. Methods: A prospective multi-institutional study used anonymized peripheral blood from 315 cancer patients [stage I (n=62), stage II (n=72), stage III (n=69) & stage IV (n=106)] from subjects with breast (n=59), esophageal (n=27), lung (n=59), renal cell carcinoma (n=37), prostate (n=74), pancreas cancers (n=59). CAMLs were isolated by the CellSieve™ microfiltration technique at 5 institutions and stained for cytokeratin 8, 18, & 19, CD14 and CD45. After imaging, a size based threshold ≥50µm was used to separate the patient cohorts, based on previously published assessments. Results: At least one CAML was found in 92% of cancer patients (n=289/315), with the lowest sensitivity in stage 1 (86%), followed by stage 2 (90%), stage 3 (97%) and stage 4 (95%). The property of CAML size was then evaluated. Overall, CAMLs of <50µm in size had superior PFS (HR: 3.8; 95% CI 2.8-5.3; p<0.001) and OS (HR=3.8; 95% CI 2.6-5.7;p<0.001) than those with ≥50 µm CAMLs. By each stage individually, stage 1 (PFS HR=7.5; 95% CI 3.2-17.7; p<0.001), stage II (PFS; HR=4.1; 95% CI 1.9-8.8; p<0.001), stage III (PFS HR=3.1; 95% CI 1.7-5.7; p=0.007), and stage IV (PFS HR=2.7; 95% CI 1.6-4.4; p<0.001). Conclusions: In this first large scale prospective study of the clinical utility of CAMLs, it appears that they are present in every stages of invasive solid tumor malignancies, with prevalence increasing with stage. Interestingly decreased CAML size was found to be associated with improved outcome and longer PFS. These data suggest that CAML detection and size assessment constitute a new real time predictor of progression, and survival in both early and late stage disease indicating the need for additional validation studies. Citation Format: Daniel L. Adams, Steven H. Lin, R. Katherine Alpaugh, Thai Ho, Jeffrey R. Marks, Stuart S. Martin, Susan Tsai, Saranya Chumsri, Cha-Mei Tang, Massimo Cristofanilli, Raymond Bergan. Cancer associated macrophage-like (CAMLs) cells in blood predict progression and survival for all stages of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2631.

Abstract Purpose: p95HER2 is a truncated form of HER2 that confers resistance to trastuzumab in vitro, but clinical results have been conflicting to date. Given that p95HER2 levels correlate with total HER2 expression levels, which confer better outcomes, we sought to evaluate the p95HER2/HER2 ratio in the North Central Cancer Treatment Group N0337 and N98-32-52 trials. Experimental Design: The HERmark assay and VeraTag technology (Monogram Biosciences) were used to measure total HER2 and p95HER2 expression levels in 91 patient samples. Results: In the multivariate model, increasing total HER2 level was significantly associated with longer (OS; HR, 0.33; P = 0.002) and decreasing p95HER2 level was significantly associated with longer OS (HR, 4.2; P = 0.01). Total HER2 expression level was significantly associated with longer progression-free survival (PFS) (HR, 0.57; P = 0.04), whereas p95HER2 level was not (HR, 1.7; P = 0.25). However, there was a positive association between p95HER2 and total HER2 expression levels (R2 = 0.48; P < 0.001). Consistent with our hypothesis, the ratio of p95HER2/HER2 was significantly associated with worsening PFS (HR, 1.7; P = 0.04) and OS (HR, 2.8; P = 0.002). Patients with the highest tertile of p95HER2/HER2 values had significantly less favorable PFS (HR, 1.8; P = 0.06) and OS (HR, 2.3; P = 0.02). Conclusions: A high p95HER2/HER2 ratio identified patients with metastatic breast cancer with poor outcomes on trastuzumab-based therapies. Further investigation of the p95HER2/HER2 ratio as a potential prognostic or predictive biomarker for HER2-targeted therapy is warranted. Clin Cancer Res; 24(13); 3053–8. ©2018 AACR.

TPS17 Background: Emerging studies suggest that breast cancer, particularly triple negative breast cancer (TNBC), may be sensitive to immunotherapy. However, the response rate of single agent immune checkpoint blockade agent in TNBC is rather low. Previous genomic study in residual tumor after neoadjuvant chemotherapy showed inverse correlations between MEK activation signature and the amount of tumor infiltrating lymphocytes (TILs) in residual disease samples as well as poor outcome. Preclinical study also showed that the combination of MEK inhibitor and anti-PD-L1 antibody in mouse model can eradicate TNBC tumors. Methods: This is a single arm, Phase I/II trial of Pembrolizumab (P) in combination with Binimetinib (B) in patients with unresectable locally advanced or metastatic TNBC. This trial is currently opened for accrual at Mayo Clinic in Florida and Minnesota. Patients with TNBC defined as ER ≤ 10% and PR ≤ 10% who received ≤ 3 prior lines with measurable disease will be enrolled. The primary objective of the Phase I part is to determine the maximum tolerated dose of B in combination with P and for the Phase II part is objective response rate (ORR) by RECIST criteria. The secondary endpoints include ORR by irRECIST, progression free survival, and overall survival. The total sample size is 15-38 patients with 6-12 patients in Phase I with 2 dose levels and 9-26 patients in Phase II. Simon’s Two-Stage Optimal Design is used to test the null hypothesis that this two-drug combination has an ORR of at most 15% vs. the alternative hypothesis that it has an ORR of at least 35%. Patients will receive single agent B for 2 weeks prior to starting P. A mandatory biopsy will be performed before starting B and an optional biopsy will be performed after 2 weeks of B. Tumor tissue will be evaluated for the amount and phenotypes of TILs, PD-L1 expression, and gene expression analysis using PanCancer Immune Profiling Panel, and PDJ amplification. Peripheral blood will be evaluated for circulating immunoregulatory cells, cytokine profiling, circulating tumor cells (CTCs), as well as p-ERK and PD-L1 expression on CTCs. Clinical trial information: NCT03106415.

Abstract Background: Circulating cancer associated macrophage-like cells (CAMLs) are cancer specific giant cells circulating in the blood of patients with solid tumors. Since their discovery, few studies have been done to elucidate their lineage or phenotypic identity. The difficulty in classifying CAMLs is exemplified by their expression of multiple heterogeneous markers that defy conventional identification. Recently, we described a restaining method (QUAS-R) to screen individual cells using an array of up to 15 biomarkers. We used this method to screen CAMLs isolated from 152 cancer patient samples in 4 types of solid tumors to classify CAMLs by phenotypic immunostaining. These data suggest that CAMLs are a morphologically diverse and phenotypically heterogeneous population of cancer specific giant cells with overlapping myeloid, epithelial, and endothelial phenotypes. Methods: This multi-institutional study used peripheral blood samples from 152 cancer patients (stage I-IV) from breast (n=42), lung (n=39), renal cell carcinoma (36) and prostate (n=35). Blood was processed by the CellSieve™ microfiltration technique at 5 institutions and stained for cytokeratin 8, 18 & 19, EpCAM, and CD45. After identification/imaging, the QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used to quench fluorescence signal of cells and then restain with vimentin/CD146/CD144, CD14/CD11b/CD41, CD11c/CD68/TIE2, or CD34/CD41/CD61. After staining, QUAS-R was again used to remove the fluorescence and samples restained with a third panel. Each patient sample was stained, quenched and restained with the above mentioned panels. Results: In agreement with a number of studies, CAMLs were found in 86% of cancer patients (n=131/152), with increased detection from stage 1 (71%), followed by stage 2 (94%), stage 3 (88%) to stage 4 (88%). Breast cancer had the most CAMLs per sample (14.1 cells/7.5mL), followed by prostate (6.8), renal cell carcinoma (4.9) and lung (3.2). CD34 was most prevalent, found on 88% of CAMLs, followed by cytokeratin (81%), CD41 (79%), CD61 (78%), CD45 (75%), CD14 (72%), vimentin (63%), EpCAM (56%), CD146 (53%), CD68 (44%), CD11c (38%), TIE-2 (25%) and CD11b (0%). Based on our results, CAMLs seem to express overlapping phenotypes from a variety of lineages i.e. macrophage (CD14/CD68/CD11c), epithelial (cytokeratin/EpCAM), endothelial (CD146/TIE-2) and megakaryocyte (CD41/CD61). Conclusions: Although identification of CAMLs is straightforward by morphological criteria (size and nuclear profile), their identification and lineage remains undetermined. We report the first mass screening of CAMLs to determine phenotypic expression. These data suggest that CAMLs cannot be grouped into any known cell subtype based on their expression profiles and represent a heterogeneous and variably differentiated population whose biological consequences in cancer remain under investigation. Citation Format: Daniel L. Adams, R Katherine Alpaugh, Thai H. Ho, Steven H. Lin, Jeffrey R. Marks, Raymond Bergan, Stuart S. Martin, Saranya Chumsri, Cha-Mei Tang, Massimo Cristofanilli. Multiplex phenotyping of circulating cancer associated macrophage-like cells in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3798. doi:10.1158/1538-7445.AM2017-3798

Abstract Estrogen receptor-positive (HR+) breast cancer is the most common form of breast cancer that is accountable for the majority of breast cancer mortality. Currently, the cyclin dependent kinase inhibitor palbociclib in combination with endocrine therapy represents the new standard first and second line therapy for patients with metastatic HR+ breast cancer. While palbociclib has been shown to significantly improved progression free survival in combination with aromatase inhibitor (AI) and fulvestrant, resistance will inevitably occur in patients with metastatic breast cancer. However, the mechanisms of resistance to the combination of palbociclib and AI remain largely unknown. It is also unclear whether resistance mechanisms would be the same if palbociclib was given as first line treatment in combination with letrozole vs. if palbociclib was given as second line treatment after letrozole resistance had already occurred. To address this, previously well established and well characterizied letrozole-sensitive MCF7Ca cells and letrozole-resistant LTLTCa cells were subjected to continuous, long term treatment with increasing doses of letrozole and palbociclib until resistance to both drugs was achieved (MCF7Calet+palb and LTLTCapalb, respectively). Preliminary MTT cell viability assays indicate palbociclib IC50s of 750 nM in MCF7Ca and LTLTCa cells vs. 21 uM in palbociclib and letrozole-resistant cells. First line and second line palbociclib resistance correlated with changes in morphology, protein expression, and cancer stem cell characteristics. Under phase contrast microscopy, first line and second line palbociclib-resistant MCF7Ca and LTLTCa cells were larger in size, more irregular in shape, and tended not to grow in epithelial cell-like groups compared to palbociclib-sensitive MCF7Ca and letrozole cells, with second-line palbociclib resistant cells exhibiting these characteristics the most. Western blot showed that ER protein expression in ER+/HER2- MCF7Ca cells decreased with first line palbociclib and letrozole resistance, and that both ER and HER2 protein expression were decreased with second line palbociclib resistance in ERlow/HER2+ LTLTCa cells. Lastly, mammosphere assays demonstrated increasing percentage of cancer stem cells with letrozole resistance alone (4 per 1000 cells plated MCF7Ca vs. 58 per 1000 planted LTLTCa) and with palbociclib and palbociclib resistance (317 per 1000 cells plated MCF7Ca and 202 per 1000 cells plated LTLTCa cells). Overall, these results indicate that 1) resistance to palbociclib, whether as first line or second line treatment has significant effects on breast cancer cells that may be relevant to patient diagnosis and treatment. Citation Format: Armina A. Kazi, Antony Sare, Saranya Chumsri, Angela Brodie. Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2053. doi:10.1158/1538-7445.AM2017-2053

Abstract Background: Trastuzumab (H), a monoclonal antibody against HER2, has revolutionized the treatment for HER2-positive breast cancer. Besides inhibiting downstream signaling of HER2, several studies showed that H also exerts its anti-tumor activity via immune-related mechanisms. While H is quite effective in preventing recurrence, significant numbers of patients still develop recurrence despite adjuvant H-based chemotherapy. In this study, we analyzed immune-related genes that were associated with poor outcome in N9831. Methods: NanoStringTM technology was used to quantify mRNA of immune-related genes in baseline samples from 1,280 patients in N9831. 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). Cox proportional hazard ratio (HR) was used to determine the association of each immune-related gene with recurrence free survival (RFS). Different immune subset signatures, including CD45, CD8, cytotoxic-cells, and T-cells were analyzed using algorithms developed by NanoString. Results: With the median follow up of 10.6 years, we identified a total 77 genes that were associated with improved outcome in arm C. Among these 77 genes, there were 20 tumor necrosis factor (TNF)-related genes. Of those, only OX40 (TNFRSF4) and its ligand TNFSF4 have interaction p < 0.10. Interestingly, we found uneven distribution of OX40 expression in the N9831 specimens. Approximately, 9.5% of HER2-positive breast cancer expressed OX40 at distinctly low level. Low expression of OX40 was significantly associated with HR positivity (OX40 low 61% vs. OX40 high 51%, p 0.003) and larger tumor size but not patients’ age, tumor grade, and lymph node status. Low expression of OX40 was significantly associated with low mTIL-CD45, CD8, cytotoxic-cells, and T-cells immune signature scores. Tumors with low OX40 expression had significantly lower levels of CTLA4 (p 9.11e-71) and PD-L1 expressions (p 2.49e-92). Low expression of OX40 is associated with poorer outcome among patients treated with H in both sequential (HR 0.88, 95% CI 0.79-0.98, p 0.022) and concurrent arms (HR 0.86, 95%CI 0.76-0.98, p 0.027) but not in chemotherapy only arm (HR 0.9, 95%CI 0.90-1.09, p 0.8). Similar findings were observed with its ligand TNFSF4 in arm A (HR 1.02, 95%CI 0.92-1.15, p 0.68), arm B (HR 0.87, 95%CI 0.76-0.99, p 0.04), and arm C (HR 0.81, 95%CI 0.68-0.95, p 0.01). Conclusion: Our study suggests that pre-existing expression of OX40 and its ligand TNFSF4 are prognostic and may also be predictive of adjuvant H benefit. Patients with distinctly low level of OX40 had poor outcome despite adjuvant H-based adjuvant chemotherapy. Our study provides a rationale to further evaluate the strategy to increase immune activation to improve outcome in this group of patients. Citation Format: Saranya Chumsri, Daniel J. Serie, Afshin Mashadi-Hossein, Sarah Warren, Alvaro Moreno-Aspitia, Geraldo Colon-Otero, Keith L. Knutson, Edith A. Perez, E. Aubrey Thompson. Correlation of immune co-stimulatory molecule OX40 and outcome in trastuzumab treated HER2-positive breast cancer patients in the NCCTG-N9831 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2017-1682

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 Introduction: A and T are associated with the development of cardiac dysfunction. The kinetics of cardiac biomarkers early after A or T treatment (Rx) is undefined. Methods: We studied hs-TnT (detection limit: 3 ng/ml, myocardial infarction threshold 14 ng/ml) and nt-proBNP (detection limit: 5 pg/ml) levels just prior to (baseline), and on days +1, +2, +3 and +7 during the first and second cycle in breast cancer pts treated with A (n = 11) or T (n = 11). Baseline and peak values for each cycle, baseline to baseline, peak to peak, time to peak, and baseline corrected area under the curve (AUC) were calculated. Study No. NCT01771549 Results: Group (grp) A pts were younger, 51 ± 18 years, than T grp pts, 60 ± 13 years. Three pts in A grp and 4 pts in T grp used antihypertensives. Baseline ejection fraction (EF) was 52-68% (A grp) and 56-69% (T grp). Hs-TnT was undetectable in 5/11 A grp, and 2/11 T grp pts at baseline. By cycle 2 day +1, all A pts had a detectable level of hs-TnT (p = 0.03) compared to 7/11 T pts. Median time to peak level for hs-TnT was 1.5 days for the A grp and 1.2 days for the T grp (NS). In the A grp, significant transient increase in nt-proBNP were also seen (Table). In the T grp, a significant rise in nt-proBNP was not seen in cycle 1 but was seen on cycle 2 day +1 and cycle 2 peak. AUC values were positive for hs-TnT in A pts but not in T pts, and were positive for nt-proBTNP for both A and T. Only 1 pt in A grp had symptomatic EF decline (47%) three months from start of A. None of T grp pts had EF decline. Conclusion: A, but not T was associated with increase in pre-Rx (cycle 2 only), peak, and AUC hs-TnT levels for cycles 1 and 2 with peak values occurring 1.5 days post-Rx. This study evaluating serial kinetics of cardiac biomarker in pts receiving A or T therapy, suggests that assessment of pre- and day +2 post-Rx values could be a means of quantifying cumulative myocardial injury over the chemotherapy course. *Roche Diagnostics Corp, Indianapolis, IN provided assay reagent kits. hs-TnT, ng/mlA, cycle 1A, cycle 2P-valT, cycle 1T, cycle 2P-valBaseline4.69.3<0.0027.75.0NSPeak9.6**16.1†<0.0028.06.8NSTime to peak, days1.61.5NS1.11.3NSAUC above baseline7.1±13.427±19<0.001-1.4±7.2-5±17NSnt-proBNP, pg/mlA, cycle 1A, cycle 2P-valT, cycle 1T, cycle 2P-valBaseline9490NS12480NSPeak361*359*NS209206†NSTime to peak, days1.81.9NS2.21.8NSAUC above baseline423499NS78**-32**<0.5*p<0.001,† p ≤0.01††p = 0.002NS = statistically non-significant (p>0.05)**p<0.03 Citation Format: Pooja Advani, Jonathan Hoyne, Alvaro Moreno-Aspitia, Marcia Dubin, Shelly Brock, Caroline Harlow, Tammy Wollett, Saranya Chumsri, Joseph Blackshear. A prospective pilot study of kinetics of high sensitivity troponin T (hs-TnT) and amino terminal (nt-proBNP) in breast cancer patients (pts) treated with doxorubicin (A) or trastuzumab (T). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3930.

BACKGROUND Activating, nonamplification ERBB2 mutations ( ERBB2 mut) are not detected by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), but are detected by DNA sequencing and may predict clinical responses to human epidermal growth factor receptor (HER2)‐targeted therapy. The authors queried 5605 advanced/metastatic breast cancers (mBC) to uncover the frequency of ERBB2 mut genomic alterations. Clinical responses to anti‐HER2 therapeutics were identified. METHODS DNA was extracted from 40 µm of formalin‐fixed paraffin‐embedded (FFPE) sections. Comprehensive genomic profiling (CGP) was used to evaluate up to 315 genes (592× mean coverage depth). Results were analyzed for base substitutions, short indels, copy number changes, and selected rearrangements. RESULTS Of 5605 cases, 698 (12.5%) featured ERBB2 alterations, including 596 (10.6%) ERBB2 amplifications ( ERBB2 amp) and 138 (2.4%) ERBB2 mut; 38 cases (0.7%) had co‐occurring ERBB2amp and ERBB2mut. ERBB2mut predominantly affected the kinase (124 cases; 90%) or extracellular (15 cases; 11%) domains. Both primary BC (52 cases; 38%) and metastatic site biopsies (86 cases; 62%) were found to harbor ERBB2 mut, which were distributed across carcinoma not otherwise specified (NOS) (69 cases; 50%), invasive ductal carcinoma (IDC) (40 cases; 29%), invasive lobular carcinoma (ILC) (27 cases; 20%), and mucinous mBC (2 cases; 1%). Genes commonly coaltered with ERBB2 were tumor protein 53 ( TP53 ) (49%); phosphatidylinositol 3‐kinase catalytic subunit alpha ( PIK3CA ) (42%); cadherin 1, type 1 ( CDH1 ) (37%); MYC (17%); and cyclin D1 protein ( CCND1 ) (16%). CDH1 mutations were enriched in ERBB2 mut mBC ( P <0.0006) and associated with recurrent mBC. Selected patients with ERBB2 mut, without ERBB2 amp, who responded to anti‐HER2 targeted therapies are presented herein. CONCLUSIONS Within this large series, 1.8% of cases harbored ERBB2 mut, which are undetectable by standard‐of‐care IHC or FISH tests. Metastatic BC driven by ERBB2 mut respond to anti‐HER2 targeted therapies, and expanding clinical trials designed to detect ERBB2 mut by CGP and optimize targeted treatments are warranted. Cancer 2016 . © 2016 American Cancer Society . Cancer 2016;122:2654–2662. © 2016 American Cancer Society.