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Background Patients with primary hyperparathyroidism (PHPT) treated surgically occasionally have normalized calcium, but persistently high parathyroid hormone (PTH). We hypothesized that a possible explanation for this phenomenon is an underlying hyperplasia rather than adenoma. Methods Retrospective cohort of patients who underwent parathyroidectomy for PHPT with biopsy of a normal‐appearing parathyroid gland were included. Cellularity level of each biopsy and of the adenoma's rim was determined. Results Forty‐seven patients were included. Of them, 19 (40%) had postoperative normocalcemia but elevated PTH. There was no correlation between cellularity either in the rim or of the normal‐appearing parathyroid gland and postoperative PTH. The postoperative high PTH group had higher preoperative PTH (P = 0.001) and larger adenomas (P = 0.025). Conclusions High PTH levels after successful parathyroidectomy in patients with primary hyperparathyroidism do not appear to result from underlying hyperplasia. A possible alternative explanation is that these patients have a higher preoperative burden of disease., Patients with Primary hyperparathyroidism treated surgically occasionally have normalized calcium, but persistently high PTH. We hypothesized that a possible explanation for this phenomenon is an underlying hyperplasia rather than adenoma.? Contrary to our hypothesis, there was no correlation between the cellularity of the normal‐appearing parathyroid glands and the parathyroid tissue surrounding the adenoma with PTH levels after surgery.

High-risk Human Papillomavirus (HPV) infections have recently emerged as an independent risk factor in head and neck squamous cell carcinoma (HNSCC). There has been a marked increase in the incidence of HPV-induced HNSCC subtype, which demonstrates different genetics with better treatment outcome. Despite the favourable prognosis of HPV-HNSCC, the treatment modality, consisting of high dose radiotherapy (RT) in combination with chemotherapy (CT), remains similar to HPV-negative tumours, associated with toxic side effects. Epidermal growth factor receptor (EGFR) is overexpressed in over 80% of HNSCC and correlates with RT resistance. EGFR inhibitor Cetuximab is the only FDA approved targeted therapy for both HNSCC subtypes, however the response varies between HNSCC subtypes. In HPV-negative HNSCC, Cetuximab sensitises HNSCC to RT improving survival rates. To reduce adverse cytotoxicity of CT, Cetuximab has been approved for treatment de-escalation of HPV-positive HNSCC. The results of several recent clinical trials have concluded differing outcome to HPV-negative HNSCC. Here we investigated the role of EGFR in HPV-positive HNSCC response to RT. Remarkably, in HPV-positive HNSCC cell lines and in vivo tumour models, EGFR activation was strongly indicative of increased RT response. In response to RT, EGFR activation induced impairment of DNA damage repair and increased RT response. Furthermore, EGFR was found to downregulate HPV oncoproteinE6 expression and induced p53 activity in response to RT. Collectively, our data uncovers a novel role for EGFR in virally induced HNSCC and highlights the importance of using EGFR-targeted therapies in the context of the genetic makeup of cancer.

Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Using pre-clinical HNC models, we demonstrated that treatment with the MEK1/2 blocker trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the anti-tumor immunity of CD8+T cells. Further activation of CD8+T cells by supplementation with anti-programmed death-1 (αPD-1) antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived colony-stimulating factor-1 (CSF-1), which reduced the abundance of two CSF-1R+CD11c+myeloid-derived suppressor cell (MDSC) populations in the tumor microenvironment (TME). In contrast, prolonged treatment with trametinib abolished the anti-tumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition (EMT) in response to trametinib restored CSF-1 expression and re-created an immune-suppressive TME. These findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to immunotherapies by using targeted therapies to interfere with the host-tumor interaction.Graphical abstract

Most Phase II and III clinical trials in head and neck cancer (HNC) combine two or more treatment modalities, which are based, in part, on knowledge of the molecular mechanisms of innate and acquired resistance to monotherapy. In this review, we describe the range of tumor-cell autonomously derived (intrinsic) and tumor-microenvironment-derived (extrinsic) acquired-resistance mechanisms to various FDA-approved monotherapies for HNC. Specifically, we describe how tumor cells and the tumor microenvironment (TME) respond to radiation, chemotherapy, targeted therapy (cetuximab), and immunotherapies [programmed cell death 1 (PD-1) inhibitors] and adapt to the selective pressure of these monotherapies. Due to the diversity of adaptive responses to monotherapy, monitoring the response to treatment in patients is critical to understand the path that leads to resistance and to guide the optimal therapeutic drug combinations in the clinical setting. We envisage that applying such a rationale-based therapeutic strategy will improve treatment efficacy in HNC patients.

An understanding of the mechanisms underlying acquired resistance to cetuximab is urgently needed to improve cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Here, we present a clinical observation that MET pathway activation constitutes the mechanism of acquired resistance to cetuximab in a patient with HNSCC. Specifically, RNA sequencing and mass spectrometry analysis of cetuximab-sensitive (CetuxSen ) and cetuximab-resistant (CetuxRes ) tumors indicated MET amplification and overexpression in the CetuxRes tumor compared to the CetuxSen lesion. Stimulation of MET in HNSCC cell lines was sufficient to reactivate the MAPK pathway and to confer resistance to cetuximab in vitro and in vivo. In addition to the direct role of MET in reactivation of the MAPK pathway, MET stimulation abrogates the well-known cetuximab-induced compensatory feedback loop of HER2/HER3 expression. Mechanistically, we showed that the overexpression of HER2 and HER3 following cetuximab treatment is mediated by the ETS homologous transcription factor (EHF), and is suppressed by MET/MAPK pathway activation. Collectively, our findings indicate that evaluation of MET and HER2/HER3 in response to cetuximab in HNSCC patients can provide the rationale of successive line of treatment.

We hypothesize that the biomechanical properties of cells can predict their viability, with Young's modulus representing the former and cell sensitivity to ultrasound representing the latter. Using atomic force microscopy, we show that the Young's modulus stiffness measure is significantly lower for superficial cancer cells (squamous cell carcinomas and melanoma) compared with noncancerous keratinocyte cells. In vitro findings reveal a significant difference between cancerous and noncancerous cell viability at the four ultrasound energy levels evaluated, with different cell lines exhibiting different sensitivities to the same ultrasound intensity. Young's modulus correlates with cell viability (R 2 = 0.93), indicating that this single biomechanical property can predict cell sensitivity to ultrasound treatment. In mice, repeated ultrasound treatment inhibits tumor growth without damaging healthy skin tissue. Histopathological tumor analysis indicates ultrasound‐induced focal necrosis at the treatment site. Our findings provide a strong rationale for developing ultrasound as a noninvasive selective treatment for superficial cancers.

Most head and neck cancer (HNC) patients are resistant to cetuximab, an antibody against the epidermal growth factor receptor. Such therapy resistance is known to be mediated, in part, by stromal cells surrounding the tumor cells, however, the mechanisms underlying such a resistance phenotype remain unclear. To identify the mechanisms of cetuximab resistance in an unbiased manner, RNA-sequencing (RNA-seq) of HNC patient-derived xenografts (PDXs) was performed. Comparing the gene expression of HNC-PDXs before and after treatment with cetuximab indicated that the transforming growth factor-beta (TGF-beta) signaling pathway was upregulated in the stromal cells of PDXs that progressed on cetuximab treatment (CetuximabProg-PDX). However, in PDXs that were extremely sensitive to cetuximab (CetuximabSen-PDX), the TGF-beta pathway was downregulated in the stromal compartment. Histopathological analysis of PDXs showed that TGF-beta-activation was detected in cancer-associated fibroblasts (CAFs) of CetuximabProg-PDX. These TGF-beta-activated CAFs were sufficient to limit cetuximab efficacy in vitro and in vivo. Moreover, blocking the TGF-beta pathway using the SMAD3 inhibitor, SIS3, enhanced cetuximab efficacy and prevented the progression of CetuximabProg-PDX. Altogether, our findings indicate that TGF-beta-activated CAFs play a role in limiting cetuximab efficacy in HNC.

Simple Summary Over the past decades, the multi-drug approach for treating various types of cancers was highly recommended. The aim of this study was to demonstrate the advantage of treating different carcinoma types with several combinations of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs). We showed that patients treated with the combination of lenvatinib and pembrolizumab showed an enhanced response to therapy compared to other treatments. Our ex-vivo studies exhibited reduced proliferation and PD-L1 levels for PDX explants treated with this combination compared to untreated explants or those treated with a single treatment. Based on the results of this study, we propose that the combination of levatinib and pembrolizumab shows great potential as a treatment option for patients with non-small cell lung carcinoma and malignant pleural mesothelioma, as previously suggested by recent clinical trials. Abstract The new era of cancer treatments has made immune checkpoint inhibitors (ICIs) and emerging multikinase inhibitors (TKIs) the standards of care, thus drastically improving patient prognoses. Pembrolizumab is an anti-programmed cell death-1 antibody drug, and lenvatinib is a TKI with preferential antiangiogenic activity. We present, to our knowledge, the first reported series of cases consisting of patients with metastatic non–small cell lung cancer and malignant pleural mesothelioma who were treated with several types of chemotherapy combinations and ICIs followed by disease progression. They were subsequently treated with combined immunotherapy and TKI treatment, resulting in a near complete response within a very short time. Clinical responses were supported by in vitro testing of each patient’s lymphocytic response to pembrolizumab after pre-exposure of target cancer cells to lenvatinib.

Background Acquisition of resistance to anticancer therapies is associated with increased expression of immunosuppressor modulators that enable tumor cells to escape from the antitumor immunity machinery. Objective To understand the mechanisms of immune escape from MEK inhibitors in head and neck cancer (HNC). Methods We have developed several mitogen-activated protein kinase (MAPK) pathway-driven cancers in immunocompetent mice. Specifically, a 4NQO (tobacco surrogate) carcinogenesis induced HNC model, novel KRASG12-driven HNC model using CRISPR technology, and HRASV12 model using a lentiviral vector. These models were used to study mechanisms of immune escape from trametinib, an MAPK pathway inhibitor. Results All of these HNC models are sensitive to trametinib in vitro and in vivo; however, over time, resistance was developed, and tumor progression was detected. In this work, we found that chronic treatment with trametinib induced epithelial-mesenchymal transition and upregulation of the tumor-derived immunosuppressor modulators like PD-L1. The tumor ecosystem was also altered during the acquisition of resistance because upon short treatment with trametininb during stable disease, tumors were enriched with infiltrated CD8+ T, whereas upon progression the CD8+ T cells in the tumors showed an exhausted phenotype with high levels of programmed cell death protein 1 (PD-1). Blocking the immune escape mechanisms using anti PD-1/ programmed death ligand 1 (PD-L1) drugs together with trametinib induced tumor eradication and most mice were cured. Conclusions Our findings suggest that administration of blockers of the PD1/PD-L1 axis enhance the antitumor activity of MAPK-targeted drugs and delay the appearance of resistance.

AXL overexpression is a common resistance mechanism to anti-cancer therapies, including the resistance to BYL719 (Alpelisib) - the p110α isoform specific inhibitor of phosphoinositide 3-kinase (PI3K) - in esophagus and head and neck squamous cell carcinoma (ESCC, HNSCC respectively). However, the mechanisms underlying AXL overexpression in resistance to BYL719 remain elusive. Here we demonstrated that the AP-1 transcription factors, c-JUN and c-FOS, regulate AXL overexpression in HNSCC and ESCC. The expression of AXL was correlated with that of c-JUN both in HNSCC patients and in HNSCC and ESCC cell lines. Silencing of c-JUN and c-FOS expression in tumor cells downregulated AXL expression and enhanced the sensitivity of human papilloma virus positive (HPVPos) and negative (HPVNeg) tumor cells to BYL719 in vitro. Blocking of the c-JUN N-terminal kinase (JNK) using SP600125 in combination with BYL719 showed a synergistic anti-proliferative effect in vitro, which was accompanied by AXL downregulation and potent inhibition of the mTOR pathway. In vivo, the BYL719-SP600125 drug combination led to the arrest of tumor growth in cell line-derived and patient-derived xenograft models, and in syngeneic head and neck murine cancer models. Collectively, our data suggests that JNK inhibition in combination with anti-PI3K therapy is a new therapeutic strategy that should be tested in HPVPos and HPVNeg HNSCC and ESCC patients.

Despite of remarkable progress made in the head and neck cancer (HNC) therapy, the survival rate of this metastatic disease remain low. Tailoring the appropriate therapy to patients is a major challenge and highlights the unmet need to have a good preclinical model that will predict clinical response. Hence, we developed an accurate and time efficient drug screening method of tumor ex vivo analysis (TEVA) system, which can predict patient-specific drug responses. In this study, we generated six patient derived xenografts (PDXs) which were utilized for TEVA. Briefly, PDXs were cut into 2 × 2 × 2 mm3 explants and treated with clinically relevant drugs for 24 h. Tumor cell proliferation and death were evaluated by immunohistochemistry and TEVA score was calculated. Ex vivo and in vivo drug efficacy studies were performed on four PDXs and three drugs side-by-side to explore correlation between TEVA and PDX treatment in vivo. Efficacy of drug combinations was also ventured. Optimization of the culture timings dictated 24 h to be the time frame to detect drug responses and drug penetrates 2 × 2 × 2 mm3 explants as signaling pathways were significantly altered. Tumor responses to drugs in TEVA, significantly corresponds with the drug efficacy in mice. Overall, this low cost, robust, relatively simple and efficient 3D tissue-based method, employing material from one PDX, can bypass the necessity of drug validation in immune-incompetent PDX-bearing mice. Our data provides a potential rationale for utilizing TEVA to predict tumor response to targeted and chemo therapies when multiple targets are proposed.

Uncontrolled growth of ovarian cancer cells is the fifth leading cause of female cancer deaths. Here, we describe the role of scattering-induced absorption in monitoring the cancer treatment efficiency in real-time. We measure the optical interaction between the evanescent fields of microfiber and ovarian cancer inter-cellular medium at different treatment stages. By introducing the roughness of silica glass, we demonstrate that the treatment of tumor cells with GDC0032, a beta-sparing PI3K, induces both cells growth arrest and alter the spectral lines in a dose-dependent manner. The data support observed affected tumor cells with decrease in proliferation and their spatial distribution. Spectral absorption signatures are correlated with optical micrographs and western blot tests. These observations are mediated by surface scatterers out of silica glass material, form an essential step toward the development of early detection of response to cancer therapy.

AXL is a tyrosine kinase membrane receptor that signals via PI3K, MAPK, and protein kinase C (PKC), among other pathways. AXL has oncogenic potential and interacts with other membrane receptors, depending on their relative abundance and availability. The increased expression of AXL in cancer is often the result of pharmacologic selective pressure to a number of chemotherapies and targeted therapies and acts as a mechanism of acquired drug resistance. This resistance phenotype, frequently accompanied by epithelial-to-mesenchymal transition, can be reversed by AXL inhibition. In tumors with high levels of EGFR, including lung, head and neck, and triple-negative breast cancer, AXL dimerizes with this receptor and initiates signaling that circumvents the antitumor effects of anti-EGFR therapies. Likewise, AXL overexpression and dimerization with EGFR can overcome PI3K inhibition by activating the phospholipase C-γ-PKC cascade that, in turn, sustains mTORC1 activity. The causative role of AXL in inducing drug resistance is underscored by the fact that the suppression of AXL restores sensitivity to these agents. Hence, these observations indicate that AXL is selectively expressed in tumor cells refractory to therapy and that cotargeting AXL in this setting would potentially overcome drug resistance. The use of AXL inhibitors should be considered in the clinic. Clin Cancer Res; 22(6); 1313–7. ©2016 AACR.

Activating alterations in PIK3CA, the gene coding for the catalytic subunit of phosphoinositide-3-kinase (PI3K), are prevalent in head and neck squamous cell carcinoma (HNSCC) and thought to be one of the main drivers of these tumors. However, early clinical trials on PI3K inhibitors (PI3Ki) have been disappointing due to the limited durability of the activity of these drugs. To investigate the resistance mechanisms to PI3Ki and attempt to overcome them, we conducted a molecular-based study using both HNSCC cell lines and patient-derived xenografts (PDXs). We sought to simulate and dissect the molecular pathways that come into play in PIK3CA-altered HNSCC treated with isoform-specific PI3Ki (BYL719, GDC0032). In vitro assays of cell viability and protein expression indicate that activation of the mTOR and cyclin D1 pathways is associated with resistance to PI3Ki. Specifically, in BYL719-resistant cells, BYL719 treatment did not induce pS6 and pRB inhibition as detected in BYL719-sensitive cells. By combining PI3Ki with either mammalian target of rapamycin complex 1 (mTORC1) or cyclin D1 kinase (CDK) 4/6 specific inhibitors (RAD001 and abemaciclib, respectively), we were able to overcome the acquired resistance. Furthermore, we found that PI3Ki and CDK 4/6 inhibitors have a synergistic anti-tumor effect when combined in human papillomavirus (HPV)-negative/PIK3CA-WT tumors. These findings provide a rationale for combining PI3Ki and CDK 4/6 inhibitors to enhance anti-tumor efficacy in HNSCC patients.

AXL overexpression is a common resistance mechanism to anti-cancer therapies, including the resistance to BYL719 (Alpelisib) – the p110α isoform specific inhibitor of phosphoinositide 3-kinase (PI3K) – in esophagus and head and neck squamous cell carcinoma (ESCC, HNSCC respectively). However, the mechanisms underlying AXL overexpression in resistance to BYL719 remain elusive. Here we demonstrated that the AP-1 transcription factors, c-JUN and c-FOS, regulate AXL overexpression in HNSCC and ESCC. The expression of AXL was correlated with that of c-JUN both in HNSCC patients and in HNSCC and ESCC cell lines. Silencing of c-JUN and c-FOS expression in tumor cells downregulated AXL expression and enhanced the sensitivity of human papilloma virus positive (HPVPos) and negative (HPVNeg) tumor cells to BYL719 in vitro. Blocking of the c-JUN N-terminal kinase (JNK) using SP600125 in combination with BYL719 showed a synergistic anti-proliferative effect in vitro, which was accompanied by AXL downregulation and potent inhibition of the mTOR pathway. In vivo, the BYL719-SP600125 drug combination led to the arrest of tumor growth in cell line-derived and patient-derived xenograft models, and in syngeneic head and neck murine cancer models. Collectively, our data suggests that JNK inhibition in combination with anti-PI3K therapy is a new therapeutic strategy that should be tested in HPVPosand HPVNegHNSCC and ESCC patients.

Genomic alterations (GA) in PIK3CA leads to the hyper-activation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway in more than 20% of ovarian cancer (OC) patients. Therefore, PI3K therapies are under clinical evaluation for this subset of patients. Evidently, in clinical trials testing the efficacy of isoform-specific inhibitors of PI3K (PI3Ki), patients having a stable disease eventually relapse, as tumors become resistant to treatment. Hence, there is an urgent clinical need to develop new therapeutic combinations to improve the efficacy of PI3Ki in PIK3CA-driven OC patients. Here we identified the molecular mechanism that limits the efficacy of the beta-sparing PI3Ki, Taselisib (GDC0032), in PIK3CA-mutated OC cell lines (IGROV1 and OAW42) that acquired resistance to GDC0032. By comparing the molecular profile of GDC0032-sensitve and -resistant OC cell lines, we found that AKT/mTOR inhibition is required for GDC0032 efficacy. In resistant cells, the sustained activation of AKT/mTOR was regulated by the upregulation of the insulin growth factor 1 receptor (IGF1R). Knockdown of IGF1R re-sensitized cells to GDC0032 in vitro, and the combination of AEW541, an IGF1R inhibitor, with GDC0032 exhibited potent anti-tumor activity in vitro and in vivo. We further demonstrated that IGF1R regulates tumor cell proliferation in IGROV1 cells, whereas in OAW42, it determines autophagy as well. Overall, our findings suggest that the dual inhibition of PI3K and IGF1R may be considered as a new therapeutic strategy in PIK3CA-driven OC.

Current technologies for optical gas imaging use one of the following methods: (1) single band-pass filter, which dictate a “man in the loop” for detection; (2) bi-channel sensors that need allocation of half of the detecting area for each band; (3) low rate and/or highly expensive multi/hyperspectral sensors. We suggest a low-cost, fast electrically-switchable notch-filter, based on liquid crystal (LC) in an infrared (IR) transparent cell which enables dynamic dual band sensing on the entire detector area. We utilize fundamental properties of the LC: its molecular IR absorption bands (rovibrational modes) are overlapping those of gaseous (hydrocarbons, carbon dioxide , and more), and the transmittance of these bands depends on the orientation of the LC molecules relative to the polarization of the incident light (ordinary or extraordinary), Applying voltage on the LC cell affects the molecules' orientation, and therefore significantly changes the band transmittance. The high frequency response of LC cells to the electrical field enable detecting dynamic gas plumes. We proved this concept experimentally using custom made polyamide-coated Germanium cells and a commercial LC, E7. The spectral response, measured by a fast spectro-radiometer, for wavelengths between 2 to 14 micrometer, shows several absorption lines overlapping those of hydro-carbonates hazardous and greenhouse gaseous that require monitoring, and we demonstrated the detection of the refrigerant gas R-134a. We are currently preforming an experimental imaging setup for gas detection based on this method, and the results will be presented in this talk.

Background: Preclinical data suggest squamous cell tumors, such as SCCHN, are driven by epidermal growth factor receptor (EGFR) overexpression. However, many patients are refractory to anti-EGFR treatment due to intrinsic and acquired resistance. PI3K/AKT/mTOR pathway activation is a potential mechanism of resistance to EGFR-directed therapy. BYL719, a selective α-isoform PI3K inhibitor, enhances cetuximab (EGFR inhibitor) activity in SCCHN cell lines. A Phase Ib/II study combining BYL719 with cetuximab in patients with recurrent/metastatic SCCHN is testing this hypothesis (Razak et al, ASCO 2014, abst 6044; NCT01602315). Here, we outline the preclinical rationale for combining BYL719 with cetuximab in the setting of cetuximab-resistant squamous cell carcinoma, highlight Phase Ib safety and efficacy data in patients with SCCHN who have received prior cetuximab, and describe the design of the Phase II portion of the study, which is assessing both cetuximab-naive and -pretreated patients. Methods: The combination of BYL719 and cetuximab was tested in vivo in two xenograft models of esophageal squamous cell carcinoma: KYSE180 (cetuximab-sensitive model) and KYSE180_CR (cetuximab-resistant, induced by long-term in vivo treatment). In the Phase Ib study, BYL719 was administered once daily (QD) in 28-day cycles with standard weekly (QW) cetuximab (400 mg/m2 on Cycle 1 Day 1; 250 mg/m2 QW thereafter) to adults with histologically/cytologically confirmed recurrent/metastatic SCCHN resistant/intolerant to platinum-based chemotherapy (prior cetuximab therapy was allowed). Results: In the KYSE180 model, the addition of BYL719 to cetuximab demonstrated an additive effect leading to tumor regression; furthermore, in the KYSE180_CR model, adding BYL719 to cetuximab completely restored sensitivity and led to similar activity as seen in the cetuximab-sensitive model, suggesting that inhibiting PIK3CA signaling may overcome resistance to cetuximab. In the Phase Ib study, as of March 10, 2014, 37 patients were treated with BYL719 300 mg QD (n=32) or 400 mg QD (n=5) and cetuximab. Frequent adverse events (>30%; all-grade/Grade 3/4) were hyperglycemia (54/24), stomatitis (38/5), and dermatitis acneiform (35/3). Best overall response (BOR) per RECIST v1.1 in the full population (n=37) was 4 partial responses (PRs), 16 stable disease (SD; of which 5 were unconfirmed PRs), 10 unknown (UNK) responses, and 1 non-complete response/non-progressive disease (PD) at 300 mg or 400 mg. Six patients had PD as BOR. The overall response rate (ORR) in the full population was 4/37 (11%) and the disease control rate (DCR) was 20/37 (54%). Within this population, 7 patients had received prior cetuximab therapy (6 in the metastatic/recurrent setting and 1 curative; BOR: 4 SD, 1 PR, 1 PD, and 1 UNK). Of these 7 patients, 1 had a confirmed PR, 2 had unconfirmed PRs, 2 had SD, and 1 had PD with BYL719 and cetuximab. One patient with prior cetuximab therapy had an UNK response due to death from tumor lysis syndrome after 1 week in the study. The ORR among patients with prior cetuximab was 1/7 (14%) and DCR was 5/7 (71%). Phase II tests the combination of BYL719 at the recommended Phase II dose of 300 mg QD in two second-line SCCHN patient populations. Patients who are cetuximab-naive are randomized to cetuximab alone or to BYL719 in combination with cetuximab, while patients who have received cetuximab and platinum therapy in the first-line setting are enrolled in a single arm to receive BYL719 with cetuximab. Conclusion: Combined inhibition of PI3Kα and EGFR by BYL719 and cetuximab, respectively, overcame cetuximab resistance in the preclinical setting, was well tolerated, and demonstrated encouraging antitumor activity in both cetuximab-naive and -pretreated patients with SCCHN. The Phase II part of the study in both patient populations is ongoing. Citation Format: Pamela Munster, Moshe Elkabets, Jill Gilbert, Albiruni R Abdul Razak, Myung-Ju Ahn, Chia-Jui Yen, Se-Hoon Lee, Hung-Ming Wang, Carla van Herpen, Wan-Teck Lim, David Demanse, Rupam Ranjan Pal, Alan Huang, Qing Sheng, Chiara Lambertini, Malte Peters, Christina Coughlin, Maurizio Scaltriti, José Baselga, George Blumenschein. Inhibition of PIK3CA with BYL719 can overcome resistance to cetuximab in squamous cell carcinoma of the head and neck (SCCHN). [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A46.

Triple negative breast cancer (TNBC) is clinically defined by lack of expression of estrogen and progesterone receptors and no overexpression/amplification of human epidermal growth factor receptor 2 (HER2). It affects approximately one-fourth of breast cancer patients and is usually characterized by an aggressive phenotype with high mitotic index and poor prognosis. No TNBC-targeted therapeutic exists and the standard of care is currently limited to cytotoxic chemotherapy. Overexpression of human epidermal growth factor receptor (EGFR) as well as inactivation or decreased expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are frequent in TNBC. Thus, inhibition of EGFR and/or the PI3K/Akt pathway are attractive therapeutic strategies for this malignancy. Preclinical evidence, however, suggests that inhibition of the PI3K/Akt axis induces compensatory expression and activation of upstream receptor tyrosine kinases, including EGFR and, most prominently, human epidermal growth factor receptor 3 (HER3). This may reduce the antitumor effects of single-agent PI3K pathway blockade. Furthermore, studies using in vitro models of resistance to the anti-EGFR antibody cetuximab suggest that HER3 expression itself can limit the sensitivity to this agent by increasing EGFR/HER3 heterodimerization and activation of downstream pathways. In this work we hypothesized that targeting both EGFR and HER3, in combination with PI3K/Akt inhibition, would result in enhanced therapeutic activity in EGFR-positive TNBC. We treated PTEN-negative EGFR-positive HCC70 and MDA-MB-468 TNBC cell lines with GDC-0068, a selective inhibitor of the Akt 1/2/3 isoforms, GDC-0941, a class I selective pan-PI3K inhibitor, MEHD7945A, an antibody targeting both EGFR and HER3 and the combinations of these inhibitors. Compared to single agents, the combination of GDC-0068 or GDC-0941 and MEHD7945A prevented the induction of EGFR and HER3 phosphorylation, enhanced the inhibition of the PI3K and ERK downstream pathways and resulted in superior inhibition of cell proliferation/viability compared to single agents. To expand our findings in vivo, we first tested the efficacy of MEHD7945A in combination with either the Akt or PI3K inhibitor in both MDA-MB-468- and HCC70-derived xenografts. While the tumors responded only modestly to single agent GDC-0068, GDC-0941 and MEHD7945A, the combination of GDC-0068 or GDC-0941 with MEHD7945A yielded superior tumor growth inhibition compared to monotherapies. We next investigated the levels of EGFR and HER3 expression/activation in HCC70 tumors collected at the end of the experiments by reverse-phase protein detection. As expected, increase of both EGFR and HER3 expression and phosphorylation induced by Akt or PI3K was prevented by the addition of MEHD7945A. We then tested the activity of the same treatments in a patient-derived xenograft (PDX) model of TNBC available in our laboratory. These tumors were characterized by undetectable levels of PTEN, high levels of EGFR and ∼70% staining for Ki67. When used as single agents, GDC-0068, GDC-0941 and MEHD7945A delayed tumor growth. However, their combinations elicited durable tumor stasis. Consistently with the cell-based xenografts, MEHD7945A counteracted EGFR and HER3 phosphorylation caused by PI3K/Akt inhibition. Collectively, these data show that targeting both EGFR and HER3 enhances the antitumor effects of PI3K/Akt inhibitors. In order to dissect the role of HER3 inhibition in these models, we compared the activity of cetuximab with MEHD7945A in combination with either GDC-0068 or GDC-0941 in HCC70 cells. Both antibodies enhanced the antiproliferative activity mediated by PI3K/Akt inhibition in cells stimulated with the EGFR ligand epidermal growth factor; however MEHD7945A was superior to cetuximab in cooperating with the antiproliferative activity of GDC-0068 and GDC-0941 in cells stimulated with the HER3 ligand heregulin. We next compared the antitumor activity of cetuximab and MEHD7945A in combination with GDC-0941 in HCC70-derived xenografts. While concomitant targeting of EGFR, HER3 and PI3K was superior to monotherapies and led to tumor shrinkage, the combination of cetuximab and GDC-0941 did not result to any further inhibition of tumor growth compared to single agent treatments. Biochemically, both MEHD7945A and cetuximab blocked EGFR phosphorylation in these xenografts; however, only MEHD7945A decreased HER3 activation. These results confirm that HER3 plays an important role in limiting the efficacy of PI3K inhibition in this setting. To investigate whether changes in EGFR and HER3 levels can affect the response to anti-EGFR therapy in TNBC patients, we measured the expression of these receptors in samples from patients enrolled in two pilot neoadjuvant clinical trials testing the antitumor activity of the anti-EGFR antibodies panitumumab (40 patients) and cetuximab (30 patients) in combination with standard chemotherapy. Of 40 patients treated with panitumumab-based therapy 19 (47.5%) achieved pathological complete response (pCR) after 24 weeks and 21 (52.5%) showed residual disease at the time of surgery. We measured EGFR and HER3 expression by immunohistochemistry in the 40 pre-treatment specimens and in the 21 residual invasive tumors excised at surgery. Patients with higher baseline EGFR expression were more likely to achieve pCR. Moreover, EGFR levels were decreased in the residual tumors of nine out of 21 non-pCR patients when compared to the paired baseline specimens (p=0.07). HER3 staining, available for 13 non-responder patients, showed higher HER3 expression in the residual lesions of 7 out of 13 non-pCR patients compared to the paired pre-treatment samples (p=0.01). Of 30 patients enrolled in the study testing the antitumor activity of cetuximab-based therapy, 9 experienced pCR. Consistently, HER3 expression was found upregulated in the residual tumors of 11 out of 19 non-pCR patients when compared to the baseline paired specimens (p=0.103). These results suggest that high EGFR expression may be required for optimal response to anti-EGFR therapeutic antibodies and that HER3 expression increases following anti-EGFR therapy in patients that do not experience complete tumor regression. In this work we provide evidence that HER3 plays a potential role in limiting the antitumor activity of both PI3K/Akt inhibitors and anti-EGFR agents. We demonstrate that simultaneous targeting of EGFR and HER3 is required to enhance the efficacy of PI3K/Akt inhibition in preclinical models of EGFR-positive TNBC. Further, our clinical findings suggest that HER3 expression is induced in TNBC patients with a lower probability of achieving tumor remission upon anti-EGFR therapy. Therefore, the rationale behind targeting simultaneously EGFR, HER3 and PI3K in TNBC is based on: 1) nearly half of TNBCs express high levels of EGFR, 2) TNBCs often express low levels of PTEN and 3) HER3 upregulation may limit their sensitivity to targeted therapy. In addition, tumor cells that express relatively high levels of these receptors can function as molecular “flags” for immune-mediated antibody dependent cytotoxicity, intrinsic characteristics of IgG type I antibodies such as MEHD7945A in vivo. Given that both pharmacokinetic and pharmacodynamic data for MEHD7945A, GDC-0068 and GDC-0941 are already available, the design of phase II clinical trials testing the activity of these possible combinations in TNBC would be straightforward. Patient enrollment may be based on high EGFR expression with the requirement of mandatory “on-treatment” biopsies to evaluate both pathway inhibition and possible HER3 upregulation upon therapy. In conclusion, we believe that simultaneous inhibition of EGFR, HER3 and the PI3K/Akt pathway has the potential to greatly expand the percentage of TNBC patients who can benefit from targeted therapy. Citation Format: Maurizio Scaltriti, Jessica Tao, Pau Castel, Nina Radosevic-Robin, Moshe Elkabets, Neil Auricchio, Dejan Juric, Frédérique Penault-Llorca, Jose Baselga. Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG01. doi:10.1158/1538-7445.AM2014-NG01

Activating mutations of the PIK3CA gene occur frequently in breast cancer and inhibitors that are specific for PI3K p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as IGF1 and neuregulin1 can activate mTOR and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α targeted drugs and delay the appearance of resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B106. Citation Format: Moshe Elkabets, Sadhna Vora, Dejan Juric, Natasha Morse, Mari Mino-Kenudson, Taru Muranen, Jessica Tao, Ana Bosch Campos, Jordi Rodon, Yasir H. Ibrahim, Violeta Serra, Vanessa Rodrik-Outmezguine, Saswati Hazra, Sharat Singh, Phillip Kim, Cornelia Quadt, Liu Manway, Alan Huang, Neal Rosen, Jeffrey A. Engelman, Maurizio Scaltriti, José Baselga. mTORC1 inhibition is required for sensitivity to PI3K p110α inhibitors in PIK3CA-mutant breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B106.

The phosphatidylinositol 3-kinase (PI3K) pathway is deregulated across a broad spectrum of human cancers. Therefore, there have been strong efforts to inhibit this pathway, with many novel targeted agents, including isoform-specific PI3K inhibitors, pan-PI3K inhibitors, and dual PI3K/mTOR inhibitors, entering the clinic. Although initial responses have been observed in PIK3CA and HER2 amplified cancers, resistance frequently emerges, thereby limiting the benefit from these drugs. We developed several PI3K inhibitor resistant cell lines by culturing sensitive PIK3CA mutant cells in gradually escalating doses of PI3K inhibitor until resistance emerges, typically over a period of several months. We then used a drug screen utilizing ~40 targeted agents to identify drugs that synergize with PI3K inhibitors in the resistant cells. Subsequently, to elucidate mechanisms of resistance to PI3K inhibitors, we used a pooled shRNA screen to identify hairpins that sensitize the resistant cells to PI3K inhibition. These two complementary screens have identified mTORC as a likely mediator of resistance to PI3K inhibition: mTORC inhibitors synergize with PI3K inhibitors in the resistant cells, and furthermore, hairpins directed against mTORC (FRAP1) sensitize resistant cells to PI3K inhibition. Other promising therapeutic targets identified by these screens that may contribute to resistance to PI3K inhibitors include IGF1R and AKT. This abstract is also presented as Poster B069. Citation Format: Sadhna Vora, Moshe Elkabets, Nayoon Kim, Carlotta Costa, Manway Liu, Li Xiaoyan, Alex Cao, Robert Schlegel, Maurizio Scaltriti, Jose Baselga, Alan Huang, Jeffrey A. Engelman. Overcoming resistance to PI3K inhibitors in breast cancer: Results of a pooled shRNA screen and combinatorial drug screen. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR06.