Targeting Aggressive B-cell Lymphomas through Pharmacological Activation of the Mitochondrial Protease OMA1.

DLBCL are aggressive, rapidly proliferating tumors that critically depend on the ATF4-mediated integrated stress response (ISR) to adapt to stress caused by uncontrolled growth, such as hypoxia, amino acid deprivation, and accumulation of misfolded proteins. Here, we show that ISR hyperactivation is a targetable liability in DLBCL. We describe a novel class of compounds represented by BTM-3528 and BTM-3566, which activate the ISR through the mitochondrial protease OMA1. Treatment of tumor cells with compound leads to OMA1-dependent cleavage of DELE1 and OPA1, mitochondrial fragmentation, activation of the eIF2α-kinase HRI, cell growth arrest, and apoptosis. Activation of OMA1 by BTM-3528 and BTM-3566 is mechanistically distinct from inhibitors of mitochondrial electron transport, as the compounds induce OMA1 activity in the absence of acute changes in respiration. We further identify the mitochondrial protein FAM210B as a negative regulator of BTM-3528 and BTM-3566 activity. Overexpression of FAM210B prevents both OMA1 activation and apoptosis. Notably, FAM210B expression is nearly absent in healthy germinal center B-lymphocytes and in derived B-cell malignancies, revealing a fundamental molecular vulnerability which is targeted by BTM compounds. Both compounds induce rapid apoptosis across diverse DLBCL lines derived from activated B-cell, germinal center B-cell, and MYC-rearranged lymphomas. Once-daily oral dosing of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first-of-its kind approach of selectively hyperactivating the mitochondrial ISR for treating DLBCL.

Quantitative Multiplexed Proteomics Could Assist Therapeutic Decision Making in Non-Small Cell Lung Cancer Patients with Ambiguous ALK Test Results.

Therapeutic guidance in non-small cell lung cancer (NSCLC) tumors that are positive for anaplastic lymphoma kinase (ALK) fluorescent in situ hybridization (FISH), but negative for ALK immunohistochemistry, is still challenging. Parallel routine screening of 4588 NSCLC cases identified 22 discordant cases. We rechecked these samples using ALK antibodies and selected reaction monitoring (SRM) quantitative multiplexed proteomics screening multiple protein targets, including ALK and MET for the ALK tyrosine kinase inhibitor (TKI), and FR-alpha, hENT1, RRM1, TUBB3, ERCC1, and XRCC1 for chemotherapy. The presence of ALK (31.8%), MET (36.4%), FR-alpha (72.7%), hENT1 (18.2%), RRM1 (31.8%), TUBB3 (72.9%), ERCC1 (4.5%), and a low level of XRCC1 (54.4%) correlated with clinical outcomes. SRM was more sensitive than the ALK D5F3 assay. Among the eight cases receiving ALK TKI, four cases with ALK or MET detected by SRM had complete or partial responses, whereas four cases without ALK or MET showed progression. Twenty-seven treatment outcomes from 20 cases were assessed and cases expressing more than half of the specific predictive proteins were sensitive to matching therapeutic agents and showed longer progression-free survival than the other cases (p < 0.001). SRM showed a potential role in therapeutic decision making in NSCLC patients with ambiguous ALK test results.

Association of high TUBB3 with resistance to adjuvant docetaxel-based chemotherapy in gastric cancer: translational study of ITACA-S.

BACKGROUND: No predictive markers for chemotherapy activity have been validated in gastric cancer (GC). The potential value of class III ß-tubulin (TUBB3) as biomarker for prognosis and resistance to taxane-based therapy was reported. METHODS: We analyzed GC samples of patients enrolled in the Intergroup Trial of Adjuvant Chemotherapy in Adenocarcinoma of the Stomach (ITACA-S), a randomized adjuvant study comparing 5-fluorouracil/leucovorin (5-FU/LV) and docetaxel-based sequential chemotherapy. TUBB3 was quantitated by selected reaction monitoring mass spectrometry and patients were stratified using a threshold of 750 attomoles per microgram (amol/µg). Cox proportional modeling and Kaplan-Meier survival analysis were used to assess the impact of TUBB3 expression on overall survival (OS) and disease-free survival. RESULTS: Patients with TUBB3 protein levels >750 and <750 amol/µg were 21.9% and 78.1%, respectively, and were well-balanced between treatment arms. TUBB3 protein levels were not prognostic. Whereas no survival differences according to the 2 arms were observed in the subgroup with low TUBB3 expression (5-year OS 47% vs 40%; p = 0.44), patients with high TUBB3 had a clinically meaningful poorer OS when receiving docetaxel-based versus 5-FU/LV chemotherapy (5-year OS 31% vs 54%; p = 0.09), with a statistically significant interaction between TUBB3 and treatment (p = 0.049). CONCLUSIONS: The quantification of TUBB3 might be considered as a negative predictive biomarker of benefit from taxane-based therapy in GC. Studies are needed to evaluate its role in the neoadjuvant setting.

MET Exon 14-altered Lung Cancers and MET Inhibitor Resistance.

PURPOSE: MET tyrosine kinase inhibitors (TKIs) can achieve modest clinical outcomes in MET exon 14-altered lung cancers, likely secondary to primary resistance. Mechanisms of primary resistance remain poorly characterized and comprehensive proteomic analyses have not previously been performed. EXPERIMENTAL DESIGN: We performed hybrid capture-based DNA sequencing, targeted RNA sequencing, cell-free DNA sequencing, selected reaction monitoring mass spectrometry (SRM-MS), and immunohistochemistry on patient samples of MET exon 14-altered lung cancers treated with a MET TKI. Associations between overall response rate (ORR), progression-free survival (PFS), and putative genomic alterations and MET protein expression were evaluated. RESULTS: Seventy-five of 168 MET exon 14-altered lung cancers received a MET TKI. Previously undescribed (zygosity, clonality, whole-genome duplication) and known (copy-number focality, tumor mutational burden, mutation region/type) genomic factors were not associated with ORR/PFS (P > 0.05). In contrast, MET expression was associated with MET TKI benefit. Only cases with detectable MET expression by SRM-MS (N = 15) or immunochemistry (N = 22) responded to MET TKI therapy, and cancers with H-score ≥ 200 had a higher PFS than cancers below this cutoff (10.4 vs. 5.5 months, respectively; HR, 3.87; P = 0.02). CONCLUSIONS: In MET exon 14-altered cancers treated with a MET TKI, a comprehensive analysis of previously unknown and known genomic factors did not identify a genomic mechanism of primary resistance. Instead, MET expression correlated with benefit, suggesting the potential role of interrogating the proteome in addition to the genome in confirmatory prospective trials.

High throughput profiling of undifferentiated pleomorphic sarcomas identifies two main subgroups with distinct immune profile, clinical outcome and sensitivity to targeted therapies.

BACKGROUND: Undifferentiated pleomorphic sarcoma (UPS) is the most frequent, aggressive and less-characterized sarcoma subtype. This study aims to assess UPS molecular characteristics and identify specific therapeutic targets. METHODS: High-throughput technologies encompassing immunohistochemistry, RNA-sequencing, whole exome-sequencing, mass spectrometry, as well as radiomics were used to characterize three independent cohorts of 110, 25 and 41 UPS selected after histological review performed by an expert pathologist. Correlations were made with clinical outcome. Cell lines and xenografts were derived from human samples for functional experiments. FINDINGS: CD8 positive cell density was independently associated with metastatic behavior and prognosis. RNA-sequencing identified two main groups: the group A, enriched in genes involved in development and stemness, including FGFR2, and the group B, strongly enriched in genes involved in immunity. Immune infiltrate patterns on tumor samples were highly predictive of gene expression classification, leading to call the group B 'immune-high' and the group A 'immune-low'. This molecular classification and its prognostic impact were confirmed on an independent cohort of UPS from TCGA. Copy numbers alterations were significantly more frequent in immune-low UPS. Proteomic analysis identified two main proteomic groups that highly correlated with the two main transcriptomic groups. A set of nine radiomic features from conventional MRI sequences provided the basis for a radiomics signature that could select immune-high UPS on their pre-therapeutic imaging. Finally, in vitro and in vivo anti-tumor activity of FGFR inhibitor JNJ-42756493 was selectively shown in cell lines and patient-derived xenograft models derived from immune-low UPS. INTERPRETATION: Two main disease entities of UPS, with distinct immune phenotypes, prognosis, molecular features and MRI textures, as well as differential sensitivity to specific anticancer agents were identified. Immune-high UPS may be the best candidates for immune checkpoint inhibitors, whereas this study provides rational for assessing FGFR inhibition in immune-low UPS. FUNDING: This work was partly founded by a grant from La Ligue.

STK11 (LKB1) mutations in metastatic NSCLC: Prognostic value in the real world.

BACKGROUND: Mutations in STK11 (STK11m) and frequently co-occurring KRAS mutations (KRASm/STK11m) are associated with poor survival in metastatic NSCLC (mNSCLC) immuno-oncology trials. There are limited data regarding the prognostic significance of these mutations in a real-world setting. METHODS: This retrospective cohort study analyzed de-identified electronic medical records from the Flatiron Clinico-Genomic database to identify patients with mNSCLC who had initiated first-line immunotherapy (IO; alone or in combination) or chemotherapy under routine care between January 1, 2013 and June 30, 2017. The primary objectives were to assess the prevalence of STK11m and KRASm/STK11m and to determine associations of these mutations with overall and progression-free survival (OS, PFS). RESULTS: Of 2407 patients with mNSCLC, STK11m and KRASm/STK11m were present in 13.6% and 6.5% of patients, respectively. Worse OS outcomes were observed in patients with STK11m versus STK11wt mNSCLC receiving IO (first-line, HR [95% CI], 1.4 [0.9-2.3; p = 0.1]; second-line [subset of first-line cohort], HR, 1.6 [1.3-2.0; p = 0.0002]) or chemotherapy (first-line, HR, 1.4 [1.2-1.6; p < 0.0001]); PFS outcomes showed similar trends. KRASm/STK11m double mutations were associated with worse OS and PFS outcomes versus KRASwt/STK11wt with IO and chemotherapy, similar to the single mutation (STK11m vs STK11wt) findings. CONCLUSIONS: This large observational genomic study among patients receiving routine care highlights the negative prognostic impact of STK11m in patients with mNSCLC treated with IO or chemotherapy. These results complement previous clinical trial data and provide further evidence in the real world of a patient population that would benefit from new treatment options.

Prognostic and Predictive Impact of Circulating Tumor DNA in Patients with Advanced Cancers Treated with Immune Checkpoint Blockade.

The utility of circulating tumor DNA (ctDNA) as a biomarker in patients with advanced cancers receiving immunotherapy is uncertain. We therefore analyzed pretreatment (n = 978) and on-treatment (n = 171) ctDNA samples across 16 advanced-stage tumor types from three phase I/II trials of durvalumab (± the anti-CTLA4 therapy tremelimumab). Higher pretreatment variant allele frequencies (VAF) were associated with poorer overall survival (OS) and other known prognostic factors, but not objective response, suggesting a prognostic role for patient outcomes. On-treatment reductions in VAF and lower on-treatment VAF were independently associated with longer progression-free survival and OS and increased objective response rate, but not prognostic variables, suggesting that on-treatment ctDNA dynamics are predictive of benefit from immune checkpoint blockade. Accordingly, we propose a concept of "molecular response" using ctDNA, incorporating both pretreatment and on-treatment VAF, that predicted long-term survival similarly to initial radiologic response while also permitting early differentiation of responders among patients with initially radiologically stable disease. SIGNIFICANCE: In a pan-cancer analysis of immune checkpoint blockade, pretreatment ctDNA levels appeared prognostic and on-treatment dynamics predictive. A "molecular response" metric identified long-term responders and adjudicated benefit among patients with initially radiologically stable disease. Changes in ctDNA may be more dynamic than radiographic changes and could complement existing trial endpoints.This article is highlighted in the In This Issue feature, p. 1775.

Overcoming hypoxia-induced functional suppression of NK cells.

BACKGROUND: Natural killer (NK) cells are immune cells capable of killing virally infected cells and tumor cells without the need for antigen stimulation. Tumors, however, can create a suppressive microenvironment that decreases NK function. A feature of many tumors is hypoxia (low oxygen perfusion), which has been previously shown to decrease NK function. A high affinity NK (haNK) cell has been engineered to express a high affinity CD16 receptor as well as internal interleukin (IL)-2 for increased antibody-dependent cellular cytotoxicity (ADCC) and activation, respectively. We sought to investigate the tolerance of NK cells versus haNK cells to hypoxia. METHODS: We exposed healthy donor (HD) NK and X-irradiated haNK cells to normoxia (20% oxygen) as well as hypoxia (0% oxygen) and investigated their ability to kill prostate, breast and lung tumor cell lines after 5 hours. We also used monoclonal antibodies cetuximab (anti-EGFR) or avelumab (antiprogrammed death-ligand 1) to investigate the effects of hypoxia on NK ADCC. Genomic and proteomic analyzes were done to determine the effect of hypoxia on the expression of factors important to NK cell function. RESULTS: While HD NK cell cytolytic abilities were markedly and significantly impaired under hypoxic conditions, haNK cells maintained killing capacity under hypoxic conditions. NK killing, serial killing and ADCC were maintained under hypoxia in haNK cells. IL-2 has been previously implicated in serial killing and perforin regeneration and thus the endogenous IL-2 produced by haNK cells is likely a driver of the maintained killing capacity of haNK cells under hypoxic conditions. Activation of signal transducer and activator of transcription 3 (STAT3) is not seen in haNKs under hypoxia but is significant in HD NK cells. Pharmaceutical activation of STAT3 in haNKs led to reduced killing, implicating active STAT3 in reduced NK cell function. CONCLUSIONS: In contrast to HD NK cells, haNK cells are resistant to acute hypoxia. The potent cytolytic function of haNK cells was maintained in an environment comparable to what would be encountered in a tumor. The data presented here provide an additional mechanism of action for haNK cells that are currently being evaluated in clinical trials for several tumor types.

Characterization of MGMT and EGFR protein expression in glioblastoma and association with survival.

PURPOSE: Understanding the molecular landscape of glioblastoma (GBM) is increasingly important in the age of targeted therapy. O-6-Methylguanine-DNA methyltransferase (MGMT) promoter methylation and EGFR amplification are markers that may play a role in prognostication, treatment, and/or clinical trial eligibility. Quantification of MGMT and EGFR protein expression may offer an alternative strategy towards understanding GBM. Here, we quantify baseline expression of MGMT and EGFR protein in newly diagnosed GBM samples using mass spectrometry. We correlate findings with MGMT methylation and EGFR amplification statuses and survival. METHODS: We retrospectively identified adult patients with newly diagnosed resected GBM. MGMT and EGFR protein expression were quantified using a selected reaction monitoring mass spectrometry assay. Protein levels were correlated with MGMT methylation and EGFR amplification and survival data. RESULTS: We found a statistically significant association between MGMT protein expression and promoter methylation status (p = 0.02) as well as between EGFR protein expression and EGFR amplification (p < 0.0001). EGFR protein expression and amplification were more tightly associated than MGMT protein expression and methylation. Only MGMT promoter methylation was statistically significantly associated with progression-free and overall survival. CONCLUSIONS: Unlike EGFR protein expression and EGFR amplification which are strongly associated, only a weak association was seen between MGMT protein expression and promoter methylation. Quantification of MGMT protein expression was inferior to MGMT methylation for prognostication in GBM. Discordance was observed between EGFR amplification and EGFR protein expression; additional study is warranted to determine whether EGFR protein expression is a better biomarker than EGFR amplification for clinical decisions and trial enrollment.

Targeted multiplex proteomics for molecular prescreening and biomarker discovery in metastatic colorectal cancer.

Protein biomarkers are widely used in cancer diagnosis, prognosis, and prediction of treatment response. Here we introduce the use of targeted multiplex proteomics (TMP) as a tool to simultaneously measure a panel of 54 proteins involved in oncogenic, tumour suppression, drug metabolism and resistance, in patients with metastatic colorectal cancer (mCRC). TMP provided valuable diagnostic information by unmasking an occult neuroendocrine differentiation and identifying a misclassified case based on abnormal proteins phenotype. No significant differences in protein levels between unpaired primary and metastatic samples were observed. Four proteins were found differentially expressed in KRAS-mutant as compared to wild-type tumours (overexpressed in mutant: KRAS, EGFR; overexpressed in wild-type: TOPO1, TOP2A). Survival analyses revealed the association between mesothelin expression and poor overall survival, whereas lack of PTEN protein expression associated with lower progression-free survival with anti-EGFR-based therapy in the first-line setting for patients with RAS wild-type tumour. Finally, outlier analysis identified putative targetable proteins in 65% of patients lacking a targetable genomic alteration. Our data show that TMP constitutes a promising, novel molecular prescreening tool in mCRC to identify protein expression alterations that may impact on patient outcomes and more precisely guide patient eligibility to clinical trials with novel targeted experimental therapies.

Pazopanib or methotrexate-vinblastine combination chemotherapy in adult patients with progressive desmoid tumours (DESMOPAZ): a non-comparative, randomised, open-label, multicentre, phase 2 study.

BACKGROUND: Desmoid tumours are locally aggressive tumours associated with substantial morbidity. No systemic treatments are approved for this disease, with methotrexate-vinblastine the only chemotherapy regimen assessed in a clinical trial setting to date. VEGF overexpression is a common feature in aggressive desmoid tumours. Pazopanib is an oral antiangiogenic agent targeting VEGF receptors 1, 2, and 3, platelet-derived growth factor receptor-like protein (PDGFR) α and ß, and c-KIT tyrosine kinases. We aimed to assess antitumour activity and safety of targeted therapy or combination chemotherapy in progressive desmoid tumours. METHODS: DESMOPAZ was a non-comparative, randomised, open-label, phase 2 trial conducted at 12 centres from the French Sarcoma Group. We enrolled adults (≥18 years) with progressive desmoid tumours, normal organ function and centrally documented progressive disease according to Response Evaluation Criteria in Solid Tumors version 1.1 based on two imaging assessments obtained within less than a 6-month interval. Participants were randomly assigned (2:1) to oral pazopanib 800 mg per day for up to 1 year or to an intravenous regimen combining vinblastine (5 mg/m2 per dose) and methotrexate (30 mg/m2 per dose), administered weekly for 6 months and then every other week for 6 months. Randomisation was stratified according to inclusion centre and tumour location. The primary endpoint was the proportion of patients who had not progressed at 6 months in the first 43 patients who had received one complete or two incomplete cycles of pazopanib. This endpoint was also assessed as a prespecified exploratory endpoint in all patients who had received one complete or two incomplete cycles of methotrexate-vinblastane. Safety analyses were done for all patients who received at least one dose of allocated treatment. This trial was registered with ClinicalTrials.gov, number NCT01876082. FINDINGS: From Dec 4, 2012, to Aug 18, 2017, 72 patients were enrolled and randomly assigned (n=48 in the pazopanib group; n=24 in the methotrexate-vinblastine group). Median follow-up was 23·4 months (IQR 17·1-25·5). 46 patients in the pazopanib group and 20 patients in the methotrexate-vinblastine group were assessable for activity. In the first 43 patients assessable for the primary endpoint in the pazopanib group, the proportion of patients who had not progressed at 6 months was 83·7% (95% CI 69·3-93·2). The proportion of patients treated with methotrexate-vinblastine who had not progressed at 6 months was 45·0% (95% CI 23·1-68·5). The most common grade 3 or 4 adverse events in the pazopanib group were hypertension (n=10, 21%) and diarrhoea (n=7, 15%) and in the methotrexate-vinblastine group were neutropenia (n=10, 45%) and liver transaminitis (n=4, 18%). 11 patients (23%) had at least one serious adverse event related to study treatment in the pazopanib group, as did and six patients (27%) in the methotrexate-vinblastine group. INTERPRETATION: Pazopanib has clinical activity in patients with progressive desmoid tumours and could be a valid treatment option in this rare and disabling disease. FUNDING: GlaxoSmithKline and Novartis.

Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.

Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics.

Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and to quantify proteins of interest in a targeted fashion using formalin-fixed, paraffin-embedded (FFPE) tumor biopsies ( n = 12) selected from our clinical laboratory. DIA analysis on the tumor biopsies provided 3713 quantifiable proteins including actionable biomarkers currently in clinical use, successfully separated two gastric cancers from colorectal cancer specimen solely on the basis of global proteomic profiles, and identified subtype-specific proteins with prognostic or diagnostic value. We demonstrate the potential use of DIA-based quantitation to inform therapeutic decision-making using TUBB3, for which clinical cutoff expression levels have been established by SRM. Comparative analysis of DIA-based proteomic profiles and mRNA expression levels found positively and negatively correlated protein-gene pairs, a finding consistent with previously reported results from fresh-frozen tumor tissues.

EGFR and MET Amplifications Determine Response to HER2 Inhibition in ERBB2-Amplified Esophagogastric Cancer.

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2. Heterogeneous 89Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2-amplified EG cancer. SIGNIFICANCE: Analysis of patients with ERBB2-amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR/ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones.See related commentary by Klempner and Catenacci, p. 166.This article is highlighted in the In This Issue feature, p. 151.

Refining the selection of patients with metastatic colorectal cancer for treatment with temozolomide using proteomic analysis of O6-methylguanine-DNA-methyltransferase.

BACKGROUND: The repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) is a validated predictor of benefit from temozolomide (TMZ) in glioblastoma. However, only 10% of patients with MGMT-methylated metastatic colorectal cancer (mCRC) respond to TMZ. METHODS: Archived tumour samples (N = 41) from three phase II TMZ trials carried out in MGMT-methylated mCRC (assessed by methylation-specific polymerase chain reaction [PCR]) were stratified by MGMT status as assessed by three different methods: mass spectrometry, PCR/methyl-BEAMing and RNA-seq. The performance of each method was assessed in relation to overall response rate, progression-free survival (PFS) and overall survival (OS). RESULTS: Overall, 9 of 41 patients responded to TMZ. Overall response rates were 50% (9/18), 50% (6/12) and 35% (8/23) among patients determined likely to respond to TMZ by mass spectrometry, methyl-BEAMing and RNA-seq, respectively. Low/negative MGMT protein expressors by mass spectrometry had longer PFS than high MGMT expressors (3.7 vs 1.8 months; HR = 0.50, P = 0.014). Results for OS were similar but statistically non-significant (8.7 vs. 7.4 months; HR = 0.55, P = 0.077). No significant association between survival and MGMT status by methyl-BEAMing or RNA-seq could be demonstrated as comparable subgroups survival could not be confirmed/excluded. Specifically, the association of high versus low methyl-BEAMing MGMT hypermethylation with survival was HR = 0.783, P = 0.46 for PFS and 0.591, P = 0.126 for OS, while association of low versus high RNA-seq MGMT level with survival was HR = 0.697, P = 0.159 for PFS and HR = 0.697, P = 0.266 for OS. CONCLUSIONS: Quantitative proteomic analysis of MGMT may be useful for refining the selection of patients eligible for salvage treatment with single-agent TMZ.

Exceptional responses to pertuzumab, trastuzumab, and docetaxel in human epidermal growth factor receptor-2 high expressing salivary duct carcinomas.

BACKGROUND: Alterations in the human epidermal growth factor receptor-2 (HER2) pathway have been identified in a subset of salivary duct carcinomas. Dual HER2 inhibition with trastuzumab and pertuzumab has superior antitumor efficacy to trastuzumab monotherapy in HER2-positive breast cancer, yet its efficacy in HER2-positive salivary duct carcinoma is unknown. METHODS: We report 2 cases of exceptional responses of HER2-positive salivary duct carcinomas to dual HER2 blockade and docetaxel combination and their molecular characteristics. RESULTS: A 54-year-old man with recurrent metastatic HER2 expressing salivary duct carcinoma of the parotid gland after definitive concurrent chemoradiation achieved a complete response (CR) after 6 cycles of trastuzumab, pertuzumab, and docetaxel (TPH). A 42-year-old woman with HER2-positive salivary duct carcinoma of the parotid gland with bone and liver metastases had CR with TPH and remains in remission on maintenance trastuzumab and pertuzumab. CONCLUSION: Dual HER2 blockage resulted in CR in patients with HER expressing salivary duct carcinoma and warrants further evaluation in this patient population.

Author Correction: Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition.

In the Supplementary Information originally published with this article, a lane was missing in the ß-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.

Ado-Trastuzumab Emtansine for Patients With HER2-Mutant Lung Cancers: Results From a Phase II Basket Trial.

Purpose Human epidermal growth factor receptor 2 ( HER2, ERBB2)-activating mutations occur in 2% of lung cancers. We assessed the activity of ado-trastuzumab emtansine, a HER2-targeted antibody-drug conjugate, in a cohort of patients with HER2-mutant lung cancers as part of a phase II basket trial. Patients and Methods Patients received ado-trastuzumab emtansine at 3.6 mg/kg intravenously every 3 weeks until progression. The primary end point was overall response rate using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. A Simon two-stage optimal design was used. Other end points included progression-free survival and toxicity. HER2 testing was performed on tumor tissue by next generation sequencing, fluorescence in situ hybridization, immunohistochemistry, and protein mass spectrometry. Results We treated 18 patients with advanced HER2-mutant lung adenocarcinomas. The median number of prior systemic therapies was two (range, zero to four prior therapies). The partial response rate was 44% (95% CI, 22% to 69%), meeting the primary end point. Responses were seen in patients with HER2 exon 20 insertions and point mutations in the kinase, transmembrane, and extracellular domains. Concurrent HER2 amplification was observed in two patients. HER2 immunohistochemistry ranged from 0 to 2+ and did not predict response, and responders had low HER2 protein expression measured by mass spectrometry. The median progression-free survival was 5 months (95% CI, 3 to 9 months). Toxicities included grade 1 or 2 infusion reactions, thrombocytopenia, and elevated hepatic transaminases. No patient stopped therapy as a result of toxicity or died on study. Conclusion Ado-trastuzumab emtansine is an active agent in patients with HER2-mutant lung cancers. This is the first positive trial in this molecular subset of lung cancers. Further use and study of this agent are warranted.

Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition.

The role of KRAS, when activated through canonical mutations, has been well established in cancer1. Here we explore a secondary means of KRAS activation in cancer: focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas2-4. KRAS-amplified gastric cancer models show marked overexpression of the KRAS protein and are insensitive to MAPK blockade owing to their capacity to adaptively respond by rapidly increasing KRAS-GTP levels. Here we demonstrate that inhibition of the guanine-exchange factors SOS1 and SOS2 or the protein tyrosine phosphatase SHP2 can attenuate this adaptive process and that targeting these factors, both genetically and pharmacologically, can enhance the sensitivity of KRAS-amplified models to MEK inhibition in both in vitro and in vivo settings. These data demonstrate the relevance of copy-number amplification as a mechanism of KRAS activation, and uncover the therapeutic potential for targeting of these tumors through combined SHP2 and MEK inhibition.

Personalized therapy based on sequential molecular analysis leads to 30 months of survival in a patient with diffuse unresectable gastric linitis plastica.

INTRODUCTION: Diffuse gastric cancer is associated with poor prognosis. We report a patient with metastatic gastric linitis plastica harboring human epidermal growth factor receptor 2 (HER2) activating mutation and HER2 amplification. CASE DESCRIPTION: The patient received 5-fluorouracil/folinic acid and oxaliplatin combined with trastuzumab/pertuzumab, resulting in disease control for 8 months. Second-line therapy with nivolumab and trastuzumab/pertuzumab was well-tolerated, with macroscopic peritoneal response. Following ovarian progression and surgical resection of ovarian metastases, immunohistochemistry of PD-L1 was negative; proteomics demonstrated normal expression of HER2 and absence of PD-L1, while genomics showed HER2 amplification, suggesting mechanisms of escape to dual HER2 blockade by downregulation of HER2 and to nivolumab by the absence of PD-L1. Based upon this and nonexpression of biomarkers of taxane resistance, therapy was changed to paclitaxel. Two and a half years after diagnosis, the patient is undergoing treatment, with excellent performance status. CONCLUSIONS: Molecular analysis and personalized therapy can help optimize treatment in difficult-to-treat cancers.