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Title: Drug target activation mapping of matched triple negative primary and axillary LN metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation Background: Triple negative breast cancer (TNBC) is comprised of heterogeneous subtypes that arise from various molecular alterations that result in TNBC having the poorest overall prognosis. There is growing interest in more sensitive ways to measure estrogen receptor (ER) and HER2 levels in the ER “negative” and HER2 “low/negative” settings, as there are patients with TNBC whose tumors are actually ER and/or HER2 “expressing/active”, and who may benefit from ER- and/or HER2-targeted therapies. Given the limitations of IHC in reliably quantitating ER and HER2 in low (0-1+) expressing tumors, we utilized highly sensitive reverse phase protein array (RPPA) to quantitate expression of these therapeutic targets along with downstream signaling activation mapping in a unique pilot set of patient-matched primary (P) and axillary lymph node (LN) metastases obtained synchronously. The quantitative expression of steroid hormone receptor expression/activation (ER, glucocorticoid receptor (GR), androgen receptor (AR)) along with their signaling architecture and dynamics were analyzed. Methods: A 12 P-LN paired tumor set was chosen for analysis (N=24). Manual macrodissection was used to enrich for tumor epithelium prior to RPPA analysis that quantitatively measured 155 proteins/phosphoprotein drug targets in key signaling pathways known to be involved in steroid hormone receptor biology and tumorigenesis. Statistical analysis using paired sample t-testing or Wilcoxon rank testing was performed (p< 0.05 uncorrected). Results: Protein pathway activation mapping and unsupervised clustering analysis revealed a subset of TNBCs from 3 of the 12 (25%) patients (N=4: 1 LN and 1 P, and one patient-matched LN and P) that was characterized by high relative levels of total ER. Interestingly, these same tumors also had amongst the highest relative levels of both total AR and activated (phosphorylated) GR compared to the rest of the 20 tumor samples. Downstream signaling analysis found significant (p< 0.05) activation/phosphorylation of AKT (S473 and/or T308) associated with the ER-AR-GR/steroid hormone activated/”high” TNBC signature compared to the 20 tumors that were steroid hormone “low”. Conclusions: Functional protein pathway activation mapping of a unique study set of synchronously obtained TNBC primary and LN metastasis revealed a steroid hormone receptor expression/activation signature of co-incidentally activated/expressed ER, GR and AR, that was also characterized by increased AKT activation (p< 0.05). This steroid hormone receptor “activated” signature was not observed to be significantly differentially expressed in LN compared to P tissues in cohort or matched pair analysis, suggesting that this signature may be present as an intrinsic event in the P, and not acquired in LN metastases. Recent data from stage I-III TNBC patients treated in the neoadjuvant setting with an AKT inhibitor1 revealed that responding TNBC patients had pre-treatment tumor biopsies that were enriched for both high AR expression and high phosphoAKT (S473 and/or T308) levels. If these observations that a subset of TNBCs are steroid hormone receptor “activated” despite being ER “negative” are confirmed in larger study sets, we hypothesize that this subgroup, identified by RPPA analysis of both P and LN metastases, could be potentially sensitive to AKT inhibition in combination with agents that target steroid hormone receptors. 1Shi Z, et al. Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer. Clin Cancer Res. 2022 Mar 1;28(5):993-1003 Citation Format: Julia Wulfkuhle, Page Blas, Heather Williams, Claudius Mueller, Rosa I. Gallagher, Mariaelena Pierobon, Joyce O’Shaughnessy, Emanuel F. Petricoin. PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-08.

Breast cancer is the major leading cause of cancer-related fatalities nationally and globally. There will be 287,850 new breast cancer cases and 43,250 breast cancer related deaths in the United States in 2022. The relapsed, treatment-resistant, undruggable, and incurable breast cancers are often associated with EGFR/HER2/K-RAS/SIAH pathway activation. The EGFR/HER2/K-RAS/SIAH pathway is a major tumor-driver whose hyperactivation is associated malignant tumor growth, multidrug-resistant phenotypes, early tumor relapse, and systematic metastasis. Seven-In-Absentia (SINA) homologues (SIAH) are extraordinarily evolutionarily-conserved E3 ubiquitin ligases that play a critical gatekeeper role downstream of the EGFR/HER2/K-RAS pathway. SIAH is a major tumor vulnerability that is ideally positioned to become an attractive target for innovative targeted therapy development against metastatic breast cancer (MBC). Prior studies have shown that tumor growth was abolished in malignant tumor cell lines such as MDA-MB-231, MDA-MB-468, MiaPaCa, A459, and HeLa following SIAH inhibition in xenograft models; however, the underpinning molecular mechanisms that give rise to this striking anti-EGFR/K-RAS and anticancer phenotype remain unclear. Specific objectives: to delineate the molecular mechanism(s) of why anti-SIAH2PD targeted therapy is so effective in impeding and eradicating the stage IV and aggressive tumors, we conducted reverse phase protein array (RPPA)-based kinomic analysis to delineate how major cancer signaling pathways and EGFR/HER2/K-RAS/SIAH-dependent signaling networks are rewired and remodeled in response to anti-SIAH2 targeted therapy. Brief statement of methods: About 300 proteins/phosphoproteins were quantititatively measured by the RPPA platform to identify new tumor vulnerabilities and actionable targets, compensatory signaling network activation/inhibition in response to anti-SIAH targeted therapies in five highly malignant cancer cell lines. Doxycycline (DOX)-inducible Tet-ON MDA-MB-231, MDA-MB-468, MiaPaCa, HeLa and A459 cell lines were amplified from single cell and DOX-induced SIAH2PD expression was confirmed. Each of the cell lines was then subjected to one of four experimental conditions: Tet-ON control cells without DOX induction (group A), Tet-ON control cells with DOX induction (group B), Tet-ON-SIAH2PD cancer cells without DOX-induction (no SIAH2PD inhibitor) (group C), Tet-ON-SIAH2PD cancer cells with DOX-induction (SIAH2PD inhibitor) (group D). Reverse Phase Protein Array (RPPA) in conjunction with Principal Component Analysis (PCA) was conducted to quantify fold-changes of proteins/phosphoproteins in response to SIAH inhibition. The ratios of D/C/B/A, D/C, D/B, C/A, and B/A were calculated using GAPDH normalized data. Summary of results: Supported by statistical analyses, we identified 6 unique phospho-proteins that were either up- or down-regulated in response to SIAHLoss-of-function. Many have known roles in controlling and regulating cell growth, cell death, NFκB signaling, stress response, DNA damage, and cell attachment pathways, supporting a tumor eradication phenotype in the absence of SIAH function in these cancer cell lines. Conclusion: Cancer landscape (CScape) functional protein pathway mapping has categorized the synergistic feedforward, feedback, and compensatory signaling pathway activation/inactivation in response to SIAH blockade in these EGFR/HER2/K-RAS-driven malignant human cell lines. Further validation analysis will be conducted to gain better insight into the global cancer pathway alterations to reveal the molecular mechanism(s) of why SIAH inhibition works so effectively to shut down malignant tumor growth in the preclinical models. Citation Format: Andrew P. Howell, Julia Wulfkuhle, Rosa I. Gallagher, Emanuel F. Petricoin, Amy H. Tang. The Use of Reverse Phase Protein Arrays (RPPA), Principal Component Analysis (PCA), and CScape Pathway Mapping to Identify New Tumor Vulnerability and Actionable Targets in Human Malignant Triple-Negative Breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-13.

Purpose:Despite extensive genomic and transcriptomic profiling, it remains unknown how signaling pathways are differentially activated and how tumors are differentially sensitized to certain perturbations. Here, we aim to characterize AKT signaling activity and its association with other genomic or IHC-based PI3K/AKT pathway biomarkers as well as the clinical activity of ipatasertib (AKT inhibitor) in the FAIRLANE trial.Experimental Design:In FAIRLANE, 151 patients with early triple-negative breast cancer (TNBC) were randomized 1:1 to receive paclitaxel with ipatasertib or placebo for 12 weeks prior to surgery. Adding ipatasertib did not increase pathologic complete response rate and numerically improved overall response rate by MRI. We used reverse-phase protein microarrays (RPPA) to examine the total level and/or phosphorylation states of over 100 proteins in various signaling or cell processes including PI3K/AKT and mTOR signaling. One hundred and twenty-five baseline and 127 on-treatment samples were evaluable by RPPA, with 110 paired samples at both time points.Results:Tumors with genomic/protein alterations in PIK3CA/AKT1/PTEN were associated with higher levels of AKT phosphorylation. In addition, phosphorylated AKT (pAKT) levels exhibited a significant association with enriched clinical benefit of ipatasertib, and identified patients who received benefit in the absence of PIK3CA/AKT1/PTEN alterations. Ipatasertib treatment led to a downregulation of AKT/mTORC1 signaling, which was more pronounced among the tumors with PIK3CA/AKT1/PTEN alterations or among the responders to the treatment.Conclusions:We showed that the high baseline pAKT levels are associated with the alterations of PI3K/AKT pathway components and enriched benefit of ipatasertib in TNBC.

AKT S473 distribution based on ER, HER2 expression measured by IHC in for primary and metastatic lesions included in the validation set.

Supplementary material legend

Concordance between PIK3CA, AKT, and PTEN mutation status and AKT (S473) and p70S6 (T389) activation.

Supplementary Table from Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Supplementary Figure from Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer

HER2-targeted therapy dramatically improves outcomes in early breast cancer. Here we report the results of two HER2-targeted combinations in the neoadjuvant I-SPY2 phase 2 adaptive platform trial for early breast cancer at high risk of recurrence: ado-trastuzumab emtansine plus pertuzumab (T-DM1/P) and paclitaxel, trastuzumab and pertuzumab (THP). Eligible women have >2.5 cm clinical stage II/III HER2+ breast cancer, adaptively randomized to T-DM1/P, THP, or a common control arm of paclitaxel/trastuzumab (TH), followed by doxorubicin/cyclophosphamide, then surgery. Both T-DM1/P and THP arms ‘graduate’ in all subtypes: predicted pCR rates are 63%, 72% and 33% for T-DM1/P (n = 52), THP (n = 45) and TH (n = 31) respectively. Toxicity burden is similar between arms. Degree of HER2 pathway signaling and phosphorylation in pretreatment biopsy specimens are associated with response to both T-DM1/P and THP and can further identify highly responsive HER2+ tumors to HER2-directed therapy. This may help identify patients who can safely de-escalate cytotoxic chemotherapy without compromising excellent outcome., HER2-targeted therapy improves patient’s outcome in early breast cancer. Here, the authors present the efficacy and biomarker analysis of two HER2-targeted combinations (ado-trastuzumab emtansine plus pertuzumab and paclitaxel, trastuzumab and pertuzumab) in the context of the neoadjuvant I-SPY2 phase 2 adaptive platform trial for early breast cancer at high risk of recurrence.

Background: Most estrogen receptor alpha positive (ER+) breast cancers (BCs) express androgen receptor (AR) protein, but its function is unclear. High AR relative to ER is associated with endocrine resistance. This randomized phase II trial of neoadjuvant fulvestrant (F) with or without the anti-androgen enzalutamide (E) was designed to determine if the addition of E to F in women with ≥T2 ER+/Her2- primary BC would increase the % patients (pts) with limited residual disease at time of surgery as measured by modified preoperative endocrine predictive index (PEPI). Methods: 4 months of therapy was given prior to surgery (F 500 mg IM weeks 1, 3, 5, 9, and 13) and pts were randomized to receive E 160 mg po daily for 16 weeks, stratified by node status and T-stage. Tumor biopsies were required at study entry and after 4 weeks on therapy (Wk5). PEPI score at time of surgery was the primary endpoint for efficacy. The minimax two-stage design had 80% power with type I error rate of 0.08. Reverse phase phosphoprotein array (RPPA), IHC for ER/PR/AR/GR/Ki67, and Polaris multiplex immunofluorescence (IF) panel for myeloid lineage immune cells were performed. Average signal levels were compared across arms, PEPI score (0, >0), PEPI by arm, and Ki67 ( Results: 69 pts consented; 59 evaluable. 95% completed surgery. PEPI=0 observed in 10 (17%). Of 33 pts on FE arm, 21 had T3/T4 tumors, 91% ER+/PR+, median AR expression 80%, Ki67 15%. Of 26 patients on F arm, 19% had T3/T4 tumors, 96% ER+/PR+, median AR expression 85%, Ki67 10%. PEPI=0 was achieved more frequently on the FE arm (8, 24%) than the F arm (2, 8%) (p=0.16). Toxicity was as expected with endocrine therapy. IHC of baseline vs Wk5 biopsies showed decreased ER, PR, and Ki67 levels by Wk5 that remained decreased at time of surgery. AR and GR were significantly decreased only in the FE arm at the time of surgery. RPPA revealed that baseline EGFR (Y1604 and Y992) was higher in tumors with PEPI=0 tumors, whereas the average baseline mTOR activation was higher among pts with PEPI>0. Significant changes detected by RPPA upon treatment included a significant decrease in AR. Myeloid-derived suppressor cells (MDSC) were significantly reduced by treatment only in the FE arm. Conclusions: The combination FE had manageable side effects. PEPI=0 was achieved more frequently on the FE arm (8/33) than the F arm (2/26). Activated EGFR was higher pretreatment in tumors achieving PEPI=0. mTOR pathway was elevated pretreatment in PEPI>0 tumors (also observed with resistance to FE in ER+/HER2- metastatic disease (SABCS 2021). When comparing pretreatment tumor to Wk5, total AR by RPPA was the most differentially decreased protein in PEPI=0 tumors. AR signaling is known to support immunosuppressive cells and we observed a marked decrease in MDSCs with treatment only on the FE arm. Citation Format: Anthony D. Elias, Alyse Staley, Monica Fornier, Gregory A. Vidal, Sharon Sams, Nicole Spoelstra, Peter Kabos, Jennifer R. Diamond, Elena Shagisultanova, Rosa I. Gallagher, Julia Wulfkuhle, Emanuel Petricoin, Kathryn Zolman, Stephanie Biller, Vida Alami, Tessa McSpadden, Virginia Borges, Lyndsey S. Crump, Dexiang Gao, Jennifer K. Richer. Randomized phase II trial of preoperative fulvestrant with or without enzalutamide for ER+/Her2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT144.

Background: In previous work we leveraged the I-SPY2 trial to create treatment response predictive subtypes (RPS) incorporating tumor biology beyond clinical HR/HER2, to better predict drug responses in an expanded treatment landscape that includes platinum agents, dual HER2-targeting regimens and immunotherapy [1]. We showed that best performing schemas incorporate Immune, DRD and HER2/Luminal phenotypes, and that treatment allocation based on these would increase the overall pCR rate to 63% from 51% using HR/HER2-based treatment selection. The RPS schema has been selected for prospective evaluation in I-SPY2. Using the RPS, one would prioritize platinum-based therapy for HER2-/Immune-/DRD+, immunotherapy for HER2-/Immune+, and dual-anti-HER2 for HER2+ that are not luminal. HER2+/Luminal patients have low response rates to dual-anti-HER2 therapy but may respond better to anti-AKT. However, there is still a considerable ‘biomarker-negative’ group of resistant cancers (HER2-/Immune-/DRD-) with very low pCR rates to all tested agents, that require a new therapeutic approach. Here we characterize the protein signaling architecture of these tumors to identify new target candidates. Methods: 987 I-SPY 2 patients from 10 arms of the trial were considered for this analysis. All have gene expression, pCR and RPS; 944 have distant recurrence free survival (DRFS) data; and 736 have reverse phase protein array (RPPA) data from laser capture microdissected tumor epithelium. These data – known collectively as the I-SPY2-990 mRNA/RPPA Data Resource - were recently made public on NCBI’s Gene Expression Omnibus [GEO: GSE196096]. We focus on HER2-/Immune-/DRD- tumors, applying Wilcoxon and t-tests to identify phosphoproteins that differ between HR+HER2-/Immune-/DRD- and other HR+HER2- tumors; and between TN/Immune-/DRD- and other TNs. The Benjamini-Hochberg (BH) method is used to adjust p-values for multiple hypothesis testing. In addition, the Kaplan-Meier method is used to estimate DRFS. Results: 201/736 I-SPY 2 patients with RPPA data are classified HER2-/Immune-/DRD- (HR+HER2-: n=138; TN: n=63). Of these, 8.5% (17/201) achieved pCR. Non-responding HER2-/Immune-DRD- had worse outcomes than responders (~75% vs. ~95% DRFS at 5 years). 60/139 phospho-proteins differ significantly between HR+HER2-/Immune-/DRD- and other HR+HER2- tumors (n=122). These tumors are relatively ‘cold’, in that 90% (54/60) of the phosphoprotein activities characterizing this group are at lower levels than in the overall HR+HER2- population; including immune (e.g. pPDL1, pJAK/STAT) and proliferation (e.g., Ki67, CyclinB1, pAURK) endpoints. Phosphoproteins showing higher levels in this subset include ERBB2 (BH p=1.7E-06), Cyclin D1 (BH p=1.4E-05), pAR (BH p=1.4E-05), and ER (BH p=3E-04). Within the TN subset, only 3/139 phospho-proteins differed significantly between TN/Immune-/DRD- and other TN tumors (n=189). These were all immune-related (pPDL1, pSTAT1, and HLA DR), with lower expression in the TN/Immune-/DRD- group. Conclusion: HR+HER2- and TN patients who are Immune-Low and DRD-Low have very low pCR rates to all tested therapeutics in I-SPY2 including standard chemotherapy, platinum, and immunotherapy. Senolytics (possibly targeting Cyclin D1), HER2low agents, and AR modulators may overcome resistance in HR+HER2-/Immune-/DRD-, whereas an immune activator beyond checkpoint inhibition is suggested for TN/Immune-/DRD- patients. [1] Wolf et. al., Redefining Breast Cancer Subtypes to Guide Treatment Prioritization and Maximize Response: Predictive Biomarkers across 10 Cancer Therapies. Cancer Cell 2022 Citation Format: Denise M. Wolf, Christina Yau, Julia Wulfkuhle, Rosa I. Gallagher, Lamorna A. Brown Swigart, Gillian L. Hirst, Jean-Philippe Coppe, Mark Jesus M. Magbanua, Rosalyn Sayaman, I-SPY2 Investigators, Laura Sit, Nola M. Hylton, Angela DeMichele, Donald A. Berry, Lajos Pusztai, Douglas Yee, Laura J. Esserman, Emanuel F. Petricoin, Laura Van ’t Veer. PD5-04 Characterizing the HER2-/Immune-/DNA repair (DRD-) response predictive breast cancer subtype: the hunt for new protein targets in a high-needs population with low response to all I-SPY2 agents [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-04.

Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug–gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy.

Purpose: Immunotherapies targeting PD-1/L1 enhance pathologic complete response (pCR) rates when added to standard neoadjuvant chemotherapy (NAC) regimens in early-stage triple-negative, and possibly high-risk estrogen receptor–positive breast cancer. However, immunotherapy has been associated with significant toxicity, and most patients treated with NAC do not require immunotherapy to achieve pCR. Biomarkers discerning patients benefitting from the addition of immunotherapy from those who would achieve pCR to NAC alone are clearly needed. In this study, we tested the ability of MHC-II expression on tumor cells, to predict immunotherapy-specific benefit in the neoadjuvant breast cancer setting. Patients and Methods: This was a retrospective tissue-based analysis of 3 cohorts of patients with breast cancer: (i) primary nonimmunotherapy-treated breast cancers (n = 381), (ii) triple-negative breast cancers (TNBC) treated with durvalumab and standard NAC (n = 48), and (iii) HER2-negative patients treated with standard NAC (n = 87) or NAC and pembrolizumab (n = 66). Results: HLA-DR positivity on ≥5% of tumor cells, defined a priori, was observed in 10% and 15% of primary non-immunotherapy–treated hormone receptor–positive and triple-negative breast cancers, respectively. Quantitative assessment of MHC-II on tumor cells was predictive of durvalumab + NAC and pembrolizumab + NAC (ROC AUC, 0.71; P = 0.01 and AUC, 0.73; P = 0.001, respectively), but not NAC alone (AUC, 0.5; P = 0.99). Conclusions: Tumor-specific MHC-II has a strong candidacy as a specific biomarker of anti–PD-1/L1 immunotherapy benefit when added to standard NAC in HER2-negative breast cancer. Combined with previous studies in melanoma, MHC-II has the potential to be a pan-cancer biomarker. Validation is warranted in existing and future phase II/III clinical trials in this setting.

This prospective phase II clinical trial (Side Out 2) explored the clinical benefits of treatment selection informed by multi‐omic molecular profiling (MoMP) in refractory metastatic breast cancers (MBCs). Core needle biopsies were collected from 32 patients with MBC at trial enrollment. Patients had received an average of 3.94 previous lines of treatment in the metastatic setting before enrollment in this study. Samples underwent MoMP, including exome sequencing, RNA sequencing (RNA‐Seq), immunohistochemistry, and quantitative protein pathway activation mapping by Reverse Phase Protein Microarray (RPPA). Clinical benefit was assessed using the previously published growth modulation index (GMI) under the hypothesis that MoMP‐selected therapy would warrant further investigation for GMI ≥ 1.3 in ≥ 35% of the patients. Of the 32 patients enrolled, 29 received treatment based on their MoMP and 25 met the follow‐up criteria established by the trial protocol. Molecular information was delivered to the tumor board in a median time frame of 14 days (11–22 days), and targetable alterations for commercially available agents were found in 23/25 patients (92%). Of the 25 patients, 14 (56%) reached GMI ≥ 1.3. A high level of DNA topoisomerase I (TOPO1) led to the selection of irinotecan‐based treatments in 48% (12/25) of the patients. A pooled analysis suggested clinical benefit in patients with high TOPO1 expression receiving irinotecan‐based regimens (GMI ≥ 1.3 in 66.7% of cases). These results confirmed previous observations that MoMP increases the frequency of identifiable actionable alterations (92% of patients). The MoMP proposed allows the identification of biomarkers that are frequently expressed in MBCs and the evaluation of their role as predictors of response to commercially available agents. Lastly, this study confirmed the role of MoMP for informing treatment selection in refractory MBC patients: more than half of the enrolled patients reached a GMI ≥ 1.3 even after multiple lines of previous therapies for metastatic disease., This prospective trial assessed the role of multi‐omic molecular profiling in informing treatment selection for metastatic breast cancer patients. Molecular profiles were collected in 14 business days and targetable alterations for commercial agents were identified in 92% of patients; 56% of patients reached a growth modulation index ≥ 1.3 after multiple lines of previous therapies. This work confirms the unique role for multi‐omic molecular profiling in guiding treatment selection for metastatic patients.

Purpose: Despite extensive genomic and transcriptomic profiling, it remains unknown how signaling pathways are differentially activated and how tumors are differentially sensitized to certain perturbations. Here, we aim to characterize AKT signaling activity and its association with other genomic or IHC-based PI3K/AKT pathway biomarkers as well as the clinical activity of ipatasertib (AKT inhibitor) in the FAIRLANE trial. Experimental Design: In FAIRLANE, 151 patients with early triple-negative breast cancer (TNBC) were randomized 1:1 to receive paclitaxel with ipatasertib or placebo for 12 weeks prior to surgery. Adding ipatasertib did not increase pathologic complete response rate and numerically improved overall response rate by MRI. We used reverse-phase protein microarrays (RPPA) to examine the total level and/or phosphorylation states of over 100 proteins in various signaling or cell processes including PI3K/AKT and mTOR signaling. One hundred and twenty-five baseline and 127 on-treatment samples were evaluable by RPPA, with 110 paired samples at both time points. Results: Tumors with genomic/protein alterations in PIK3CA/AKT1/PTEN were associated with higher levels of AKT phosphorylation. In addition, phosphorylated AKT (pAKT) levels exhibited a significant association with enriched clinical benefit of ipatasertib, and identified patients who received benefit in the absence of PIK3CA/AKT1/PTEN alterations. Ipatasertib treatment led to a downregulation of AKT/mTORC1 signaling, which was more pronounced among the tumors with PIK3CA/AKT1/PTEN alterations or among the responders to the treatment. Conclusions: We showed that the high baseline pAKT levels are associated with the alterations of PI3K/AKT pathway components and enriched benefit of ipatasertib in TNBC.

Using pre-treatment gene expression, protein/phosphoprotein and clinical data from the I-SPY2 neoadjuvant platform trial (NCT01042379), we create alternative breast cancer subtypes incorporating tumor biology beyond clinical hormone-receptor (HR) and Human Epidermal Growth Factor Receptor-2 (HER2) status to better predict drug responses. We assess predictive performance of mechanism-of-action biomarkers from ~990 patients treated with 10 regimens targeting diverse biology. We explore >11 subtyping schemas and identify treatment-subtype pairs maximizing pathologic complete response (pCR) rate over the population. The best performing schemas incorporate Immune, DNA-repair and HER2/Luminal phenotypes. Subsequent treatment allocation increases overall pCR rate to 63% from 51% using HR/HER2-based treatment selection. pCR gains from reclassification and improved patient selection are highest in HR+ subsets (>15%). As new treatments are introduced, the subtyping schema determines the minimum response needed to show efficacy. This data platform provides an unprecedented resource and supports the usage of response-based subtypes to guide future treatment prioritization.

The AKT inhibitor MK2206 (M) was evaluated in I-SPY 2 and graduated in the HER2+, HR−, and HR− HER2+ signatures. We hypothesized that AKT signaling axis proteins/genes may specifically predict response to M and tested 26 phospho-proteins and 10 genes involved in AKT-mTOR-HER signaling; in addition, we tested 9 genes from a previous study in the metastatic setting. One hundred and fifty patients had gene expression data from pretreatment biopsies available for analysis (M: 94, control: 56) and 138 had protein data (M: 87, control: 51). Logistic modeling was used to assess biomarker performance in pre-specified analysis. In general, phospho-protein biomarkers of activity in the AKT-mTOR-HER pathway appeared more predictive of response to M than gene expression or total protein biomarkers in the same pathway; however, the nature of the predictive biomarkers differed in the HER2+ and TN groups. In the HER2+ subset, patients achieving a pCR in M had higher levels of multiple AKT kinase substrate phospho-proteins (e.g., pmTOR, pTSC2). In contrast, in the TN subset responding patients had lower levels of AKT pathway phospho-proteins, such as pAKT, pmTOR, and pTSC2. Pathway mutations did not appear to account for these associations. Additional exploratory whole-transcriptome analysis revealed immune signaling as strongly associated with response to M in the HER2+ subset. While our sample size is small, these results suggest that the measurement of particular AKT kinase substrate phospho-proteins could be predictive of MK2206 efficacy in both HER2+ and TN tumors and that immune signaling may play a role in response in HER2+ patients.

The inability to propagate human prostate epithelial cells indefinitely has historically presented a serious impediment to prostate cancer research. The conditionally reprogrammed cell (CRC) approach uses the combination of irradiated J2 mouse fibroblasts and a Rho kinase inhibitor such as Y27632 to support the continuous culture of cells derived from most epithelial tissues, including the prostate. Due to their rapid establishment and overall ease of use, CRCs are now widely used in a variety of basic and preclinical settings. In addition, CRCs were successfully used to clinically treat respiratory papillomatosis. Although both normal and tumor-derived prostate CRCs have been used to study the basic biology of prostate cancer and to test new therapies, certain limitations exist. We have previously reported that prostate CRCs form functional prostate glands when implanted under the mouse renal capsule. However in conventional culture, the prostate CRCs exist in an adult stem-like, transient amplifying state and consequently do not adequately recapitulate several important features of a differentiated prostate epithelium. To address these limitations, we previously described a transwell dish-based model that supported the culturing of prostate CRCs and the collection of cells and cell extracts for molecular and genetic analyses. Using normal and tumor-derived prostate CRCs, we describe the combined effects of the multi-dimensional transwell platform and defined culture media on prostate cellular proliferation, differentiation and signaling.

BackgroundAnti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples.MethodsPD-L1 expression was measured using five antibody clones (22C3, 28–8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment.ResultsMedian-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: −0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response.ConclusionsContinuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

De novo resistance and rapid recurrence often characterize responses of B-cell malignancies to ibrutinib (IBR), indicating a need to develop drug combinations that block compensatory survival signaling and give deeper, more durable responses. To identify such combinations, we previously performed a combinatorial drug screen and identified the Bcl-2 inhibitor venetoclax (VEN) as a promising partner for combination with IBR in Mantle Cell Lymphoma (MCL). We have opened a multi-institutional clinical trial to test this combination. However, analysis of primary samples from patients with MCL as well as chronic lymphocytic leukemia (CLL) revealed unexpected heterogeneous de novo resistance even to the IBR+VEN combination. In the current study, we demonstrate that resistance to the combination can be generated by microenvironmental agonists: IL-10, CD40L and, most potently, CpG-oligodeoxynucleotides (CpG-ODN), which is a surrogate for unmethylated DNA and a specific agonist for TLR9 signaling. Incubation with these agonists caused robust activation of NF-κB signaling, especially alternative NF-κB, which led to enhanced expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, thus decreasing dependence on Bcl-2. Inhibitors of NF-κB signaling blocked overexpression of these anti-apoptotic proteins and overcame resistance. Inhibitors of Mcl-1, Bcl-xL, or survivin also overcame this resistance, and showed synergistic benefit with the IBR+VEN combination. We conclude that microenvironmental factors, particularly the TLR9 agonist, can generate de novo resistance to the IBR+VEN combination in CLL and MCL cells. This signaling pathway presents targets for overcoming drug resistance induced by extrinsic microenvironmental factors in diverse B-cell malignancies.

Background: The goal of I-SPY 2 is to rapidly test novel therapies in addition to standard of care in high-risk breast cancer patients. It has resulted in increasing response rates, where pCR rates in TNBC and HR-HER2+ subsets have reached ~60% and ~75%, respectively. Yet, there remains a sizeable subset of non-responders, especially among HR+ patients. Identification of ‘universal’ resistance mechanisms may guide rational selection of agents to improve these patient's outcomes. Thus, we analyzed reverse phase protein array (RPPA) based quantitative protein/phosphoprotein data across arms to assess whether there are common mechanisms rendering these cancers resistant to all agent classes tested to date. Methods: 736 patients (260 HR+HER2-, 252 TN, 142 HR+HER2-, and 82 HR-HER2+; over 8 arms: 194 Ctr, 105 neratinib (N), 63 veliparib/carboplatin (VC), 128 AMG386 (anti-ANG1/2), 87 MK2206 (anti-AKT), 43 TH/pertuzumab (P), 49 TDM1/P, and 67 pembrolizumab (Pembro)) with pCR and RPPA data at the pre-treatment time point were considered for this analysis. 141 RPPA endpoints representing key cancer pathways were assessed for association with pCR using logistic regression modeling, with HR, HER2 and treatment arm as covariates (likelihood ratio test; p Citation Format: Julia Wulfkuhle, Denise Wolf, Christina Yau, Lamorna Brown-Swigart, Rosa I Gallagher, Gillian Hirst, Laura Sit, Smita Asare, I-SPY 2 TRIAL Investigators, Nola Hylton, Angela DeMichele, Douglas Yee, Jo Chien, Hope Rugo, John Park, Kathy Albain, Rita Nanda, Debu Tripathy, Richard Schwab, Don Berry, Laura Esserman, Laura van t' Veer, Emanual Petricoin, III. Identification of biomarkers associated with therapeutic resistance: Quantitative protein/phosphoprotein analysis of ~750 patients across 8 arms of the neoadjuvant I-SPY 2 TRIAL for high-risk early stage 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 PD9-04.

Background: In the I-SPY 2 TRIAL, HER2- patients were randomized to receive standard chemotherapy or chemotherapy plus the oral PARP inhibitor veliparib in combination with carboplatin (V+C), which graduated in the HR-/HER2- arm. Exploratory analysis of protein signaling was performed to identify biomarker candidates that correlated with pCR in the HER2- population. We evaluated 110 key signaling proteins using reverse phase protein microarray (RPPA) data from pre-treatment LCM purified tumor epithelium. Methods: Of 115 patients, 97 (V+C: 61 controls: 36) had RPPA and pCR data. RPPA data was correlated to pCR in both the treated and control patients using parametric (t-test) or non-parametric (Wilcoxon) statistical analysis, depending on data distribution. Only analytes whose pre-treatment levels were associated with response in the V+C but not the control arm were identified (P Results: 11 protein/phosphoprotein markers were significantly associated with pCR in the V+C arm but not in controls. Two were positive predictors of response: YAP S127 p= 0.03 and LC3B p=0.04. Negative predictors of response included Cyclin D1 p=0.001, and a number of phosphorylated RTKs: ROS Y2274 p=0.03, IGF1R Y1135/Y1136-IR Y1150/Y1151 p=0.03, ERBB4 Y1284 p=0.002, total HER2 p=0.04, and total IGF1R p=0.01. Moreover, a number of AKT-mTOR pathway proteins were found to be negative predictors of V+C response: ACC S79 p=0.005, p70S6K S371 p=0.01, and B-RAF S445 p=0.01. Conclusion: Our sample size is too small to draw definitive conclusions and the results are exploratory. Coordinated RTK-mTOR pathway activation appears to be a hallmark signature of lack of response to veliparib in HER2- tumors. We also found that HER2 levels were correlated paradoxically with lack of response in this HER2- population, suggesting potential added clinical value of quantitative HER2 measurement techniques. Such exploratory results merit evaluation in larger trials with HER2- breast cancer patients. Citation Format: Wulfkuhle JD, Yau C, Wolf DM, Gallagher RI, Deng J, Brown-Swigart L, Hirst G, I-SPY 2 Trial Investigators, Rugo H, Olopade OI, Esserman L, Berry D, van't Veer L, Petricoin EF. Prediction of complete pathologic response to veliparib/carboplatin plus standard neoadjuvant therapy in HER2 negative breast cancer: Exploratory protein pathway marker results from the I-SPY 2 trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-48.

Background: We have previously described that TNBC patients whose tumors have both HER2 Y1248 phosphorylation (pHER2) “high” and phospho-EGFR Y1173 (pEGFR) “high” have increased response (pCR) to neratinib in the I-SPY2 TRIAL. We hypothesize that the paradoxical finding of a response prediction signature comprised of HER2 activation in a HER2 IHC/FISH-negative population means there must be a ligand-driven biochemical event responsible for the HER2 phosphorylation because HER2 mutations were also not found to be significant. Exploratory analysis of additional cellular signaling events and protein expression levels in pre-treatment, LCM-purified tumor epithelium by reverse phase protein microarray (RPPA) included semi-quantitative measurement of total levels of estrogen receptor alpha (ERα), which has been previously shown to be able to act as a membrane non-genomic signaling molecule through direct interaction with various tyrosine kinases including EGFR and HER2. Since ERα has been previously shown to act as a ligand and co-stimulate (activate) HER2 and EGFR when present at low levels, we investigated whether or not RPPA-measured ERα levels in the TNBC cohort analyzed to date were higher in tumors with both pHER2 “high” and pEGFR “high” levels and thus provide evidence explaining how HER2-EGFR activation is occurring in TNBC. Methods: Using RPPA analysis, we measured 118 analytes in lysates of LCM tumor epithelium obtained from the pre-treatment biopsy samples of 86 TNBC (Allred=0) patients in the I-SPY2 TRIAL analyzed to date. Cutpoints for pEGFR and pHER2 were determined previously by ROC analysis for pCR correlation in the neratinib treated TNBC population, and used here to dichotomize the pHER2 and pEGFR data in the larger TNBC population. Wilcoxon Rank Sum testing was performed using the continuous variable total ERα data and compared the TNBC that were both pHER2 and pEGFR “high” (N=39) to the rest of the TNBC population (N=47). Total ERα values were then divided into “high” and “low” groups based on the TNBC population median value in order to determine frequency/percentages within each class. Our study is exploratory with no claims for generalizability of the data, and calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Results: Total ERα values were obtained in 84/86 TNBC tumors analyzed. Total levels of ERα were higher (p< 0.006) in TNBC tumors with pHER2 and pEGFR “high” levels. 68% (26/38) of tumors in the pHER2 and pEGFR “high” group had ERα levels above the population median compared to 35% (16/46) in the rest of the TNBC population. Conclusion: Our exploratory analysis reveals that ERα levels are significantly higher in TNBC with pHER2 and pEGFR activation and may be behaving as a direct signaling ligand in TNBC and driving HER2-EGFR signaling. This ERα-pHER2/pEGFR association was missed by current ER and HER2 clinical laboratory testing techniques, and if validated in larger independent study sets could suggest that utilization of new protein-based techniques defining ER more quantitatively could be helpful to understand tumor biology and therapeutic response prediction, especially in the context of TNBC that are ostensibly ER negative. Citation Format: Gallagher RI, Yau C, Wolf DM, Dong T, Hirst G, Brown-Swigart L, ISPY-2 TRIAL Investigators, Buxton M, DeMichele A, van't Veer L, Yee D, Paoloni M, Esserman L, Berry D, Park J, Petricoin EF, Wulfkuhle JD. Quantitative ERα measurements in TNBC from the I-SPY 2 TRIAL correlate with HER2-EGFR co-activation and heterodimerization [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 P3-05-02.

Purpose In the I-SPY 2 TRIAL (Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2), the pan–erythroblastic oncogene B inhibitor neratinib was available to all hormone receptor (HR)/human epidermal growth factor receptor 2 (HER2) subtypes and graduated in the HR-negative/HER2-positive signature. We hypothesized that neratinib response may be predicted by baseline HER2 epidermal growth factor receptor (EGFR) signaling activation/phosphorylation levels independent of total levels of HER2 or EGFR proteins. Materials and Methods Complete experimental and response data were available for between 130 and 193 patients. In qualifying analyses, which used logistic regression and treatment interaction analysis, 18 protein/phosphoprotein, 10 mRNA, and 12 DNA biomarkers that related to HER family signaling were evaluated. Exploratory analyses used Wilcoxon rank sum and t tests without multiple comparison correction. Results HER pathway DNA biomarkers were either low prevalence or nonpredictive. In expression biomarker analysis, only one gene ( STMN1) was specifically associated with response to neratinib in the HER2-negative subset. In qualifying protein/phosphoprotein analyses that used reverse phase protein microarrays, six HER family markers were associated with neratinib response. After analysis was adjusted for HR/HER2 status, EGFR Y1173 (pEGFR) showed a significant biomarker-by-treatment interaction ( P = .049). Exploratory analysis of HER family signaling in patients with triple-negative (TN) disease found that activation of EGFR Y1173 ( P = .005) and HER2 Y1248 (pHER2) ( P = .019) were positively associated with pathologic complete response. Exploratory analysis in this pEGFR/pHER2–activated TN subgroup identified elevated levels of estrogen receptor α ( P < .006) in these patients. Conclusion Activation of HER family phosphoproteins associates with response to neratinib, but only EGFR Y1173 and STMN1 appear to add value to the graduating signature. Activation of HER2 and EGFR in TN tumors may identify patients whose diseases respond to neratinib and implies that there is a subset of patients with TN disease who paradoxically exhibit HER family signaling activation and may achieve clinical benefit with neratinib; this concept must be validated in future studies.

Background: We hypothesize that response to the pan-ERBB inhibitor, neratinib (N), may be predicted by pre-treatment HER2-EGFR signaling. In the I-SPY 2 TRIAL, N graduated in the HR-/HER2+ signature. All patients received at least standard chemotherapy. For HER2+ patients, N was administered in place of trastuzumab. We evaluated 18 HER family signaling proteins as biomarkers of N response using reverse phase protein microarray (RPMA) data from pre-treatment LCM purified tumor epithelium. Methods: 168 patients (N: 106, concurrent controls: 62) had RPMA and pCR data. 18 biomarkers relating to HER family signaling were evaluated: AKT S473, AKT T308, EGFR, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFR Y992, ERBB2, ERBB2 Y1248, ERBB3 total, ERBB3 Y1289, ERK1/2 T202/Y204, Heregulin, mTOR, mTOR S2448, PI3K p85 Y458/p55 Y199, PTEN S380, and SHC Y317. We assessed association between biomarker and response in the N and control arms alone (likelihood ratio test), and relative performance between arms (biomarker x treatment interaction) using a logistic model. Analysis was also performed adjusting for HR/HER2 status. In an exploratory analysis, we selected the marker with the greatest interaction (phosphorylated EGFR (Y1173)) to dichotomize patients optimally based on the data and assessed it in the context of the graduating signature by adding the EGFR Y1173-High patients to the HR-/HER2+ subtype and evaluating the treatment effect in this ‘biomarker-positive’ group. Our study is exploratory with no claims for generalizability of the data and does not account for multiplicities. Statistical calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Results: 7 HER pathway markers (EGFR Y1068, EGFR Y1173, EGFR Y992, ERBB2 total, ERBB2 Y1248, ERBB3 Y1289, SHC Y317) are associated with response in the N but not the control arm. However, the difference in performance between arms did not reach significance by permutation testing. Adjusting for HR/HER2 status, EGFR Y1173 shows a significant biomarker x treatment interaction (p = 0.049). In an exploratory analysis, we dichotomized patients by their EGFR Y1173 levels and evaluated the distribution of pCR rates (Table 1). Neratinib (n=106)Control (n=62)EGFR Y1173 Low (n=31)EGFR Y1173 High (n=75)EGFR Y1173 Low (n=29)EGFR Y1173 High (n=33)HR-/HER2+ (n=28)0 / 412 / 181 / 11 / 5Not HR-/HER2+ (n=140)3 / 2724 / 575 / 285 / 28Table 1 OR between EGFR Y1173 groups in the N relative to control arm is 10.1. When EGFR Y1173 High patients are added to the graduating HR-/HER2+ subset, the OR associated with treatment is 3.2 and is comparable to that in the HR-/HER2+ signature (OR: 2.1), while increasing the prevalence of biomarker-positive patients by ∼50%. Evaluation of EGFR Y1173 under the I-SPY 2 Bayesian model is pending. Conclusion: Our sample size is too small to draw definitive conclusions. Our exploratory analysis reveals that HER family phosphoproteins associate with response to N, but only phosphorylated EGFR Y1173 appears to add value to the graduating signature. Given that this biomarker would expand the patient population that may benefit, it merits evaluation in other ongoing trials of neratinib. Citation Format: Julia D Wulfkuhle, Christina Yau, Denise M Wolf, Rosa I Gallagher, Ashish Sanil, Lamorna Brown-Swigart, Susan Flynn, Gillian Hirst, I-SPY 2 TRIAL Investigators, Meredith Buxton, Angela DeMichele, Nola Hylton, William F Symmans, Laura van't Veer, Douglas Yee, Melissa Paoloni, Laura Esserman, Donald Berry, Minetta C Liu, John W Park, Emanuel F Petricoin III. Evaluation of HER family protein signaling network as a predictive biomarker for pCR for breast cancer patients treated with neratinib in the I-SPY 2 trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-15.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Reverse phase protein array (RPPA) technology evolved from the advent of miniaturized immunoassays and gene microarray technology. Reverse phase protein arrays provide either a low throughput or high throughput methodology for quantifying proteins and their post-translationally modified forms in both cellular and non-cellular samples. As the demand for patient tailored therapies increases so does the need for precise and sensitive technology to accurately profile the molecular circuitry driving an individual patient's disease. RPPAs are currently utilized in clinical trials for profiling and comparing the functional state of protein signaling pathways, either temporally within tumors, between patients, or within the same patients before/after treatment. RPPAs are generally employed for quantifying large numbers of samples on one array, under identical experimental conditions. However, the goal of personalized cancer medicine is to design therapies based on the molecular portrait of a patient's tumor, which in turn result in more efficacious treatments with less toxicity. Therefore, RPPAs are also being validated for low throughput assays of individual patient samples. This review explores RPPA technology in the cancer research field, concentrating on its role as a fundamental tool for deciphering protein signaling networks and its emerging role in personalized medicine.

The primary objective was to determine if multi-omic molecular profiling (MMP) informed selection of approved cancer treatments could change the clinical course of disease for patients with previously treated metastatic breast cancer (MBC) (i.e., produce a growth modulation index (GMI) ≥1.3). GMI was calculated as the ratio of progression free survival on MMP-selected therapy/time to progression on last prior treatment. To meet the primary objective at least 35 % of the subjects should demonstrate a GMI ≥1.3. Secondary endpoints included determining the response rate (according to RECIST 1.1), the percent of patients with non-progression at 4 months, and overall survival in patients whose therapy is selected by molecular profiling and proteomic analysis. Eligible patients had MBC, with ≥3 prior lines of therapy. A multi-omic based approach was performed incorporating multiplexed immunohistochemistry, c-DNA microarray, and phosphoprotein pathway activation mapping by reverse phase protein array. MMP was performed on fresh core biopsies; results were generated and sent to a Treatment Selection Committee (TSC) for review and treatment selection. Three sites enrolled 28 patients, of which 25 were evaluable. The median range of prior treatment was 7 (range 3-12). The MMP analysis and treatment recommendation were delivered within a median of 15.5 days from biopsy (range 12-23). The TSC selected MMP-rationalized treatment in 100 % (25/25) of cases. None of the MMP-based therapies were the same as what the clinician would have selected if the MMP had not been performed. GMI ≥1.3 was reported in 11/25 (44 %) patients. Partial responses were noted in 5/25 (20 %), stable disease in 8/25 (32 %) and 9/25 (36 %) had no progression at 4 months. This pilot study demonstrates the feasibility of finding possible treatments for patients with previously treated MBC using a multiplexed MMP-rationalized treatment recommendation. This MMP approach merits further investigation.

2631 Background: Response to immune checkpoint inhibitors has been associated with immune activation and mutational load within a tumor. Previous results in other tumors have implicated MHC II protein tumor cell expression as a response predictor to immune checkpoint inhibitors. In the I-SPY 2 TRIAL, the anti-PD1 therapeutic antibody pembrolizumab (P) was available to HER2-negative subtypes and graduated in both the HR+/HER2- and TNBC signatures. Pre-specified biomarker analysis was performed to test tumor MHC II expression as a predictor of response to P in the I-SPY 2 TRIAL based on its central role in tumor antigen presentation. Methods: 156 patients (P: 67, controls: 89) had RPPA and pCR data. RPPA-based quantitative data for pan-MHC II protein isotypes HLA-DR/DP/DQ/DX and HLA-DR protein isotype was obtained from LCM-enriched tumor epithelium, and protein levels were assessed for association with pCR in the P and control arms separately using the Wilcoxon Rank Sum test (p < 0.05). Analysis was also performed in the HR+ and HR- subgroups. Markers were analyzed individually; p-values are descriptive and were not corrected for multiple comparisons. Results: Across all P- treated patients, the HLA class II molecules –DR and -DR/DP/DQ/DX had a positive association with response to P (p = 0.014 and p = 0.001). Expression of HLA-DR/DP/DQ/DX also had a positive association with response to P in HR+ tumors. Neither of these associations were seen in the control arm samples. Conclusions: The observation of elevation of MHC II protein expression in HER2- responding patients treated with P suggests that activation of antigen peptide exchange facilitated by these molecules in T and B cells may enhance response to P treatment.

Cheiracanthium inclusum (Hentz), a hunting spider, feeds on eggs of Helicoverpa zea (Boddie) and other moths. This study investigated whether C. inclusum can use chemical compounds present in H. zea scales and egg residues as kairomones to find these non-motile prey items. In a series of no- choice tests, spiders were presented with a piece of florist paper containing scales alone, scales + egg residues, or untreated controls. Next, spiders were presented with solvent extracts of either scales or eggs. Polar and non-polar solvents were used in the extractions. Contact with scales alone, scales + egg residues, and non-polar solvent extracts of both scales and eggs resulted in retention and/or induction of local searching behavior. Extracts made with polar solvents induced no apparent response, indicating that the chemostimulatory compounds are lipophilic. These results show that C. inclusum responds to kairomones left by ovipositing H. zea and use these chemical cues to detect and locate H. zea eggs.

12099Background: In the I-SPY 2 TRIAL, the allosteric AKT inhibitor MK2206 was available to all HR/HER2 subtypes and graduated in the HR-/HER2+ signature. Qualifying biomarker analysis was performe...

12103Background: Trebananib (T), an angiopoietin 1/2 neutralizing peptibody that inhibits interaction with TIE2 receptors, was available to all HR/HER2 subtypes in the I-SPY2 TRIAL. The agent did n...

The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.

Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (BTK), has been FDA approved for Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, IBR responses are incomplete in most cases, and of short duration for MCL and higher-risk CLL subtypes. We hypothesize that intrinsic resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be co-targeted with BTK inhibition to achieve deeper and more durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generated synergistic cytotoxicity with IBR in MCL lines as well as circulating leukemic B cells in 13/19 CLL and 4/5 MCL patient samples treated ex vivo (Jayappa KD et al. ASH 2015; Portell CA et al. ASH 2014). However, the sensitivity to VEN or IBR+VEN was highly variable among patient samples, and did not correlate with the diagnostic genetic lesions in the CLL/MCL cells or clinical characteristics of the patients. Here, we show that resistance to IBR+VEN can be induced by interaction with soluble factors found in the in vivo "macroenvironment" of the circulating cancer cells. To gain insight into changes in signaling pathways that might underlie mechanisms of drug synergy and resistance, we analyzed drug resistant MCL lines treated with IBR, VEN, and the combination by Reverse Phase Protein Arrays. We observed downregulation of PI3K-Akt, MAPK, JAK-STAT, and NOTCH signaling after IBR and IBR+VEN treatment, cleavage of caspases and PARP after VEN-treatment, and greatly enhanced cleavage of caspases and PARP after IBR+VEN treatment. A notable exception was significantly increased phosphorylation on p65 S536 of the NF-kB pathway at longer times after IBR+VEN treatment, indicating a possible role of NF-kB signalling in generating resistance to IBR and VEN in cells that survived treatment. To determine whether extrinsic factors in the cancer cell environment might be able to induce a drug-resistant phenotype, we co-cultured or pre-incubated PBMC with a diverse panel of stromal cells, cytokines, and other agonists. We found that the combination of soluble CD40L, IL-10, and CpG DNA (agonist mix) generated nearly complete resistance to IBR+VEN in CLL and MCL patient samples, with CpG DNA being the most effective single agent. Every sample treated with agonist mix displayed increased resistance to IBR+VEN drug combination, suggesting that responsiveness transcends genetic heterogeneity and reflects the developmental lineage of the cancer cells. We investigated whether the extrinsic agonists induce drug resistance by activating the NF-κB pathway, by analyzing nuclear localization of NF-kB transcription factors RelA and RelB using ImageStream and cell fractionation. We observed robust activation of alternative-NF-kB signaling in primary CLL and MCL cells treated with agonist mix, with little effect on canonical NF-κB. PKC, MAPK and PI3K-Akt signaling also showed evidence of activation by agonist mix. Agonist mix treatment also caused significant overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, but not Bcl-2. Inhibitors of NF-κB blocked RelB translocation and overexpression of Mcl-1, Bcl-xL and survivin. Inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame drug resistance induced by agonist mix. Inhibitors of the other activated pathways (MAPK, PI3K-Akt, PKC) did not block agonist-induced drug resistance at pharmacologically relevant concentrations. To determine whether extra-nodal agonists in the blood of patients could generate resistance to IBR and VEN, we analyzed drug cytotoxicity in CLL patient PBMCs cultured with autologous plasma. We found that autologous plasma was able to induce increased resistance to IBR+VEN in 3/7 CLL samples and this resistance was blocked by treatment with an NF-kB inhibitor. In conclusion, soluble agonists in the patient macroenvironment of circulating CLL/MCL cells can generate resistance to IBR+VEN by activating alternative-NF-kB signaling and over-expression of multiple anti-apoptotic proteins. Inhibitors of NF-kB or of the upregulated anti-apoptotic proteins overcame IBR+VEN resistance generated by these extrinsic factors. We suggest that survival signals generated by extra-nodal agonists in the patient macroenvironment generate drug resistance in CLL and MCL, and that improved responses could occur with interventions that block these survival signals. Disclosures Portell: AbbVie: Research Funding; Roche/Genentech: Research Funding; Infinity: Research Funding; Acerta: Research Funding. Wulfkuhle:Theranostics Health, LLC: Equity Ownership. Petricoin:Perthera, Inc.: Consultancy, Equity Ownership; Ceres Nanosciences, Inc.: Consultancy, Equity Ownership, Patents & Royalties; Avant Diagnostics, Inc.: Equity Ownership. Williams:University of Virginia: Employment; Janssen and Pharmacyclics: Research Funding.

Laser capture microdissection (LCM) is a technique that allows the precise procurement of enriched cell populations from a heterogeneous tissue under direct microscopic visualization. LCM can be used to harvest the cells of interest directly or can be used to isolate specific cells by ablating the unwanted cells, resulting in histologically enriched cell populations. The fundamental components of laser microdissection technology are (a) visualization of the cells of interest via microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. Laser energy supplied by LCM instruments can be infrared (810 nm) or ultraviolet (355 nm). Infrared lasers melt thermolabile polymers for cell capture, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes the unique features of the Arcturus(XT) laser capture microdissection instrument, which incorporates both infrared capture and ultraviolet cutting technology in one instrument, using a proteomic downstream assay as a model.

Laser capture microdissection (LCM) is a technique that allows the precise procurement of enriched cell populations from a heterogeneous tissue under direct microscopic visualization. LCM can be used to harvest the cells of interest directly or can be used to isolate specific cells by ablating the unwanted cells, resulting in histologically enriched cell populations. The fundamental components of laser microdissection technology are (a) visualization of the cells of interest via microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. Laser energy supplied by LCM instruments can be infrared (810 nm) or ultraviolet (355 nm). Infrared lasers melt thermolabile polymers for cell capture, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes the unique features of the Arcturus(XT) laser capture microdissection instrument, which incorporates both infrared capture and ultraviolet cutting technology in one instrument, using a proteomic downstream assay as a model.

The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

BACKGROUND: While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2-4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. CONCLUSIONS: Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

1085 Background: In the I-SPY 2 trial, the pan-ERBB inhibitor, neratinib (N) arm was open to all HR/HER2 subtypes but graduated in the HR-/HER2+ signature. Exploratory analysis of protein signaling...

Background: Profiling protein signaling activation in cancer is critical as these proteins represent targets for new molecular therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane- based chemotherapy that longitudinally collected biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens (T2, 1–4 days post-chemotherapy). Reverse Phase Protein Microarray technology was used to quantitatively measure the activation of 39 and 100 key signaling proteins in the T1 and T2 biopsies, respectively, including numerous drug targets for Phase I-III and FDA-cleared therapeutics. Associations between protein activation and response to chemotherapy (RCB 0/1 vs 2/3) and relapse-free survival (RFS) were evaluated for the HR+/HER2− patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p < 0.05; Cox proportional hazard model, likelihood ratio p < 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of HER family signaling and downstream AKT activation in tumors from patients who do not respond (RCB2/3) and/or whom recurred. Increased ERBB2 and ERBB4 levels were observed at T1 and T2, respectively, from patients who did not respond to neoadjuvant therapy. Dynamic changes in ERBB3 and AKT phosphorylation/activation were revealed between the T1 and T2 time points, which were associated with recurrence. Increased phosphorylation of MARCKS, a known PKC substrate, was seen in the T1 biopsy in the non-response cohort. Conclusions: Our analysis revealed activation/increased expression of HER family proteins along with downstream AKT activation in HR+/HER2− patients who have poor clinical response. These events, if validated, point to potential treatment options that could be rationally considered for non-responding HR+/HER2− patients with a number of investigational agents that target ERBB3 and AKT signaling in the clinic today. Increasing HER2 protein expression in a HER2− cohort may appear contradictory, however these findings may point to important subtle increases in HER2 that have important clinical considerations. Given the known clinical benefit from HER2 directed therapy in HER2− patients seen in other studies, our findings may have clinical relevance if validated. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with HR+/HER2− tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-02.

Background: Identification of alternative therapeutic strategies is critically needed for patients who do not achieve complete clinical response in the neoadjuvant setting, and in particular, from that cohort with the most aggressive disease (shortest relapse-free survival (RFS)). The I-SPY 1 TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and extensive molecular and clinical/pathological characterization of non-responding patients. Methods: Tumor epithelium was procured by Laser Capture Microdissection (LCM) from frozen surgical specimens of 27 patients who did not achieve complete pathological response in the I-SPY1 TRIAL. Reverse Phase Protein Microarray (RPPA) technology was used to measure the levels of 120 key signaling proteins including drug targets for Phase I-III and FDA-approved therapeutics. Cox proportional hazard univariate and multivariate analyses were used to evaluate RFS correlates. Significance threshold was p = Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-01.