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Purpose: Neratinib plus capecitabine (N+C) demonstrated significant progression-free survival (PFS) benefit in NALA (NCT01808573), a randomized phase III trial comparing N+C with lapatinib + capecitabine (L+C) in 621 patients with HER2-positive (HER2+) metastatic breast cancer (MBC) who had received ≥2 prior HER2-directed regimens in the metastatic setting. We evaluated correlations between exploratory biomarkers and PFS. Patients and Methods: Somatic mutations were evaluated by next-generation sequencing on primary or metastatic samples. HER2 protein expression was evaluated by central IHC, H-score, and VeraTag/HERmark. p95 expression (truncated HER2) was measured by VeraTag. HRs were estimated using unstratified Cox proportional hazards models. Results: Four hundred and twenty samples had successful sequencing: 34.0% had PIK3CA mutations and 5.5% had HER2 (ERBB2) mutations. In the combined patient populations, PIK3CA mutations trended toward shorter PFS [wild-type vs. mutant, HR = 0.81; 95% confidence interval (CI), 0.64–1.03], whereas HER2 mutations trended toward longer PFS [HR = 1.69 (95% CI, 0.97–3.29)]. Higher HER2 protein expression was associated with longer PFS [IHC 3+ vs. 2+, HR = 0.67 (0.54–0.82); H-score ≥240 versus Conclusions: PIK3CA mutations were associated with shorter PFS whereas higher HER2 expression was associated with longer PFS. Higher HER2 protein expression was also associated with a greater benefit for N+C compared with L+C.

Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P < 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P < 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Introduction: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified or mutated across various cancer types. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. All three TKIs bind and inhibit the kinase domain of HER2 but differ both in the mechanism of binding and in specificity for other HER family members. Direct comparisons to differentiate the pre-clinical efficacy of the three TKIs have been limited to small-scale studies and novel biomarkers of response to further define appropriate patient populations are required. Methods: In this study, the anti-proliferative effects of the three TKIs were compared using a 115-cancer cell line panel, including 12 breast cancer cell lines and 22 cell lines harbouring point mutations or amplifications of EGFR, HER2, or HER3. Hierarchical clustering analysis was carried out to compare the IC50 “fingerprint” of the three TKIs to 168 other anti-cancer agents. Novel markers of TKI sensitivity and resistance were identified through cross-analysis of each drug response profile with mutation, copy number variation, and gene expression data. Results: All three TKIs were effective against HER2-positive breast cancer models; neratinib showed the most potent activity, followed by tucatinib and lapatinib respectively (Table 1). Neratinib displayed the greatest anti-proliferative activity in HER2-mutant and EGFR-mutant cell lines. Clustering analysis revealed that the anti-proliferative profile of tucatinib was most similar to trastuzumab, while neratinib and lapatinib were most like other HER family inhibitors. Mutation and gene expression analysis identified potential markers of response for each TKI. High expression of four genes (HER2, VTCN1, CDK12, and RAC1) correlated with response to all three TKIs. DNA damage repair genes were significantly associated with resistance to the HER2-targeted TKIs. BRCA2 mutation was correlated with neratinib and tucatinib response, and high gene expression of ATM, BRCA2, and BRCA1 were all associated with neratinib resistance. Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed possible mechanisms that may be exploited using combinatorial strategies involving CDK inhibitors, immunotherapies, and targeting DNA repair pathways. Table: IC50 values for neratinib, lapatinib, and tucatinib in the HER2+ breast cancer cell linesIC50 values (nM)Cell linesNeratinibLapatinibTucatinibAU-56520294125BT-4745926229HCC195413814262122MDA-MB-453306228445928SKBR3715222 Citation Format: Neil T Conlon, Jeffrey J Kooijman, Suzanne JC van Gerwen, Winfried R Mulder, Guido JR Zaman, Irmina Diala, Lisa D Eli, Alshad S Lalani, John Crown, Denis Collins. Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-06.

Background: HER2 mutations are oncogenic in hormone receptor positive (HR+) metastatic breast cancer (MBC), and may confer resistance to prior endocrine therapy but retain sensitivity to neratinib. Neratinib is an oral, irreversible, pan-HER tyrosine kinase inhibitor with clinical activity either as a single agent or in combination with fulvestrant in HER2-mutated, HER2-non-amplified MBC. Genomic analyses suggest that acquired resistance to neratinib can occur via additional HER2 alterations, which may alter HER2-pathway signaling. We investigated whether dual HER2-targeted therapy could improve clinical benefit in a cohort of patients with HER2-mutant, HR+ MBC treated with neratinib + trastuzumab + fulvestrant (N+T+F) from SUMMIT - a phase 2 basket trial (NCT01953926). Methods: Patients with HR+ MBC with known or suspected pathogenic HER2 mutation(s) identified by genomic sequencing were eligible to receive N+T+F (oral neratinib 240 mg/day, i.v. trastuzumab 8 mg/kg initially followed by 6 mg/kg every 3 weeks, and i.m. fulvestrant 500 mg on days 1&15 of month 1, then on day 1 every 4 weeks). Loperamide prophylaxis was mandatory during the first 2 treatment cycles. There was no restriction on the number of prior lines of systemic treatment for MBC. Efficacy endpoints: confirmed objective response rate and clinical benefit rate (RECIST v1.1); duration of response; progression-free survival. Results: As of 22-May-2020, 46 patients were enrolled in the N+T+F cohort and received at least 1 dose of study medication (safety population). 14 unique HER2 allelic variants were identified: 8 kinase domain missense; 1 extracellular domain missense; 2 transmembrane domain missense; 2 exon-20 insertion; 1 exon-19 deletion. The most common HER2 mutant variant was L755S (n=15, 33%) Median number of prior systemic regimens for metastatic disease was 4 (range 0-10); 34 (74%) patients had received prior fulvestrant, and 31 (67%) patients had received prior cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor therapy. 16 (35%) patients had ductal histology, 29 (63%) had lobular carcinoma, and 1 (2%) had mixed ductal and lobular carcinoma. At this time, 30/46 patients had RECIST measurable disease and are efficacy evaluable (ongoing patients who did not have the opportunity for their first post-baseline tumor assessment were excluded); clinical activity - see Table. Diarrhea was the most commonly reported adverse event (80% any grade) with 15 (33%) patients reporting grade 3 diarrhea (no grade 4 diarrhea). 10 patients (22%) had a neratinib dose reduction due to diarrhea but no patients discontinued treatment due to diarrhea. Conclusions: The combination of N+T+F demonstrated encouraging clinical activity in heavily pre-treated HER2-mutant, HR+, HER2-non-amplified MBC, including patients who had previously received either fulvestrant and/or CDK4/6 inhibitor-based therapies. While the rate of grade 3 diarrhea was higher than that observed with single-agent neratinib in SUMMIT, this was manageable through loperamide prophylaxis, and no patients discontinued study treatment due to diarrhea. SUMMIT has recently been amended to evaluate N+T+F, T+F and F (1:1:1 randomization) and continues to enroll patients. RECIST measurable and efficacy evaluable patients (n=30)Confirmed objective response,a n (%)12 (40)CR0PR12ORR, % (95% CI)40 (23-59)Best overall response, n (%)18 (60)CR0PR18Best overall response rate, % (95% CI)60 (41-77)Medianb DOR, months (95% CI)8.4 (4.1-NE)Clinical benefit,c n (%)14 (47)CR or PR12SD ≥24 weeks2CBR, % (95% CI)47 (28-66)Medianb PFS, months (95% CI)8.3 (4.2-12.5)aORR is defined as either a CR or a PR that is confirmed no less than 4 weeks after the criteria for response are initially met; bKaplan-Meier analysis; cCBR is defined as confirmed CR or PR or SD for ≥24 weeks; CR, complete response; CBR, clinical benefit rate; DOR, duration of response; NE, not estimable; ORR, objective response rate; PFS, progression-free survival; PR, partial response; SD, stable disease. Citation Format: Komal Jhaveri, Cristina Saura, Angel Guerrero-Zotano, Iben Spanggaard, François-Clement Bidard, Jonathan W Goldman, José A García-Sáenz, Andrés Cervantes, Valentina Boni, John Crown, Adam Brufsky, Sherene Loi, Barbara Haley, Ingrid A Mayer, Stephen Chia, Janice Lu, James Waisman, Noa Efrat Ben-Baruch, Mark E Burkard, Jennifer M Suga, Lucía González-Cortijo, Bruno Perrucci, Feng Xu, Sofia Wong, Jie Zhang, Lisa D Eli, Alshad S Lalani, Hans Wildiers. Latest findings from the breast cancer cohort in SUMMIT - a phase 2 ‘basket’ trial of neratinib + trastuzumab + fulvestrant for HER2-mutant, hormone receptor-positive, metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-05.

BackgroundHuman epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations.MethodsIn this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data.ResultsAll three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity inHER2-mutant andEGFR-mutant cells. High expression ofHER2,VTCN1,CDK12, andRAC1correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance.BRCA2mutations were correlated with neratinib and tucatinib response, and high expression ofATM,BRCA2, andBRCA1were associated with neratinib resistance.ConclusionsNeratinib was the most effective HER2-targeted TKI againstHER2-amplified, -mutant, andEGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

Background: HER2 mutations define a subset of metastatic breast cancers (MBCs) with a unique mechanism of oncogenic addiction to HER2 signaling. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been shown to have encouraging clinical activity when combined with fulvestrant in HER2-mutant, hormone receptor-positive (HR+) MBC [Smyth et al. SABCS 2018]. Genomic analyses suggest that acquired resistance to neratinib may occur by the acquisition of additional HER2 alterations, which may amplify HER2 pathway signaling [Won et al. AACR 2019]. We therefore explored whether dual HER2-targeted therapy may improve clinical benefit in this setting. Here we describe initial results from a cohort of patients with HER2-mutant, HR+ MBC treated with neratinib + trastuzumab + fulvestrant (N+T+F) from the phase 2 SUMMIT ‘basket’ trial (NCT01953926). Methods: Patients with HR+ MBC and known oncogenic driver HER2 mutations identified by genomic sequencing were eligible to receive combination treatment with oral neratinib 240 mg daily, intravenous trastuzumab 8 mg/kg initially followed by 6 mg/kg every 3 weeks, and intramuscular fulvestrant 500 mg on days 1 and 15 of month 1, then on day 1 every 4 weeks (N+T+F). Loperamide prophylaxis was mandatory during cycle 1. There was no restriction on the number of prior lines of systemic treatment for MBC. Efficacy endpoints included: confirmed objective response rate and clinical benefit rate - all defined according to RECIST v1.1 - as well as duration of response and progression-free survival. Genomic profiling from fresh/archival tumor tissues and/or plasma cfDNA was performed retrospectively by next-generation sequencing (MSK-IMPACT). Results: As of 01-May-2019, 19 patients were enrolled into the N+T+F cohort and received study treatment (safety population). 20 HER2 mutations were identified in the 19 patients: 14 kinase domain missense mutations, 3 extracellular domain missense mutations, and 3 exon-20 insertion mutations. Median number of prior systemic regimens for metastatic disease was 4 (range 0-10) and histologies were evenly split between lobular and ductal carcinomas. While the majority of patients remain on study treatment (n=15), only 13 of the 19 enrolled patients are efficacy evaluable at this time (having had ≥1 post-baseline tumor assessment). Clinical activity is summarized in the Table. Diarrhea was the most commonly reported adverse event (84.2% any grade) with 5 patients reporting Grade 3 diarrhea (there were no Grade 4 diarrhea events). Three patients (15.8%) reduced neratinib dose due to diarrhea but no patient discontinued treatment due to diarrhea. Conclusions: The combination of N+T+F resulted in an encouraging response rate and was a well-tolerated regimen in predominantly heavily pretreated HER2-mutant HR+ breast cancers. Based on a pre-planned interim analysis, the cohort has been expanded to enroll a total of 50 patients. Updated efficacy and safety data will be presented. Neratinib + trastuzumab + fulvestrant(n=13)Confirmed objective response rate, % (95% CI)39 (13.9-68.4)Complete response0Partial response5 (39)Duration of responses range, months4.2*-10.4*Median progression-free survivala,b, months (95% CI)NA (1.9-NA)*Response ongoing; aKaplan-Meier analysis; bincludes all patients enrolled (n=19); NA, not applicable. Citation Format: Hans Wildiers, Valentina Boni, Cristina Saura, Mafalda Oliveira, Komal Jhaveri, Helen Won, François-Clément Bidard, Adam M Brufsky, Mark E Burkard, Andrés Cervantes, Carlos Fernández-Martos, Barbara Haley, Sherene Loi, Iben Spanggaard, Stefano Panni, Janice Lu, Melanie E Dujka, Feng Xu, Sonia Macia, Lisa D Eli, Alshad S Lalani, Sarina Piha-Paul, Funda Meric-Bernstam, David B Solit, David M Hyman. Neratinib + trastuzumab + fulvestrant for HER2-mutant, hormone receptor-positive, metastatic breast cancer: Updated results from the phase 2 SUMMIT ‘basket’ trial [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 P1-19-08.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib. Significance: HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer. This article is highlighted in the In This Issue feature, p. 161

Human epidermal growth factor receptor (EGFR) 2 (HER2) is overexpressed/amplified in about 25% of all breast cancers, and EGFR is overexpressed in up to 76% and amplified in up to 24% of triple-negative breast cancers (TNBC). Here, we aimed to identify inhibitors that may enhance the anti-tumor activity of neratinib for HER2+ breast cancer and TNBC. By conducting a non-biased high-throughput RNA interference screening, we identified PI3K/AKT/mTOR and MAPK as two potential inhibitory synergistic canonical pathways. We confirmed that everolimus (mTOR inhibitor) and trametinib (MEK inhibitor) enhances combinatorial anti-proliferative effects with neratinib under anchorage-independent growth conditions (p &lt, 0.05). Compared to single agent neratinib, the combination therapies significantly enhanced tumor growth inhibition in both SUM190 HER2+ breast cancer (neratinib plus everolimus, 77%, neratinib plus trametinib, 77%, p &lt, 0.0001) and SUM149 TNBC (neratinib plus everolimus, 71%, neratinib plus trametinib, 81%, 0.0001) xenograft models. Compared to single-agent neratinib, everolimus, or trametinib, both everolimus plus neratinib and trametinib plus neratinib significantly suppressed proliferation marker Ki67 and enhanced antitumor efficacy by activating the apoptosis pathway shown by increased Bim and cleaved-PARP expression. Taken together, our data justify new neratinib-based combinations for both HER2+ breast cancer and TNBC.

Background: Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that inhibits PI3K/Akt and MAPK signaling pathways after HER2 receptor activation. The ExteNET study showed that neratinib significantly improved 5-year invasive disease-free survival (iDFS) in women who completed trastuzumab-based adjuvant therapy for early breast cancer (EBC). We assessed the prognostic and predictive significance of PIK3CA alterations in patients in ExteNET. Methods: Participants were women aged ≥ 18 years (≥ 20 years in Japan) with stage 1–3c (modified to stage 2–3c in February 2010) operable breast cancer, who had completed (neo)adjuvant chemotherapy plus trastuzumab ≤ 2 years before randomization, with no evidence of disease recurrence or metastatic disease at study entry. Patients were randomized to oral neratinib 240 mg/day or placebo for 1 year. Formalin-fixed, paraffin-embedded primary tumor specimens underwent polymerase chain reaction (PCR) PIK3CA testing for two hotspot mutations in exon 9, one hot-spot mutation in exon 20, and fluorescence in situ hybridization (FISH) analysis for PIK3CA amplification. The primary endpoint (iDFS) was tested with log-rank test and hazard ratios (HRs) estimated using Cox proportional-hazards models. Results: Among the intent-to-treat population (n = 2840), tumor specimens were available for PCR testing (991 patients) and PIK3CA FISH (702 patients). Overall, 262 samples were PIK3CA altered: 201 were mutated (77%), 52 (20%) were amplified, and 9 (3%) were mutated and amplified. iDFS was non-significantly worse in placebo-treated patients with altered vs wild-type PIK3CA (HR 1.34; 95% CI 0.72–2.50; P = 0.357). Neratinib’s effect over placebo was significant in patients with PIK3CA-altered tumors (HR 0.41; 95% CI 0.17–0.90, P = 0.028) but not PIK3CA wild-type tumors (HR 0.72; 95% CI 0.36–1.41; P = 0.34). The interaction test was non-significant (P = 0.309). Conclusions: Although there was a greater absolute risk reduction associated with neratinib treatment of patients with PIK3CA-altered tumors in ExteNET, current data do not support PIK3CA alteration as a predictive biomarker of response to neratinib in HER2-positive EBC. Trial registration: ClinicalTrials.gov , NCT00878709 . Trial registered April 9, 2009.

Càncer de coll uterí; Assaig clínic; Mutant HER2 Cáncer de cuello uterino; Ensayo clínico; Mutante HER2 Cervical cancer; Clinical trial; HER2 mutant Objective: Somatic HER2 mutations occur in ~5% of cervical cancers and are considered oncogenic and associated with poor prognosis. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, is active in multiple HER2-mutant cancers. SUMMIT is a phase II basket trial investigating the efficacy and safety of neratinib in solid tumors. Methods: Patients with HER2-mutant, persistent, metastatic/recurrent cervical cancer with disease progression after platinum-based treatment for advanced/recurrent disease received oral neratinib 240 mg/day with mandatory loperamide prophylaxis during cycle 1. The primary endpoint was confirmed objective response rate (ORR). Secondary endpoints included: response duration (DOR); clinical benefit rate (CBR); progression-free survival (PFS); overall survival (OS); safety. Results: Sixteen eligible patients were enrolled; 10 (62.5%) had endocervical adenocarcinoma. The most common HER2 mutation was S310F (63% of patients). Three of 12 RECIST-measurable patients had confirmed partial responses (ORR 25%; 95%CI 5.5-57.2%); 3 had stable disease ≥16 weeks (CBR 50%; 95%CI 21.1-78.9%). DOR for responders were 5.6, 5.9, and 12.3 months. Median PFS was 7.0 months (95%CI 0.7-18.3 months); median OS was 16.8 months (95%CI 4.1-NE months). Diarrhea (75%), nausea (44%), and decreased appetite (38%) were the most common adverse events. One patient (6%) reported grade 3 diarrhea. There were no grade 4 events, and no diarrhea-related treatment discontinuations. Conclusions: Neratinib monotherapy showed evidence of activity in heavily pretreated patients with HER2-mutant cervical cancer, with no new safety signals. Given the few effective options for cervical cancer after platinum-based therapy failure, neratinib warrants further investigation in this molecularly defined patient population. This work was supported by Puma Biotechnology Inc . 10880 Wilshire Blvd, Suite 2150, Los Angeles, CA 90024, USA.

Background: Neratinib is a pan-HER tyrosine kinase inhibitor that blocks the PI3K/Akt and MAPK signaling pathways downstream from HER2. The international, randomized, placebo-controlled phase III ExteNET trial showed that a 1-year course of neratinib after trastuzumab-based adjuvant therapy significantly improved 2-year invasive disease-free survival (iDFS) in early-stage HER2+ breast cancer (HR 0.67; 95% CI 0.50–0.91; p=0.0091) [Chan et al. Lancet Oncol 2016]. Furthermore, the effects of neratinib on iDFS were shown to be durable at 5 years' follow-up (HR 0.73; 95% CI 0.57–0.92; p=0.008) [Martin et al. ESMO 2017]. PIK3CA alterations are common in HER2+ breast cancers, and in general are associated with a worse prognosis. We sought to assess the prognostic and predictive significance of PIK3CA alterations in an exploratory substudy of the ExteNET trial. Methods: ExteNET is an international, multi-center, randomized, double-blind, placebo-controlled phase III trial (Clinicaltrials.gov: NCT00878709). Patients received oral neratinib 240 mg/day or placebo for 1 year. Of the intent-to-treat (ITT) population (n=2840), primary formalin-fixed paraffin-embedded (FFPE) tumor specimens were available from 991 patients for PIK3CA mutation testing by RT-PCR for two hot-spot mutations in exon 9 (E542K, E545K/D) and one hot-spot mutation in exon 20 (H1047R). 702 FFPE tumor slides underwent FISH analysis for PIK3CA amplification with a ratio of ≥2.2 considered as amplified. Primary endpoint: iDFS. iDFS events were tested by 2-sided log-rank tests, and HR (95% CI) were estimated using Cox proportional-hazards models. Data cut-off: March 2017. Results: Baseline demographics and disease characteristics between treatment arms of the correlative cohort (n=1201) were balanced. Overall, 21.2% (n=210) of primary tumors harbored one of the specified PIK3CA mutations, and 8.7% (n=61) were PIK3CA FISH-amplified. Patients with PIK3CA-altered tumors (i.e. PIK3CA mutations or FISH-amplified) had fewer iDFS events with neratinib compared with placebo (HR 0.41; 95% CI 0.17-0.90, p=0.028). The interaction test was not significant (p=0.1842). Results of the various correlative analyses within treatment arms are shown in the table. NeratinibPlacebo iDFS iDFS 2-sidedPopulationnevents, nnevents, nHR (95% CI)P valueaITT142011614201630.73 (0.57–0.92)b0.008bCorrelative cohort59345608700.67 (0.45–0.96)0.0317PIK3CA-mutation positive1047106170.43 (0.17–1.01)0.056PIK3CA-mutation negative38527396420.66 (0.40-1.06)0.089PIK3CA-amplified3312840.20 (0.01-1.33)0.106PIK3CA-non-amplified31629325360.85 (0.52-1.39)0.521PIK3CA-altered1308132200.41 (0.17-0.90)0.028a. Log-rank test; b. Stratified analysis Conclusions: One year of neratinib treatment after trastuzumab-based adjuvant therapy significantly improves iDFS after 5 years in patients with early-stage HER2+ breast cancer. From this modest-sized exploratory cohort, it appears that PIK3CA may be a biomarker for differential sensitivity to neratinib after 1 year of trastuzumab in the adjuvant setting.These exploratory results should be validated in a larger subset. Citation Format: Chia SKL, Martin M, Holmes FA, Ejlertsen B, Delaloge S, Moy B, Iwata H, von Minckwitz G, Mansi J, Barrios CH, Gnant M, Tomašević Z, Denduluri N, Šeparović R, Kim S-B, Hugger Jakobsen E, Harvey V, Robert N, Smith II J, Harker G, Lalani AS, Zhang B, Eli LD, Buyse M, Chan A. PIK3CA alterations and benefit with neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: Correlative analyses of the phase III ExteNET 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 PD3-12.

Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.

Background: We previously showed preclinical and clinical activity of combined inhibition of EGFR and PARP with lapatinib and veliparib in triple negative breast cancer (Nowsheen et al. PLoS One 2012;7(10):e46614; Reasor et al. J Clin Onc 2018; 36(15_suppl):1095), due to an induced homologous recombination repair defect with EGFR inhibition sensitizing cells to PARP inhibition. In this study, we tested the in vitro and in vivo activity of the irreversible pan-HER tyrosine kinase inhibitor, neratinib, with the PARP inhibitor olaparib or niraparib. Methods: Human triple negative breast cancer (TNBC) cells MDA-MB231 (BRCA wt), MDA-MB453 (BRCA wt), and MDA-MB436 (BRCA1 mutant) and Mouse TNBC 4T1 cells (BRCA wt) were used. Cell proliferation and colony formation assays were performed to assess the in vitro activity of various concentrations of neratinib and olaparib. Markers of DNA damage were measured via gamma-H2AX western blot analysis. In vivo effects of combination treatments on tumor metastasis were tested using intracardiac injection of tumor cells labelled with luciferase and mice were monitored for survival. Results: Combining neratinib with olaparib or niraparib significantly decreased proliferation compared to either agent alone in MDA-MB231, MDA-MB453, MDA-MB436 and 4T1 cells. Similar results were observed using colony formation assays. Cytotoxicity was associated with increased gamma-H2AX levels, indicating persistent DNA damage. Mice treated with 20mg/kg/day of neratinib and 100mg/kg/day of olaparib after MDA-MB231-Luc intracardiac injection exhibited reduced tumor metastases compared to either agent alone, resulting in better survival (p=0.0088). Conclusions: Combining neratinib with PARP inhibition induces synthetic lethality resulting in increased anti-tumor activity in TNBC in cell lines and in xenografts. Future clinical trials testing these combinations are warranted. Citation Format: Chuan Xing, Zhuo Zhang, Deborah Della Manna, Irmina Diala, Lisa Eli, Alshad S. Lalani, Eddy Shih-Hsin Yang. Neratinib induces synthetic lethality with PARP inhibitors in triple negative breast cancer cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1181.

Hundreds of somatic ERBB4 mutations have been described in cancer tissues with very limited information about their functional significance. However, analyses of individual mutants have indicated that activating ERBB4 mutations, such as ERBB4 K935I, do exist. Understanding the functional consequences of ERBB4 mutations is needed in order to assess the relevance of targeting ERBB4 in human cancers with matched therapies such as neratinib. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that binds to and potently inhibits ERBB4 kinases activity in-vitro, and is currently approved clinically for the treatment of early stage and metastatic HER2+ breast cancers. We set up to address the transforming potential of individual ERBB4 variants by selecting 18 mutations (Table 1) from cBioPortal data (www.cbioportal.org) using the following criteria: 1) the mutations were recurrent, 2) they were analogous to activating mutations described for other oncogenic ERBB family members and/or 3) their position at receptor dimerization interfaces suggested functional relevance. Retroviral pBABE-puro-gateway vector was used to achieve functional, but still near-physiological expression levels of ERBB4 in different cell backgrounds. A screen for optimal cellular background to assess the transforming potential of ERBB4 variants indicated that 1) IL-3 independent growth of mouse lymphoid Ba/F3 cells, 2) focus formation analysis of mouse NIH-3T3 fibroblasts, and 3) ligand-induced proliferation of human mammary epithelial MCF-10A provided read-outs with robust differences when the effects of the positive control mutant ERBB4 K935I was compared to wild-type ERBB4. These models are now being used to address the transforming potential of the 18 mutations (Table 1). Table 1.Somatic ERBB4 mutations chosen for functional analyses.R106CE452KR711CL798RG870RK1223TS303FR524CG741EV840IG907ES1289AR393WR544WS774GR847HR992CR1304W Citation Format: Peppi Suominen, Deepankar Chakroborty, Kari J. Kurppa, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, Klaus Elenius. Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2420.

9068 Background: The phase 2 SUMMIT basket trial (NCT01953926) demonstrated efficacy of neratinib in patients with EGFR exon 18-mutant NSCLC [Boni et al. WCLC 2020]. Neratinib also has documented activity in HER2+ metastatic breast cancer with CNS metastases [Saura et al. SABCS 2020 & J Clin Oncol 2020]. Here we report neratinib efficacy in a subgroup of patients with EGFR exon 18-mutant NSCLC and CNS involvement from SUMMIT. Methods: Patients with EGFR exon 18-mutant NSCLC were treated with single-agent neratinib (240 mg po daily). Prior EGFR tyrosine kinase inhibitors (TKIs), chemotherapy, and checkpoint inhibitors (IO) were allowed. Patients with stable, asymptomatic CNS metastasis were enrolled. Study endpoints: objective response rate (ORR) at week 8 (±1 week); ORR (RECIST 1.1 confirmed); duration of response (DOR); clinical benefit rate (CBR); progression-free survival (PFS); safety; biomarkers. Results: Baseline characteristics of 11 patients with EGFR exon 18-mutant NSCLC: median age 67 (range 56–83) years; ECOG PS 0/1 (45%/55%). Prior lines of therapies: 2 (range 1–3): EGFR TKIs (91%); chemotherapy (55%); IO (27%). 3/11 patients had baseline CNS metastasis and received radiation 8–22 months prior to study enrollment. Best CNS response in these 3 patients was stable disease with overall individual PFS of 1.9 (censored), 6.9 and 9.1 months and OS of 2.6 (censored), 17.7 (censored), and 17.9 months. Efficacy is summarized in Table. Efficacy summary: TKI-pretreated EGFR exon 18-mutant NSCLC cohort receiving neratinib monotherapy. Conclusions: Activity of single-agent neratinib was observed in prior TKI-exposed patients with EGFR exon 18-mutant NSCLC. Despite the small sample size of only 3 patients with baseline CNS metastases, findings suggest a potential role for neratinib as a systemic treatment option for patients with NSCLC and difficult-to-treat uncommon mutations with CNS involvement. The SUMMIT trial continues to enroll patients with EGFR exon 18-mutant NSCLC. Clinical trial information: NCT01953926. [Table: see text]

310 Background: FLC, a rare liver cancer of young adults, has no effective systemic therapies. Surgical resection is used extensively with non-curative intent. FLC is associated with a DNAJB1- PRKACA chimeric transcript that produces a fusion protein with retained kinase activity and increased expression of several oncogenic signaling pathways including, but not limited to, HER2 ( ERBB2). Methods: N (240 mg oral daily) was studied in FLC pts in the SUMMIT study (NCT01953926); and later under compassionate use for N-based combinations (combo): P (2 mg/kg q3w), E (7.5 mg daily), and V (240 mg q2w) in doublet or triplet regimens. Eligible pts: ≥12y; histologically confirmed FLC; adequate organ function; any number of prior therapies. Primary endpoint: objective response rate (ORR; RECIST v1.1). Secondary endpoints: duration of response; clinical benefit rate (CBR); safety (CTCAE v4.0); somatic and germline sequencing (MSK IMPACT). Results: As of 03-Sep-2020, 15 pretreated pts received N in SUMMIT (confirmed ORR 0%; CBR 13%). Efficacy data for 5 pts from SUMMIT and 2 more pts receiving combo under compassionate use (4 male, 3 female, median age 26 years, median 0 [range 0–4] prior systemic therapies) are in shown in the table. The most common adverse events (AE) with single-agent N (n = 5) were diarrhea (grade 1 80%; grade 2 20%) and nausea (grade 1 60%); other AEs were grade ≤1 in ≤20% of pts. Conclusions: N monotherapy had limited benefit as a single agent in FLC pts. Several case studies evaluating N-based combo with checkpoint inhibitors administered under compassionate use demonstrated that NP led to 1 PR, and the triplet of NPE to prolonged SD. These are case-limited observations but are critical and worth evaluating further in upcoming clinical trials given the continued lack of a standard of care therapy for pts with FLC. Clinical trial information: NCT01953926. [Table: see text]

Purpose: Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer (MBC) is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. We and others previously described acquired activating hotspot HER2 (ERBB2) mutations in ~5% of ER+ MBC that conferred resistance to multiple ER-targeting therapies, including the selective estrogen receptor degrader fulvestrant. These tumors could be re-sensitized to fulvestrant in vitro through addition of the irreversible pan-HER tyrosine kinase inhibitor neratinib, suggesting a possible clinical combination strategy for patients. Although some HER2 mutations are relatively more frequent in tumors, there is a “long tail” of rare HER2 mutations that have not been characterized but remain clinically important for patients whose tumors harbor them. Therefore, there is biological and clinical value in prospectively characterizing all possible missense mutations in HER2. Methodology: Since only activating HER2 mutations conferred resistance to fulvestrant (and not passenger or inactivating mutations), resistance to fulvestrant in ER+ breast cancer cells can be used as a surrogate kinase assay for HER2 activity. Therefore, we are performing a saturation mutagenesis screen of HER2, using fulvestrant resistance as a readout for activating mutants. Screen optimization included: a) testing selected mutants cloned into a custom vector to identify appropriate positive and negative controls for screen QC, b) custom screen design (transduction under ER inhibition, and an empirically-determined ratio of growth in fulvestrant vs DMSO) to enable recovery of mutants of varying growth phenotypes. We also designed PCR conditions to enable efficient amplification of HER2 (the largest ORF ever tested by saturation mutagenesis) from genomic DNA, and custom-designed and built a comprehensive HER2 library that includes built-in controls such as stop codons. The saturation mutagenesis screen is currently underway, and will generate putative activating HER2 mutations (i.e., not growth-inhibited in fulvestrant versus complete media), that will be validated in a “minipool” screen. Validated hits will be further tested for sensitivity to the combination of fulvestrant+neratinib or other kinase inhibitors. Summary and conclusions: We have designed a saturation mutagenesis screen to recover a spectrum of activating HER2 mutations, the largest such library generated to date, and using resistance to the ER inhibitor fulvestrant as a novel surrogate kinase assay. This screen will generate a comprehensive reference table of HER2 mutant phenotypes in terms of response and resistance to ER and HER2 targeting agents. These findings will have translational applicability, and may suggest promising precision medicine approaches for clinical management of patients harboring somatic HER2 mutations. Citation Format: Utthara Nayar, Federica Piccioni, Xiaoping Yang, David Root, J T. Neal, Lisa D. Eli, Irmina Diala, Alshad S. Lalani, Nikhil Wagle. Phenotypic characterization of a comprehensive set of HER2 missense mutants in ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1914.

An estimated 15-20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2/ERBB2/neu). Two small-molecule tyrosine kinase inhibitors (TKIs), lapatinib and neratinib, have been approved for the treatment of HER2-positive (HER2+) breast cancer. Lapatinib, a reversible epidermal growth factor receptor (EGFR/ERBB1/HER1) and HER2 TKI, is used for the treatment of advanced HER2+ breast cancer in combination with capecitabine, in combination with trastuzumab in patients with hormone receptor-negative metastatic breast cancer, and in combination with an aromatase inhibitor for the first-line treatment of HER2+ breast cancer. Neratinib, a next-generation, irreversible pan-HER TKI, is used in the US for extended adjuvant treatment of adult patients with early-stage HER2+ breast cancer following 1 year of trastuzumab. In Europe, neratinib is used in the extended adjuvant treatment of adult patients with early-stage hormone receptor-positive HER2+ breast cancer who are less than 1 year from the completion of prior adjuvant trastuzumab-based therapy. Preclinical studies have shown that these agents have distinct properties that may impact their clinical activity. This review describes the preclinical characterization of lapatinib and neratinib, with a focus on the differences between these two agents that may have implications for patient management. Published version

Background: Mutations in ERBB2, encoding the HER2 protein, occur in up to 4% of breast cancers. A subset of ERBB2 mutations have been shown to have oncogenic potential through constitutive activation of the HER2 kinase. SUMMIT (NCT01953926) is a phase II ‘basket' study investigating the use of the irreversible pan-HER tyrosine kinase inhibitor neratinib, in combination with the estrogen degrader fulvestrant, for the treatment of patients with advanced hormone-positive breast cancers harboring ERBB2 mutations. We collected and sequenced serial samples of circulating cell-free DNA (cfDNA) from participating patients in order to identify biomarkers of response and resistance. Methods: Patients were treated with neratinib 240 mg daily and fulvestrant at the labelled 500 mg dose. Plasma was collected and cfDNA was extracted from patients at baseline and on Day 1 of each cycle (occurring every 4 weeks). Next generation sequencing (NGS) was performed on cfDNA samples collected from 12 patients using the Guardant360 test (Guardant Health, CA), which identifies select point mutations, small insertions and deletions, copy number changes, and structural rearrangements in 73 genes. Paired samples were sequenced from 9 of the 12 patients, including baseline and progression samples from 7 patients. cfDNA NGS results were compared to pretreatment tissue NGS results obtained 0-6 years prior (median: 6 months). Results: 90% (9/10) baseline cfDNA NGS detected the locally reported ERBB2 mutation identified by tissue NGS. In one patient with only a sample obtained at progression, the ERBB2 mutation was identified. No ERBB2 mutation was identified in a singular on-treatment sample obtained from a patient with continued response to therapy. Eight ERBB2 mutations were identified in the baseline cfDNA sample of one patient with a hypermutated tumor, 6 of which were not identified by tissue NGS. Emergence of at least one additional ERBB2 mutation was seen in 3/7 patients with paired baseline and progression samples, including patients with 2 and 5 acquired ERBB2 mutations. The ERBB2 mutant allele fraction (MAF) increased compared to baseline in 4/7 of patients at the time of progression and declined in 2/2 patients with continued response to therapy. cfDNA NGS detected mutations, unidentified by tissue NGS in 9/10 baseline samples and in 11/12 patients, including 5 ESR1 mutations in 3 patients. Conclusion: cfDNA NGS was sensitive for the detection of ERBB2 mutations. MAF dynamics typically tracked response to, or progression on therapy, at the time of cfDNA collection. The emergence of additional ERBB2 mutations was common at the time of progression; however, no T798 gatekeeper mutation was observed. Many subclonal mutations were identified on cfDNA NGS that were not observed on tissue NGS. Citation Format: Alison M. Schram, S. Duygu Selcuklu, Rebecca J. Nagy, Lillian M. Smyth, Lisa D. Eli, Richard E. Cutler, Alshad S. Lalani, David M. Hyman, Richard B. Lanman. Cell-free DNA sequencing in ERBB2-mutant breast cancer patients treated with neratinib and fulvestrant: Exploratory analysis from the Phase 2 SUMMIT ‘basket' trial [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 5533.

Background: Somatic HER2 (ERBB2) and HER3 (ERBB3) mutations can lead to constitutive HER2 activation in the absence of gene amplification. They occur in a variety of solid tumors with a prevalence that does not exceed 5-10% in any tumor type. Activating HER2 mutations occur in the HER2 receptor extracellular and transmembrane domains, and at multiple hotspots in the kinase domain, with no single mutation predominating. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, inhibits growth of HER2- and HER3-mutant tumors in preclinical models. We explored the clinical activity of neratinib in pts with HER2/HER3 mutant cancers in SUMMIT, a global (9 countries), multicenter, multi-histology, phase II ‘basket’ trial. Methods: Pts with advanced solid tumors and locally documented HER2/HER3 mutations received neratinib 240 mg daily with high-dose loperamide prophylaxis during cycle 1. Primary endpoint: objective response rate at week 8 (ORR8) per RECIST 1.1; pts without RECIST-measurable disease were assessed separately using FDG-PET. Retrospective confirmation of HER2/HER3 mutations, as well as a broader cancer gene profiling, was performed using both tumor and plasma-derived cfDNA. For each tumor type, using Simon’s optimal 2-stage design, a true ORR8 ≤10% was considered unacceptable (null hypothesis) whereas a true ORR8 ≥30% (alternative hypothesis) merited further study. Interim data cut: 16-Dec-2016. Clinicaltrials.gov: NCT01953926. Results: 141 (125 HER2, 16 HER3) pts have received neratinib including 125 efficacy-evaluable pts (110 HER2, 15 HER3). 21 unique cancer types were enrolled (most common: breast, lung, bladder, colorectal cancer). 30 HER2 and 12 HER3 mutations were observed, the most frequent HER2 variants involving S310, L755, A755_G776insYVMA and V777. In the HER2-mutant cohort, clinical responses were observed in tumors with S310, L755, V777, P780_Y781insGSP, and A775_G776insYVMA. When stratified by tumor type, responses were observed in pts with breast, cervical, biliary, salivary and non-small-cell lung cancers, which led to indication-specific cohort expansions. No activity was observed in the HER3-mutant cohort. Tumor and/or cfDNA from 116 pts has been sequenced centrally. Of these, the locally reported HER2/3 mutation was confirmed in 108 (93%), a discordant HER2/3 mutation detected in 5 (4%), and not detected in 3 (3%). Co-mutation patterns in different tumor types and their association with neratinib sensitivity/resistance are under review. Conclusions: SUMMIT provides the largest body of clinical data to date on the use of a pan-HER inhibitor in pts with solid tumors with somatic HER2/HER3 mutations. Responses in HER2 mutants varied by type of tumor and the specific HER2 mutation, suggesting: 1) mutant HER2 is a driver oncogene in some cancers; and 2) not all mutations generate the same level of HER2 hyperactivity and/or oncogene dependence. Citation Format: David M. Hyman, Sarina A. Piha-Paul, Jordi Rodon, Cristina Saura, Geoffrey I. Shapiro, David I. Quinn, Victor Moreno, Ingrid A. Mayer, Carlos Arteaga, Valentina Boni, Emiliano Calvo, Sherene Loi, Albert C. Lockhart, Lillian M. Smyth, Joseph Erinjeri, Maurizio Scaltriti, Gary Ulaner, Jean Torrisi, Juber Patel, Jiabin Tang, Fanli Meng, Duygu Selcuklu, Helen Won, Nancy Bouvier, Michael F. Berger, Richard E. Cutler, Feng Xu, Anna Butturini, Lisa D. Eli, Grace Mann, Alshad S. Lalani, Richard P. Bryce, Funda Meric-Bernstam, José Baselga, David B. Solit. Neratinib in HER2 or HER3 mutant solid tumors: SUMMIT, a global, multi-histology, open-label, phase 2 "basket" study [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 CT001. doi:10.1158/1538-7445.AM2017-CT001

e12554 Background: ERBB2 mutations in the absence of gene amplification are rare, with an incidence of 2-4%. Neratinib is a HER2/EGFR tyrosine kinase inhibitor being evaluated for use in ERBB2 mutated breast cancer. Neratinib has been found to have clinical activity on heavily pre-treated ERBB2 mutant breast cancer patients. We are evaluating the response and genomic profiles of 3 postmenopausal patients with metastatic ERBB2 mutant/non-amplified breast cancer receiving neratinib and fulvestrant NCT01953926, NCT01670877. Methods: Samples were collected at different points during treatment and CTCs were identified. Other representative cells were tracked but not classified as CTCs. CD45-CK+ cells with cytoplasmic and/or nuclear apoptosis were defined as CTC-Apoptotic. CD45- cells expressing little to no CK but otherwise meeting morphological criteria for CTCs are CTC-LowCK. CD45-CK+ cells with small nuclear size are CTC-SmallCK. Single CTCs will be analyzed using whole genome copy number variation to determine chromosomal alterations. Results: The patients had an average of 4.7 lines of therapy for metastatic disease before neratinib. Two had stable disease and one had progression. The patient who progressed had a rise in the number of CTCs from 37 to 52 cells/ml and drop in apoptotic cells from 5 to 0 cells/ml. Conclusions: The high CTC count of the patient who progressed may suggest more aggressive disease. The drop in the apoptotic cell count may correlate with a failure to respond to therapy. Samples have been sent for copy number variation profiling. The goal is to identify any genomic amplifications or deletions associated with clinical response and progression after targeted therapy. We hope to demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the use of fluid biopsies to monitor disease progression. [Table: see text]