Neoadjuvant anti-HER2 Therapy Research Articles

Abstract PD5-04: Tumor-infiltrating lymphocytes evaluation using machine learning image analysis based on core needle biopsy in HER2-positive breast cancer

Abstract Purpose: To evaluate tumor-infiltrating lymphocytes (TILs) quantitatively in breast cancer and to verify the utility of machine learning image analysis in TIL assessment. Methods: Stromal TILs in 105 Human epidermal growth factor receptor 2 (HER2)-positive invasive breast cancers, diagnosed from patients treated with neoadjuvant anti-HER2 therapy, were evaluated by image analysis of hematoxylin and eosin-stained slides from core needle biopsies. TIL level was determined as the number of TILs per square millimeter of stromal tissue. Associations of TIL level with clinicopathological parameters, pathological response, and clinical outcomes were assessed. Results: Median TIL level were 1287/mm2 (range, 123 - 8101/mm2). Higher TIL level was associated with higher histological grade (P = 0.02), estrogen receptor (ER) negativity (P = 0.036), and pathological complete response (pCR) (P< 0.0001). According to analysis of the receiver operating characteristic curve, a threshold TIL level of 2420/mm2 stroma best discriminated pCR from non-pCR. In multivariate analysis, high TIL levels (>2420/mm2) were significantly associated with pCR (P< 0.0001). On Kaplan-Meier analysis, prognosis was not associated with high or low TIL levels. However, high TIL levels tended to be associated with better prognosis in the HER2+/ER+ subgroup (P = 0.131), but not in the HER2+/ER- subgroup. Conclusions: A machine learning image analysis algorithm could assess TIL level as the number of TILs per square millimeter, contrary to semiquantitative evaluation. TIL level derived from this method could offer an independent predictor of pCR. Citation Format: Norie Abe, Hirofumi Matsumoto, Reika Takamatsu, Kentaro Tamaki, Naoko Takigami, Kanou Uehara, Yoshihiko Kamada, Nobumitsu Tamaki, Tokiwa Motonari, Norihiro Nakada, Hisamitsu Zaha, Naoki Yoshimi. Tumor-infiltrating lymphocytes evaluation using machine learning image analysis based on core needle biopsy in HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-04.

Quantitative digital image analysis of tumor-infiltrating lymphocytes in HER2-positive breast cancer.

As visual quantification of the density of tumor-infiltrating lymphocytes (TILs) lacks in precision, digital image analysis (DIA) approach has been applied in order to improve. In several studies, TIL density has been examined on hematoxylin and eosin (HE)-stained sections using DIA. The aim of the present study was to quantify TIL density on HE sections of core needle biopsies using DIA and investigate its association with clinicopathological parameters and pathological response to neoadjuvant chemotherapy in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The study cohort comprised of patients with HER2-positive breast cancer, all treated with neoadjuvant anti-HER2 therapy. DIA software applying machine learning-based classification of epithelial and stromal elements was used to count TILs. TIL density was determined as the number of TILs per square millimeter of stromal tissue. Median TIL density was 1287/mm2 (range, 123-8101/mm2). A high TIL density was associated with higher histological grade (P = 0.02), estrogen receptor negativity (P = 0.036), and pathological complete response (pCR) (P < 0.0001). In analyses using receiver operating characteristic curves, a threshold TIL density of 2420/mm2 best discriminated pCR from non-pCR. In multivariate analysis, high TIL density (> 2420/mm2) was significantly associated with pCR (P < 0.0001). Our results indicate that DIA can assess TIL density quantitatively, machine learning-based classification algorithm allowing determination of TIL density as the number of TILs per unit area, and TIL density established by this method appears to be an independent predictor of pCR in HER2-positive breast cancer.

Postneoadjuvant therapy: a new approach to the treatment of HER2-positive breast cancer (KATHERINE study results)

Up until recently, neoadjuvant and adjuvant treatment regimens for breast cancer (BC) were considered equivalent in their effect on long-term treatment outcomes. Despite the fact that additional information on the prognosis of patients (achievement or failure to achieve complete drug pathomorphosis) was obtained during neoadjuvant therapy, we could not change this prognosis, since there was no evidence that any variant of adjuvant therapy could improve survival of patients, who did not achieve complete morphological tumour regression. In December 2018, investigators presented the results of the first planned interim analysis of invasive disease-free survival (iDFS) of patients with early HER2-positive breast cancer, who had residual tumour after neoadjuvant anti-HER2-containing therapy, depending on the adjuvant treatment option: either trastuzumab emtansine (n = 743) or trastuzumab (n = 743). The expected 3-year iDFS in patients, who received trastuzumab emtansine as adjuvant therapy, was 88.3%, while that in the standard trastuzumab group accounted for only 77% (RR = 0.50; 95% CI 0.39–0, 64; h <0.001). The results of the KATHERINE study, which showed an improvement in the survival rates of patients with HER2-positive breast cancer, who did not achieve complete therapeutic pathomorphism (pCR) against the background of neoadjuvant anti-HER2 therapy with the use of trastuzumab emtansine in the adjuvant mode, are not just an important milestone in the treatment of HER2-positive breast cancer. These results reverse the attitude towards neoadjuvant therapy, as we were able, for the first time, to improve the treatment outcome due to the change of the therapy based on therapeutic pathomorphosis data. Thus, this approach leads to the fact that neoadjuvant therapy becomes a more effective technique, at least in HER2-positive breast cancer.

Pathological outcomes of HER2-positive non-metastatic breast cancer patients treated with neoadjuvant dual anti-HER2 therapy and taxane: An Australian experience.

Internationally, there has recently been growing interest in the use of neoadjuvant pertuzumab and trastuzumab in patients with non-metastatic HER-2 positive breast cancer following the NEOSPHERE trial in 2012. However, pertuzumab is currently not funded by the Pharmaceutical Benefits Scheme (PBS) in Australia for use in this setting. The authors sought to assess the clinical and pathological response rates at the time of surgery in patients who received neoadjuvant dual anti-HER2 and taxane therapy in a multidisciplinary breast cancer unit. A retrospective case series of all patients treated with the neoadjuvant therapy, and who had definitive surgery was conducted. Demographic data, size, grade, tumor type, receptor status prior to neoadjuvant treatment, pathological complete response (pCR) rates, and adverse effects were analyzed. Nineteen patients were included in the study. Sixty-eight percent of all patients achieved pCR, of which 54% further demonstrated no residual ductal carcinoma in situ. Eight patients (42%) had N1 disease pretreatment, of these 88% demonstrated total pCR in the axilla and the breast. Most adverse effects to treatment were manageable grade 1-2 side effects. This is the first reported Australian experience using neoadjuvant dual anti-HER2 and taxane therapy for HER-2 positive nonmetastatic breast cancer. The authors have demonstrated favorable pCR rates for invasive disease compared to the NEOSPHERE trial (68% vs 46%), with reasonable patient tolerability. Larger collaborative data sets are required to fully evaluate correlation of pCR with survival outcomes, and cost-effectiveness. National funding models need to be considered.

Impact of dual anti-HER2 therapy on pathologic complete response rate in breast cancer in a minority-enriched population.

e18111 Background: The addition of pertuzumab (P) to a neoadjuvant trastuzumab (H) plus chemotherapy combination has been shown to significantly improve the pathologic complete response rate (pCR) in localized HER2+ breast cancer; however, minorities have been under-represented in these trials. Racial/ethnic disparities have also been shown to affect outcomes of cancer treatment. This study is aimed to assess the impact of neoadjuvant dual HER2-blockade in an unselected minority-enriched population. Methods: A retrospective chart review was conducted of women with stage I to III HER2+ breast cancer who received neoadjuvant treatment between 2007 and 2017 at an academic institution and its affiliated safety net health system. Data on stage, chemotherapy, race/ethnicity, site of therapy (academic vs safety net hospital), and hormone receptor status were collected. All patients underwent surgery after completion of neoadjuvant chemotherapy. pCR was defined as ypT0/is, ypN0. Chi-squared test and univariate/multivariate logistic regression were used for statistical analysis. Results: The study population included 261 women with the following race/ethnic distribution: 37.7% Non-Hispanic Whites, 34.6% Hispanics, 20.6% Blacks, and 7% other racial/ethnic origin. Ninety-five patients (36%) received chemotherapy-H vs 166 patients (64%) received chemotherapy-HP. Patients at the safety net health system had higher stage at diagnosis compared to the academic site. Site of care and race/ethnicity did not impact the choice of neoadjuvant treatment. The pCR rate was significantly higher for the chemotherapy-HP group (55.4%) compared to the chemotherapy-H group (34.7%) (p = 0.001). There was no association between race/ethnicity, or site of treatment (academic vs safety net), and the probability of achieving pCR. Multivariate analysis showed only dual anti-HER2 therapy (OR: 2.67, CI: 1.55-4.59, p = 0.0004) and hormone-receptor negative status (OR: 2.18, CI: 1.30-3.67, p = 0.0031) to correlate with pCR. Conclusions: Neoadjuvant dual anti-HER2 therapy was more likely to result in a pCR in our minority enriched population. Our data also suggests the combination of chemotherapy-HP confers similar benefit irrespective of race/ethnicity or site of care.

HER2 cluster amplification as a factor of an especial sensitivity for anti-HER2 neoadjuvant therapy with biosimilar of trastuzumab in Russian women with breast cancer stage II-III.

e12102 Background: Effect of neoadjuvant chemotherapy (NACT) is extremely important for patients with HER2-positive breast cancer stage II-III as it correlates with long-term outcomes. However, predictive factors for achieving complete pathologic response (pCR) remain unclear. Aim of the study: Assess an impact of clinical, morphological and genetic factors on pCR achievement in patients with HER2-positive breast cancer stage II-III treated by biosimilar of trastuzumab. Methods: We studied treatment results in 73 patients with HER2-positive breast cancer (BC) stage II-III (aged 29-71, median – 51 years), treated with NACT with anti-HER2 therapy and radical surgery in “N.N. Blokhin National Medical Research Center of Oncology” оf the Ministry of Health of the Russian Federation in 2015-2018. After radical surgery pathologic response was assessed. Initially operable tumors were observed in 45,2% patients, locally advanced - in 53,4%. Luminal HER2-positive BC was diagnosed in 41,1%, non-luminal HER2-positive in 58,9%. Ki67 was high (≥ 20%) in 91,5%. NACT included anthracycline and non-antracycline regimens with addition of biosimilar of trastuzumab ± pertuzumab. Radical mastectomy was performed in 78,8% patients, 21,2% had breast-conserving surgery. We studied biopsies obtained before the start of treatment in all women, HER2 amplification was detected by HER2 IQFISH pharmDx (DAKO) kit in accordance with ASCO/CAP 2018 guidelines. In 87,1% HER2+ status was defined as ASCO/CAP 2018 category 1; cluster amplification was detected in 30,1% patients. We analyzed the rate of bpCR и tpCR achievement depending on clinical, pathological data and amplification of HER2. Results: Pathologic complete response in primary tumor (bpCR) was achieved in 57,4%, both in primary tumor and lymph nodes (tpCR) – in 48,9% patients; bpCR achievement depended on age, NACT regimen, addition of pertuzumab and HER2 copy number ( &lt; 0,05). The highest rate of bpCR was noted in women aged 50 and older (71,9%, p = 0,026); in patients received TCH±Р regimen (80,0%, p = 0,045); with addition of pertuzumab (88,9%, p = 0,049); and if cluster amplification was detected (81%, p = 0,013). Cluster amplification was the only one significant predictive factor for achieving tpCR: with cluster amplification - 68,8%, without - 38,7% (p = 0,049). Conclusions: Cluster amplification of HER2 is the most significant factor of especial sensitivity for NACT with biosimilar of trastuzumab in HER2-positive BC stage II-III.

P179 - Positive HER3 could be a marker of poor response to neoadjuvant dual anti-HER2 therapy in breast cancer

P179 - Positive HER3 could be a marker of poor response to neoadjuvant dual anti-HER2 therapy in breast cancer

Changing pattern of recurrences in patients with early HER2-positive breast cancer receiving neoadjuvant chemotherapy in the era of dual anti-HER2 therapy

BackgroundOver the last 10 years, there has been a major treatment revolution for early human epidermal growth factor receptor 2 (HER2)–positive breast cancer. We aimed to explore the outcome of...

Abstract P3-10-17: mRNA expression of HER2 by PCR as a strong predictor of pathologic response to neoadjuvant antiHER2 therapy

Abstract Background: Pathologic response to neoadjuvant therapy is the most important prognostic factor so new agents should aim to improve it. HER2 expression in breast cancer provides prognostic and predictive information of response to antiHER2 therapy. The quantification of HER2 expression by mRNA is one of the most potent markers of cellular dependence to HER2 pathway. Methods: From a series of 160 patients with early or locally advanced HER2 positive breast cancer who received neoadjuvant treatment with chemotherapy and antiHER2 drugs, mRNA expression of HER2 was analyzed in 69 specimens and it was correlated to pathologic complete response (pCR). Results: Median age was of 52.5 years (29-86), tumor size of 33mm (10-111), 39 (53%) patients had nodal involvement. Median ki67 expression was of 45% (17-80), 28 (38%) tumors were estrogen receptor (ER) positive and 46 (62%) negative. We also analyze the expression of tumor-infiltrating-lymphocytes (TILs) by immunohistochemistry in 43 samples because their expression is also a strong predictive of pathologic response. A total of 31/69 patients (45%) achieved a pCR with a median of mRNA expression of 7,55 (3,8 – 11) in comparison with 2.03 (1,2 -2,8) in patients without pCR (p: 0,002). In the univariable analysis, variables correlated to pCR were estrogen receptor=0 (OR 0,186 IC 95% 0,063-0,549, p: 0,002), mRNA (OR 5,689 IC 95% 1,854-17,452, p: 0,002) and TILs (OR 15,167 IC 95% 3,223-71,38, p: 0,001) and in the multivariable analysis, TILs and mRNA expression were found to be independent predictive factors for pCR. The quantification of HER2 by mRNA obtained an impressive response specially in ER negative patients with a rate of pCR of 100% in samples of mRNA expression upper of 3,6 and ER negative. However, patients with mRNA expression lower than 3,6 with a pCR rate of 32%, could reach to an 80% rate if they had high TILs expression. Conclusions: Quantification of HER2 expression by mRNA is a strong predictive factor of pCR (OR 5,689); achieving a 100% pCR in ER negative tumors. TILs are also a predictive factor for pCR (OR 15,167) and its combination with mRNA expression could improve pCR rates; thus, patients with mRNA expression lower than 3,6, if they had a higher TILs expression could reach to an 80% pCR. Citation Format: Gasol Cudós A, Morales Murillo S, Veas Rodriguez J, Velasco Sánchez A, Serrate López A, Melé Olivé J, Canosa Morales C. mRNA expression of HER2 by PCR as a strong predictor of pathologic response to neoadjuvant antiHER2 therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-17.

Role of clustered HER2/neu amplification as a marker for a special sensitivity to neoadjuvant anti-HER2 therapy with trastuzumab in patients with stage II-III breast cancer

To evaluate the influence of clinical and morphological factors and HER2 copy numbers on pathologic complete response (pCR) rates in patients with HER2-positive stage II-III breast cancer (BC). Treatment results were studied in 73 patients with HER2-positive Stage II-III BC, who received treatment at the N.N. Blokhin National Medical Research Center of Oncology in 2015 to 2018. Treatment included neoadjuvant chemotherapy (NACT) with HER2-blockade and radical surgery followed by the evaluation of a pathologic response in the primary tumor and regional lymph nodes. The patients` age varied from 29 to 71; its median was 51.5; 45.2% of patients had primary operable stages (T1-3N0-1) and 54.8% had locally advanced tumors. All the patients had grade 2-3 anaplasia; luminal HER2-positive BC was diagnosed in 41.4% of patients; hormone-negative tumors were seen in 58.9%; 91.5% of patients had Ki-67 ≥20% in 75.3% of patients, preoperative systemic therapy included anthracycline-containing regimens (4AC + 4 x paclitaxel 175 mg/m2/12 × weekly administrations of paclitaxel 80 mg/m2; trastuzumab therapy was simultaneously performed with the administration of taxanes in the standard regimen) and anthracycline-free regimen TCH ± Pertuzumab regimen in 24.7% of cases. After NACT patients underwent surgery (radical mastectomy in 78.1%, breast-sparing treatment in 21.9%) with the assessment of morphological findings. Biopsy specimens obtained before the treatment was restudied; HER2 amplification was detected using a Dako HER2 IQFISH pharmDx kit according to its instruction and the 2018 ASCO/CAP guidelines. In 87.1% of cases, the HER2-positive status corresponded to the first category of the 2018 ASCO/CAP criteria for HER2-positive BC; clustered HER2 amplification was found in 30.1% of cases. The authors analyzed the frequency of bpCR and tpCR attainment by various clinical and morphological factors, as well as the impact of a HER2 amplification level on pCR rates. A breast pCR (bpCR) was achieved in 57.4% patients; bpCR and lymph node CR (lnCR) were noted in 48.9% patients. The rates of bpCR significantly depended on female age, chemotherapy regimen, addition of Pertuzumab, and HER2 copy number. That of bpCR in women less than 35 years of age, in those aged 36-50 years, and in those aged older than 50 years was 22.2, 57.7 and 71.9%, respectively (p=0.026). The maximum bpCR rate observed with the TCH±P regimen was 80.0%, that with anthracycline-containing regimes was 52.8% (p=0.045), and the addition of Pertuzumab increased complete response rates up to 88.9% (that with Trastuzumab was 54.2% (p=0.049). The relationship of bpCR rates to the detection of cluster amplification turned out to be highly significant (81% in its detection and 48.9% in its absence (p=0.013). In addition, clustered HER2 amplification was the only significant predictive factor for complete regression in the primary tumor and lymph nodes: in its presence, the tpCR rate reached 68.8% versus 38.7%. Clustered amplification of the HER2 gene is the most significant factor of sensitivity to anti-HER2 therapy for Stage II-III BC, and is associated with the maximum rate of both bpCR and total pCR. Further study of this factor may assist in optimizing the treatment algorithm for HER2 + BC.

Impact of timing of trastuzumab initiation on long-term outcome of patients with early-stage HER2-positive breast cancer: the \u201cone thousand HER2 patients\u201d project

BackgroundThe optimal timing of (neo)adjuvant trastuzumab initiation with respect to chemotherapy and surgery remains undefined.MethodsRetrospective analysis of a large institutional database of HER2-positive patients who received anti-HER2 therapy. We included all Stage I to III patients treated with trastuzumab with a minimum follow up of 3 years. The date of first breast biopsy was recorded as initial diagnosis.ResultsA total of 506 patients [adjuvant: 386 (76%)-neo-adjuvant: 120 (24%)] were included. The median time-to-first-trastuzumab (TFT) from diagnosis was 12 weeks (range 1.9–122.3). Median follow-up is 73.3 months (range 1.4–176.3). TFT was significantly shorter in the neo-adjuvant than in the adjuvant cohort (median: 4.4 vs. 14 weeks, p < 0.00001). Despite the neo-adjuvant cohort having significantly more node-positive patients (75 vs. 53%, p < 0.0001), DFS rate (neo-adjuvant: 12.5 vs. adjuvant: 18%, p = 0.094) was numerically superior in neo-adjuvant patients. A TFT ≤ 12 weeks was associated with significantly superior DFS and OS over TFT > 12 weeks. Early concomitant regimens were associated with superior DFS over delayed-concomitant and sequential regimens.ConclusionsInitiating trastuzumab more than 12 weeks from diagnosis has a negative impact on clinical outcome. Neo-adjuvant anti-HER2 therapy could be the optimal strategy to treat early stage HER2-positive breast cancer.

Abstract PD3-06: Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC)

Abstract Introduction: Previous studies have shown that HER2+ breast cancer is biologically heterogenous and intrinsic subtypes can be identified (luminal A, luminal B, HER2-enriched [HER2-E] and basal-like). HER2-E predominates and is associated with higher response rates following anti-HER2-based chemotherapy or dual HER2 blockade (only with lapatinib and trastuzumab). We explored the ability of intrinsic subtypes to predict pCR in pts treated with anti-HER2 neoadjuvant therapy. Methods: KRISTINE (NCT02131064) is an open-label, phase 3 study of neoadjuvant trastuzumab emtansine + pertuzumab (T-DM1+P) vs docetaxel + carboplatin + trastuzumab + pertuzumab (TCH+P) in pts with HER2+ EBC. Treatment-naive pts with stage II–IIIC HER2+ EBC were randomized to receive 6 cycles of T-DM1+P or TCH+P and assessed for the primary endpoint, pCR (ypT0/is, ypN0). HER2 and hormone receptor (HR) status were centrally assessed. Gene expression (RNA) was assessed by a custom 800-gene codeset on the nCounter platform. Intrinsic subtypes were assessed with the research-based PAM50 classifier. Results: KRISTINE randomized 444 pts (data cutoff, Dec 3, 2015; TCH+P, n=221; T-DM1+P, n=223). PAM50 results were available for 354 pts (79.7% of the intent-to-treat [ITT] population). Baseline characteristics and efficacy in the PAM50 population were similar to that of the ITT population. The HER2-E subtype represented 54.8% of the samples (Table 1). Differences were observed by HR status. Almost all luminal tumors (131/132) were identified within HR+ disease. Of HR+ tumors, 32% were identified as HER2-E. In the TCH+P arm, the pCR rate was 72.1% for HER2-E vs 32.8% in the other subtypes combined (Table 2.) In the T-DM1+P arm, the pCR rate was 62.2% for HER2-E vs 26.9% in the other subtypes combined. No major differences were observed in pCR rates within the HER2-E subtype according to HR status. Further multivariable analyses assessing differences between treatment arms and treatment benefit across subtypes is ongoing. Table 1. Intrinsic subtypesn (%)ITT (n=354)HR- (n=143)*HR+ (%) (n=200)*HER2-E194 (54.8)123 (86.0)64 (32.0)Luminal A60 (16.9)1 (0.7)57 (28.5)Luminal B74 (20.9)074 (37.0)Basal-like26 (7.3)19 (13.3)5 (2.5)*Central HR status unknown for n=11 Table 2. pCR by intrinsic subtype TCH+PT-DM1+P npCR, n (%)npCR, n (%)pCR difference (95% CI)HER2-E10475 (72.1)9056 (62.2)-9.89 (-23.11, 3.32)HER2-E and HR+ *3724 (64.9)2715 (55.6)-9.31 (-33.56, 14.94)HER2-E and HR- *6448 (75.0)5937(62.7)-12.29 (-28.56, 3.98)Other subtypes combined6722 (32.8)9325 (26.9)-5.9 (-20.36, 8.45)Luminal A254 (16.0)3510 (28.6)12.57 (-8.18, 33.32)Luminal B3211 (34.4)4212 (28.6)-5.80 (-27.19, 15.58)Basal-like107 (70.0)163 (18.8)-51.25 (-85.49, -17.01)*Central HR status unknown for n=7 Conclusions: In this analysis from the KRISTINE study, HER2-E was the most common intrinsic subtype and was associated with the highest pCR rate with both regimens. Results are consistent with previous findings. The luminal A and B subtypes were well associated with HR+ status. A sizeable subgroup of the HER2-E subtype was HR+ (32%), and pCR rates within the HER2-E subtype seemed independent of HR status. Citation Format: Prat A, Slamon D, Hurvitz SA, Press MF, Lewis Phillips G, Lopez Valverde V, Kiermaier A, Helms H-J, Martin M, de Haas SL. Association of intrinsic subtypes with pathological complete response (pCR) in the KRISTINE neoadjuvant phase 3 clinical trial in HER2-positive early breast cancer (EBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-06.

Abstract GS1-04: Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial

Abstract Background In the NeoALTTO trial, the combination of trastuzumab and lapatinib for the neoadjuvant treatment of HER2 positive early breast cancer patients (pts) nearly doubled the rate of pathological complete response (pCR) compared with either anti-HER2 treatment alone. In the same trial, pCR was shown to be a surrogate for long-term outcome. Expression of ERBB2, ESR1 and immune signatures were the main drivers of pCR (Fumagalli et al. JAMA Oncol 2016). The main aim of the current study was to investigate the relevance of copy number aberrations (CNAs) for pCR and event-free survival (EFS). Methods For 271 out of the 455 pts enrolled in the study with sufficient tumour content, DNA was hybridized on CytoScan HD arrays. The log2 ratio intensities and B allele frequencies were segmented jointly using the multitrack segmentation algorithm. Integer level estimates of total copy number and major allele were obtained using GAP. Recurrent CNAs were found with GISTIC2. The genome instability index (GII) was defined as the median absolute deviation of the normalized copy number. Tumor infiltrating lymphocytes (TILs) and gene expression were obtained as described previously. The correlations between differents parameters were assessed using Spearman correlations (ρ). The Mann-Whitney test was used to relate binary and numerical features. EFS analysis was performed using the Cox proportional hazard model. Results CNAs estimates were obtained for 185 out of 271 pts. CNA distribution was similar to the ones observed in HER2+ pts from the METABRIC and the TCGA datasets. There were 64% of diploid pts, 26% of triploid and 9% of tetraploid or more. Aneuploidy level was not associated with pCR or EFS. Of interest, there were many significant differences in CNA profiles between ER+ and ER- pts. Those differences mirror those observed between ER+ and ER- among HER2- pts in TCGA (ρ=63%) and METABRIC (ρ=56%). ERBB2 amplification was predictive of high pCR (p=0.0007), albeit less so than ERBB2 expression, and ceased to be significant correcting for ERBB2 expression. The pCR rate increased with the GII (p=0.03), independently of ERBB2 amplification. The effect was stronger in ER+ patients (p=0.01). GISTIC analysis revealed 159 recurrent CNA regions. Amplification of 2 regions on 6q23-24 was significantly associated with higher pCR (p=0.00005 and p=0.00087, FDR=0.006 and 0.05). The most significant segment of 6q23-24 contained 39 genes, some whose expression level also predicted pCR (e.g. MAP3K5, p=10&amp;lt;sup style="font-family: Calibri;"&amp;gt;-4). Gene ontology analysis of the genes correlated with this segment highlighted the category 'response to interferon-alpha' (p=4.3x10&amp;lt;sup style="font-family: Calibri;"&amp;gt;-7). No amplified region or gene was found to be predictive of EFS, after multiple testing correction. Conclusions The amplification of ERBB2 was shown to be predictive of pCR, however its expression was more predictive. High genomic instability was associated with higher rate of pCR in ER+ subgroup. Of interest, a novel amplified region, involving chromosome 6 was shown to be predictive of pCR, which may warrant further investigation. Citation Format: Sotiriou C, Rothé F, Maetens M, Fumagalli D, Brown DN, Salgado R, Bradbury I, Pusztai L, Harbeck N, Ignatiadis M, Sarp S, de la Pena L, de Azambuja E, Huober J, Nuciforo P, Baselga J, Piccart M, Loi S, Venet D. Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-04.

Abstract PD3-04: Complete pathologic response rate to neoadjuvant chemotherapy increases with increasing HER2 ratio in HER2 over-expressing breast cancer: Analysis of the National cancer database (NCDB)

Abstract Background: HER2-positive (HER2+) breast cancer is an aggressive subtype that overexpresses human epidermal growth factor receptor 2 promoting cancer cell growth. Monoclonal antibodies targeting the HER2 receptor have improved survival for this patient population, and current NCCN guidelines recommend consideration of neoadjuvant anti-HER2 therapy (NAC) in Stage 2 &amp; 3 HER2+ breast cancer. Pathologic complete response (pCR) to NAC has correlated with longer disease free survival in multiple trials. Per ASCO-CAP guidelines tumors are considered HER2+ if HER2 copy number≥ 6/cell, HER2/CEP17 ratio≥ 2, or ratio&amp;lt;2 &amp; HER2 copy number ≥6/cell. We hypothesize that patients with higher HER2 ratios will have higher rates of pCR after NAC. Methods: The National Cancer Database is supported by the American College of Surgeons and the American Cancer Society containing de-identified patient treatment data from over 1,500 US facilities. We performed a retrospective review comparing pCR rates after NAC based on HER2 ratio. Patients were excluded if they were HER2 negative, did not undergo NAC, or if the HER2 ratio was not recorded. Chi-squared and Fisher's exact test were used to compare pCR versus partial response between deciles of HER2 ratios. Results: The NCDB included 237,118 patients with HER2 equivocal or HER2+ breast tumors. 29,291 of these patients underwent NAC, and HER2 ratios were recorded in 14,597 of the NAC cases. The majority (98%) of included cases were from 2010-2014. A pCR was noted in 9,752 patients and 11,402 patients had a partial response. No response was observed in 1,735 patients and 6,402 patients had a response but the degree was not recorded. HER2 ratios were significantly different between pCR vs. partial response groups, p &amp;lt;0.001. We identified a direct relationship between increasing HER2 ratio and response to NAC. For ratios 2-2.9, 23.6% achieved pCR and 44.7% had a partial response. For ratio of 5-5.9, 40.7% achieved pCR and even higher rates of pCR were noted for ratios 8-8.9; 49.5% achieved pCR. While both estrogen receptor (ER) positive and ER negative tumors demonstrated this trend, ER negative tumors had higher rates of pCR (ER negative pCR range 37.6% to 59.4% vs ER positive pCR range 16.9% to 42.3%, p&amp;lt;0.01). Conclusion: Contrary to current dogma, not all HER2+ tumors respond similarly to NAC. We demonstrate a linear relationship between HER2 ratio and pCR in over 14,000 patients. Those with HER2 ratios ≥5.0 were more likely to achieve pCR compared to patients with ratio ≤4.9. The NCDB reflects current clinical practice across the country not restricted to confines of clinical trials, and in this population higher HER2 ratios are predictive of pCR after NAC. Response to NAC by Her2 Ratio- Complete vs Partial Response Response to NAC p ValueHER2 Ratio Complete Response- pCR (N) Partial Response (N) 1.00- 1.99141819.5%343047.2%&amp;lt;0.01 2.00- 2.9951423.6%97444.7%&amp;lt;0.01 3.00- 3.9928328.7%41942.4%&amp;lt;0.01 4.00- 4.9926533.2%30638.2%&amp;lt;0.01 5.00- 5.9929940.7%24333.1%&amp;lt;0.01 6.00- 6.9929241.0%25435.5%&amp;lt;0.01 7.00- 7.9924746.2%17432.5%&amp;lt;0.01 8.00- 8.9918749.5%12132.0%&amp;lt;0.01 9.00- 9.87 and greater44143.9%31431.3%&amp;lt;0.01TOTAL 394627.0%623542.7%&amp;lt;0.01 Citation Format: Greenwell K, Hussain L, Ho C, Dunki-Jacobs E, Lee D, Bramlage M, Bills G, Mehta A, Jones J, Jackson A, Wexelman B. Complete pathologic response rate to neoadjuvant chemotherapy increases with increasing HER2 ratio in HER2 over-expressing breast cancer: Analysis of the National cancer database (NCDB) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-04.

Evaluation of Immune Reaction and PD-L1 Expression Using Multiplex Immunohistochemistry in HER2-Positive Breast Cancer: The Association With Response to Anti-HER2 Neoadjuvant Therapy

BackgroundImmune reaction with tumor-infiltrating lymphocytes (TILs) has been extensively investigated in breast cancer. Programmed cell death 1 and its ligand (PD-L1) are key physiologic suppressors of cytotoxic immune reaction. However, the combination of TILs with PD-L1 expression has not been well studied in breast cancer. Patients and MethodsA multi-color immunohistochemical multiplex assay simultaneously detecting PD-L1, CD8, and CD163 was performed on biopsy whole sections from 123 HER2-positive (HER2+) breast cancers, including 64 treated with anti-HER2 neoadjuvant therapy and subsequent resection. ResultsPD-L1 expression was identified in 88 cases (72%) including 21 (17%) in tumor cells and 67 (55%) in immune cells. PD-L1 expression was positively associated with high Nottingham grade, high nuclear grade, and a high level of CD8+ and CD163+ cells. Among the 64 patients who received neoadjuvant therapy, 39 had pathologic complete remission (pCR) and 25 had incomplete response. Multivariate analysis showed progesterone receptor negativity, HER2/chromosome 17 centromere (CEN17) ratio and intratumoral CD8+ cells were significantly associated with pCR. Furthermore, all patients with intratumoral CD8+ cells but no PD-L1 expression achieved pCR. ConclusionOur data have shown that examination of intratumoral CD8+ cells together with PD-L1 expression proves useful in predicting response to anti-HER2 targeted therapy in patients with HER2+ breast cancer.

HER2-Overexpressing Breast Cancers Amplify FGFR Signaling upon Acquisition of Resistance to Dual Therapeutic Blockade of HER2.

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or pertuzumab has been shown to be superior to single-agent trastuzumab. However, a significant fraction of HER2-overexpressing (HER2+) breast cancers escape from these drug combinations. In this study, we sought to discover the mechanisms of acquired resistance to the combination of lapatinib + trastuzumab.Experimental Design: HER2+ BT474 xenografts were treated with lapatinib + trastuzumab long-term until resistance developed. Potential mechanisms of acquired resistance were evaluated in lapatinib + trastuzumab-resistant (LTR) tumors by targeted capture next-generation sequencing. In vitro experiments were performed to corroborate these findings, and a novel drug combination was tested against LTR xenografts. Gene expression and copy-number analyses were performed to corroborate our findings in clinical samples.Results: LTR tumors exhibited an increase in FGF3/4/19 copy number, together with an increase in FGFR phosphorylation, marked stromal changes in the tumor microenvironment, and reduced tumor uptake of lapatinib. Stimulation of BT474 cells with FGF4 promoted resistance to lapatinib + trastuzumab in vitro Treatment with FGFR tyrosine kinase inhibitors reversed these changes and overcame resistance to lapatinib + trastuzumab. High expression of FGFR1 correlated with a statistically shorter progression-free survival in patients with HER2+ early breast cancer treated with adjuvant trastuzumab. Finally, FGFR1 and/or FGF3 gene amplification correlated with a lower pathologic complete response in patients with HER2+ early breast cancer treated with neoadjuvant anti-HER2 therapy.Conclusions: Amplification of FGFR signaling promotes resistance to HER2 inhibition, which can be diminished by the combination of HER2 and FGFR inhibitors. Clin Cancer Res; 23(15); 4323-34. ©2017 AACR.

Extracellular Matrix/Integrin Signaling Promotes Resistance to Combined Inhibition of HER2 and PI3K in HER2+ Breast Cancer.

PIK3CA mutations are associated with resistance to HER2-targeted therapies. We previously showed that HER2+/PIK3CAH1047R transgenic mammary tumors are resistant to the HER2 antibodies trastuzumab and pertuzumab but respond to PI3K inhibitor buparlisib (TPB). In this study, we identified mechanisms of resistance to combined inhibition of HER2 and PI3K. TPB-resistant tumors were generated by treating HER2+/PIK3CAH1047R tumor-bearing mice long term with the drug combination. RNA sequencing of TPB-resistant tumors revealed that extracellular matrix and cell adhesion genes, including collagen II (Col2a1), were markedly upregulated, accompanied by activation of integrin β1/Src. Cells derived from drug-resistant tumors were sensitive to TBP when grown in vitro, but exhibited resistance when plated on collagen or when reintroduced into mice. Drug resistance was partially reversed by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoate. Inhibition of integrin β1/Src blocked collagen-induced resistance to TPB and inhibited growth of drug-resistant tumors. High collagen II expression was associated with significantly lower clinical response to neoadjuvant anti-HER2 therapy in HER2+ breast cancer patients. Overall, these data suggest that upregulation of collagen/integrin/Src signaling contributes to resistance to combinatorial HER2 and PI3K inhibition. Cancer Res; 77(12); 3280-92. ©2017 AACR.

Comparison of neoadjuvant anti-HER2 therapy for breast cancer in public versus private health care: Time to shorten the distance.

e18045 Background: In the neoadjuvant setting (NAC) of HER2-positive breast cancer (BC), the addition of pertuzumab (P) to trastuzumab (T) and chemotherapy (chemo) has increased pathological complete response (pCR) from 40-50% to over 70%. Recent data has shown that pCR is a reliable surrogate marker of disease-free (DFS) and overall survival (OS). In Brazil, there is an important gap between cancer drugs approved in the public health care system (PHCS) vs. the private setting. Although P approval for NAC (Aug/2016) and metastatic setting (Sep/2015) in the private health system, it is not approved for any indication in the PHCS. Likewise, T is not approved for use as NAC at the PHCS. We aim to compare private practice experience of T + P + chemo in the NAC vs. HER2-positive advanced BC patients treated at the PHCS. Methods: Retrospective analysis of 95 patients diagnosed with locally advanced HER2-positive BC by immunohistochemistry 3+ or FISH according to ASCO/CAP definition. We evaluated 17 consecutive patients at the COAEM private cancer center in Brazil since Feb/2014 (cohort 1). Neoadjuvant therapy was T and P plus docetaxel-based chemo. A cohort of 78 patients with locally advanced HER2-positive BC treated in a PHCS cancer center was analyzed (cohort 2). They received standard NAC regimen according to PHCS guidance: anthracycline and taxane-based therapy without anti-HER2 therapy. Results: Cohort 1: 17 patients with median age of 50 years (range 34-70); Four patients had clinical stage IIA; 2 patients IIB; 7 patients IIIA; 3 patients IIIB; one patient IIIC. The pCR rate was 70.5% (12 patients). Cohort 2: 78 patients with median age of 48 years (range 26-79). One patient IIB; 33 patients IIIA; 37 patients IIIB; 7 patients IIIC. The pCR rate was 16.7% (13 patients); 29 patients (37.2%) had clinical complete response. Only 26 patients (33.3%) received T in the adjuvant setting. Conclusions: Neoadjuvant P and T combined with chemo has shown pCR rates superior to 70% in clinical trials, which is a surrogate marker for DFS and OS in HER2-positive BC. The absence of anti-HER2 therapy in the PHCS must be urgently reviewed not just in light of double-blockage strategy but also in T monotherapy use.

Abstract P5-16-28: A single-institution clinical experience with neoadjuvant docetaxel/carboplatin/trastuzumab/pertuzumab (TCHP): A safety and efficacy analysis

Abstract Background: Pertuzumab, a monoclonal antibody targeting subdomain II of HER2 and blocking dimerization , was approved by the FDA in 2013 for use in combination with trastuzumab and docetaxel as neoadjuvant therapy for pts with HER2+, locally advanced, inflammatory or early-stage breast cancer (&amp;gt;2cm and/or node-positive). This accelerated approval was based on results from the NeoSphere and the TRYPHAENA trials. In NeoSphere, pathologic complete response in the breast and nodes [pCR] was 39.3% after 4 cycles of neoadjuvant pertuzumab/trastuzumab/docetaxel. In TRYPHAENA, pCR was 63.6% among 76 patients treated with 6 cycles of neoadjuvant TCHP(47.5% in pts with ER+and/or PR+/HER2+ tumors and 81.1% in those with ER-/PR-/HER2+ tumors). Aside from TRYPHAENA, we have limited information on clinical outcomes, with neoadjuvant TCHP. Here we report our institutional experience at UF Health Cancer Center, Orlando (UFHCC) with Neoadjuvant TCHP in patients with operable or locally advanced breast cancer. Patients and Methods: After IRB approval, electronic medical record search was performed in order to identify HER2+ patients with tumors T2-T4/N0-3 or Tany/N1-3, treated with neoadjuvant TCHP between 10/13 and 5/16. Information from chart review included patient and tumor characteristics at the time of diagnosis , details of neoadjuvant chemotherapy plus anti-HER2 therapy, clinical, radiologic and pathologic assessment of tumor response to neoadjuvant TCHP, type of breast and axillary nodal surgery, surgical outcomes as well as disease outcomes. Results: 76 patients (75 female, 1 male) met the inclusion criteria; median age: 52 yrs; 83% of pts presented with clinical stage II and 17% with clinical stage III; 62% were ER+ and/or PR+ and 38% were ER-/PR-negative. 49 patients received all planned 6 cycles without dose reduction. The remaining 27 patients required dose reduction due to rash, diarrhea, nausea, vomiting, neuropathy or neutropenia; 5 patients requested dose reduction due to poor quality of life and fatigue; 2 patients required dose delay due to asymptomatic cardio-toxicity with ≥ 10% drop in EF. None had symptomatic CHF; 37% of patients underwent breast conserving surgery, 7% unilateral mastectomy and 55% bilateral mastectomy. Surgical lymph node assessment was performed after neoadjuvant chemotherapy and included sentinel lymph node biopsy (SLNB) in 74%, axillary dissection (ALND) in 8% or both in 18% of pts. Overall pCR rate (ypT0/is, ypN0) was 63.2%. pCR rate was 53.1% in pts with ER+ and/or PR+ tumors and 79.3% in those with ER-/PR- tumors. pCR by stage was 61% for Stage IIA, 65% for Stage IIB, 67% for Stage IIIA and 71% for Stage IIIC. Toxicity profile was consistent with what has been observed in the TRYPHAENA trial with fatigue, nausea, vomiting and neuropathy being the more commonly noted grade 3/4 toxicities. With median follow up of 18 months, all patients are disease-free with no documented recurrences observed. Conclusion: Our clinical experience with neoadjuvant TCHP confirms the efficacy and safety data from the TRYPHAENA trial in a single-institution, tertiary care center setting. Citation Format: Tariq R, Ajaz B, Shah N, Mamounas E, Moroose R. A single-institution clinical experience with neoadjuvant docetaxel/carboplatin/trastuzumab/pertuzumab (TCHP): A safety and efficacy analysis [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 P5-16-28.

RNA Sequencing to Predict Response to Neoadjuvant Anti-HER2 Therapy

In neoadjuvant trials, treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancers with dual HER2 blockade resulted in increased pathologic complete response (pCR) rates compared with each targeted agent alone. Amplification and/or overexpression of HER2 currently remains the only biomarker for therapeutic decisions, but it is insufficient to explain the heterogeneous response to anti-HER2 agents. To investigate the ability of clinically and biologically relevant genes and gene signatures (GSs) measured by RNA sequencing to predict the efficacy of anti-HER2 agents. The neoadjuvant NeoALTTO trial randomized 455 women with HER2-positive early-stage breast cancer to trastuzumab, lapatinib, or the combination for 6 weeks followed by the addition of weekly paclitaxel for 12 weeks, followed by 3 cycles of fluorouracil, epirubicin, and cyclophosphamide after surgery. The present substudy, which was planned in the NeoALTTO main protocol, evaluated the association of pretreatment gene expression levels defined using RNA sequencing with pCR and event-free survival (EFS). Gene expression-based biomarkers using RNA sequencing were examined for their association with response to anti-HER2 therapy and long-term outcome. Sequencing data were available for 254 (56%) of the NeoALTTO participants (mean [SD] age of substudy participants, 48.8 [11.2] years). The expression of ERBB2/HER2 was the most significant predictor of pCR, followed by HER2-enriched subtype, ESR1, treatment arm, ER immunohistochemical analysis scores, Genomic Grade Index, immune, proliferation, and AKT/mTOR GSs. Adjusting for clinicopathological variables and treatment arms, ERBB2/HER2, HER2-enriched subtype, ESR1, and Genomic Grade Index remained significant. Immune GSs were associated with higher pCR only in the combination arm (odds ratio, 2.1; 95% CI, 1.2-4.0; interaction test P = .01), while the stroma GSs were significantly associated with higher pCR in the single arms and with lower pCR in the combination arm (odds ratio, 0.46; 95% CI, 0.25-0.84; P = .009). None of the evaluated variables was associated with EFS after correction for multiple testing, but this analysis was underpowered. High levels of ERBB2/HER2 and low levels of ESR1 were associated with pCR in all treatment arms. In the combination arm, high expression of immune and stroma GSs were significantly associated with higher and lower pCR rates, respectively, and should be further explored as candidate predictive markers. clinicaltrials.gov Identifier: NCT00553358.