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Supplementary Fig 6

Additional results for dual probe EGFR and ERBB2 FISH from Patients 9, 30 and 32.

Detailed patient information who were treated with anti-EGFR therapy

Supplementary Fig 3

Four Supplementary Tables

Supplementary Fig 2

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

Supplementary Fig 5

Supplementary Table 1

Supplementary Fig 4

Three Supplementary Figures

Supplementary Figure Legends

Supplementary Figures S1-S6, Table S1-S3, and eMethods

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.

Drug resistance is a long-standing impediment to effective systemic cancer therapy and acquired drug resistance is a growing problem for molecularly-targeted therapeutics that otherwise have shown unprecedented successes in disease control. The hepatocyte growth factor (HGF)/Met receptor pathway signaling is frequently involved in cancer and has been a subject of targeted drug development for nearly 30 years. To anticipate and study specific resistance mechanisms associated with targeting this pathway, we engineered resistance to the HGF-neutralizing antibody rilotumumab in glioblastoma cells harboring autocrine HGF/Met signaling, a frequent abnormality of this brain cancer in humans. We found that rilotumumab resistance was acquired through an unusual mechanism comprising dramatic HGF overproduction and misfolding, endoplasmic reticulum (ER) stress-response signaling and redirected vesicular trafficking that effectively sequestered rilotumumab and misfolded HGF from native HGF and activated Met. Amplification of MET and HGF genes, with evidence of rapidly acquired intron-less, reverse-transcribed copies in DNA, was also observed. These changes enabled persistent Met pathway activation and improved cell survival under stress conditions. Point mutations in the HGF pathway or other complementary or downstream growth regulatory cascades that are frequently associated with targeted drug resistance in other prevalent cancer types were not observed. Although resistant cells were significantly more malignant, they retained sensitivity to Met kinase inhibition and acquired sensitivity to inhibition of ER stress signaling and cholesterol biosynthesis. Defining this mechanism reveals details of a rapidly acquired yet highly-orchestrated multisystem route of resistance to a selective molecularly-targeted agent and suggests strategies for early detection and effective intervention.

The vascular endothelial growth factor (VEGF)/VEGFR and hepatocyte growth factor (HGF)/c-MET signaling pathways act synergistically to promote angiogenesis. Studies indicate VEGF inhibition leads to increased levels of phosphorylated c-MET, bypassing VEGF-mediated angiogenesis and leading to chemoresistance. We conducted a phase 1 clinical trial with 32 patients with refractory solid tumors to evaluate the safety, pharmacokinetics, and pharmacodynamics of combinations of VEGF-targeting pazopanib and the putative c-MET inhibitor ARQ197 (tivantinib) at 5 dose levels (DLs). Patients either took pazopanib and tivantinib from treatment initiation (escalation phase) or pazopanib alone for 7 days, with paired tumor sampling, prior to starting combination treatment (expansion phase). Hypertension was the most common adverse event. No more than 1 dose limiting toxicity (DLT) occurred at any DL, so the maximum tolerated dose (MTD) was not determined; DL5 (800 mg pazopanib daily and 360 mg tivantinib BID) was used during the expansion phase. Twenty of 31 evaluable patients achieved stable disease lasting up to 22 cycles. Circulating VEGF, VEGFR2, HGF, and c-MET levels were assessed, and only VEGF levels increased. Tumor c-MET levels (total and phosphorylated) were determined in paired biopsies before and after 7 days of pazopanib treatment. Total intact c-MET decreased in 6 of 7 biopsy pairs, in contrast to previously reported c-MET elevation in response to VEGF inhibition. These results are discussed in the context of our previously reported analysis of epithelial-mesenchymal transition in these tumors. Supplementary information The online version contains supplementary material available at 10.1007/s10637-021-01138-x.

Background: Trastuzumab deruxtecan (T-DXd) has been approved for advanced or metastatic breast, gastric and lung cancers. It has been postulated that irreversible tyrosine kinase inhibitors (TKIs) increase HER2 internalization, thus enhancing T-DXd efficacy. Here we report studies evaluating the combination of TKIs with T-DXd in preclinical models of HER2-low disease. Methods: Surface HER2 was measured with flow cytometry quantifying fluorescently labelled trastuzumab on the surface of HER2-low cell lines HCC38 and HCC1171 treated with afatinib, neratinib, lapatinib or tucatinib for 12 h. HER2 receptor dynamics on cell surface was also measured in NCI-N87 (HER2 IHC 3+) and HCC38 at 24, 48, 72 and 96 h post-treatment with afatinib, and recovery was measured up to 72 h after drug washout. In vivo efficacy was established with T-DXd alone and in combination with afatinib or tucatinib in two gastric patient-derived xenograft (PDX) models, HD-2 (HER2 IHC 1+) and FU (HER2 IHC 2+), and one lung PDX model, MEDI-NSCLC-04 (HER2 IHC 1+). Results: In both HER2-low cell lines, treatment with irreversible inhibitors neratinib or afatinib led to enhanced internalization with a greater than 50% surface HER2 reduction, while treatment with the reversible inhibitors lapatinib or tucatinib led to 13-35% higher surface HER2 levels. Washout experiments indicated that receptor recovery at the plasma membrane begins between 4-12h post-washout of afatinib, with complete recovery at about 72h. Tumor shrinkage was significant in HER2-low gastric PDXs when T-DXd was combined with afatinib, but not with tucatinib. In HD-2, T-DXd 10mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 74% and -3% (with respect to T-DXd) tumor growth inhibition (TGI), respectively. In FU, T-DXd 3mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 80.3% and 24.7% TGI, respectively. Similarly, in MEDI-NSCLC-04 PDX model, benefit was observed in 5mg/kg T-DXd combination with 20mg/kg QD afatinib (95.3% TGI with respect to monotherapy T-DXd), but not with tucatinib (0.37% TGI). Intermittent schedules (QW, MWF) were also tested for afatinib in combination with T-DXd, with the MWF showing 49.2% TGI and QW showing 33.0 %TGI. In tissue samples from MEDI-NSCLC-04 efficacy study, γH2AX was detected by IHC in T-DXd and T-DXd+afatinib combination. In western blots, a decrease in HER2 as well as pHER2 was observed in response to T-DXd and 20mg/kg afatinib treatment, but not with tucatinib. Conclusions: In HER2 low gastric and lung preclinical models, T-DXd combined with irreversible pan-HER inhibitor induced superior antitumor effects compared to single agents or the combination of T-DXd with reversible inhibitors. These data support the rationale to test combination of T-DXd and pan-HER irreversible inhibitors in the clinical setting. Citation Format: Deepa Bhavsar, Laura Kazlauskas, Flavia Michelini, Matt Griffin, Matt Wilson, Fabiola Cecchi, Liz Croydon, Kim Maratea, Elisa de Stanchina, Yelena Y. Janjigian, Maurizio Scaltriti, Theresa Proia, Jerome Mettetal. Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3999.

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.

Background:No predictive markers for chemotherapy activity have been validated in gastric cancer (GC). The potential value of class III β-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 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.

Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM. Limits of quantitation were determined by spiking synthetic peptides into a human spleen matrix. In addition, 20 clinical samples were analyzed using FAIMS-PRM and the quantitation of HER2 was compared with that obtained with the Ventana immunohistochemistry assay. FAIMS-PRM improved the overall signal-to-noise ratio over that from PRM and increased assay sensitivity in FFPE tissue analysis for four (HER2, EGFR, cMET, and KRAS) of five proteins of clinical interest. FAIMS-PRM enabled sensitive quantitation of basal HER2 expression in breast cancer samples classified as HER2 negative by immunohistochemistry. Furthermore, we determined the degree of FAIMS-dependent background reduction and showed that this correlated with an improved lower limit of quantitation with FAIMS. FAIMS-PRM is anticipated to benefit clinical trials in which multiple biomarker questions must be addressed and the availability of tumor biopsy samples is limited.

[This corrects the article DOI: 10.1371/journal.pone.0054014.].

194 Background: Although regularly used in breast and gastric cancer, HER2 testing is not routinely performed in colorectal cancer (CRC). Immunohistochemistry (IHC) and in situ hybridization (ISH) scoring have not been optimized for assessment of HER2 overexpression and amplification in CRC. Varying rates of HER2 overexpression (2%-11%) have been reported in previously untreated CRC (Wang World J Gastrointest Oncol 2019) and patients with advanced/metastatic CRC with HER2 amplification or overexpression respond poorly to current standard of care therapies (Sartore-Bianchi Oncologist 2019). We characterized HER2 prevalence in a retrospective cohort analysis of clinical trial patients. Methods: The HORIZON III trial (NCT00384176) evaluated FOLFOX + bevacizumab or cediranib (AZD2171) as first-line (1L) treatment in patients with metastatic CRC (Schmoll J Clin Oncol 2012). The primary objective of this analysis of a subset of HORIZON III samples was to characterize HER2 prevalence in 1L metastatic CRC. Secondary and exploratory objectives included correlating HER2 status to other molecular findings (eg, RAS/RAF mutations), clinicopathologic characteristics, and patient outcomes. IHC was performed using a monoclonal anti-HER2 antibody PATHWAY HER2 [4B5] Ventana on primary CRC tumor sections and scored according to ASCO/CAP guidelines for gastric cancer (Bartley Arch Pathol Lab Med 2016). H&E staining was performed to determine the adequacy of tumor samples (ie, > 100 viable tumor cells per specimen). All tumors with an IHC score of 2+ were analyzed for amplification by ISH (HER2 IQFISH pharmDx dual probe kit, Agilent Technologies K573111-5). Targeted mutation panel testing was used to determine other molecular alterations. Descriptive statistics were used to summarize baseline and clinical outcome data by HER2 status. The current analysis was approved by the AstraZeneca bioethics review board. Results: Of the 1614 patients in HORIZON III, 396 met the inclusion criteria of appropriate consent, sufficient tumor sample for analysis, and a unique identifier. HER2-positive tumors (IHC3+ or IHC2+/ISH+) were identified in 2.1% of samples; 1.3% were IHC3+ and 0.8% were IHC2+/ISH+. Compared with IHC3+, IHC2+ tumors were more heterogeneous with mixed components of cells weakly expressing HER2 and lacking HER2 expression. Conclusions: This exploratory analysis of the HORIZON III study provides insights into HER2 prevalence in 1L, unselected, advanced/metastatic CRC, finding that 2.1% of tumors were HER2 positive. The demographic characteristics of patients with analyzable samples were representative of the entire HORIZON III study population; however, further assessment of KRAS status and other clinicopathological characteristics is needed. These data may inform future clinical development and support selection of patients with CRC who are likely to benefit from HER2 targeting therapies. Clinical trial information: NCT00384176 .

Biomarcadores del cáncer; Cáncer metástico colorrectal; Terapias experimentales Cancer biomarkers; Colorectal metastatic cancer; Experimental therapies Biomarcadors del càncer; Càncer metastàtic colorrectal; Teràpies experimentals 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.

Background: Metastatic breast cancer is the second leading cause of death among women globally. A better understanding of tumor biology, and the availability of high-throughput technologies, have enabled the emergence of precision medicine bringing new expectations and giving rise to molecular screening programs (MSP). SOLTI, as a collaborative Spanish network, designed AGATA, the first multi-institutional MSP ever implemented in this country. Here, we report both the primary and some of the secondary results of the pilot study. Methods: A total of 10 sites within SOLTI network in Spain participated. DNA-sequencing of 56 cancer related genes was performed using FFPE tumor samples (primary or metastatic). Each clinical case was reviewed by a multidisciplinary advisory board (MAB), which recommended, in a prospective manner, potential experimental treatments, mainly in the context of clinical trials. The primary objective was to determine the success rate of matching a DNA alteration to an experimental drug or drug class. Secondary objectives included a comprehensive molecular characterization of tumor samples by PAM50 subtyping and quantification of protein expression levels by MASS-SPEC (70 proteins panel). Results: 305 patients (pts) were screened from September 2014 to July 2017 and 260 (85.3%) were finally evaluated by the MAB. Pts characteristics were: mean age 54 years (29-80), ER+/HER2- (n=192; 74%), HER2+ (n=30; 11.5%) and TNBC (n=38; 14.5%). 163 primary tumors and 97 metastatic samples were profiled. Regarding the primary objective, 116 pts (45%) presented at least one mutation (range 1-6) that could be matched to a drug or drug class. Of these, 13 pts (11.2%) received therapy matched to their molecular profile according to the MAB recommendation and their follow-up is still on-going. No mutation was detected in 97 (37%) pts (WT), and 47 patients (18.1%) presented a mutation but no match was possible. The most common mutations were PIK3CA (34%), TP53 (22%), AKT1 (5%), ESR1 (3%) and ERBB2 (3%). Intrinsic subtype distribution in 177 samples was as follows: 34% Luminal A (n=60); 21% Luminal B (n=36); 13% HER2E (n=22); 19% Basal-like (n=34) and 13% Normal-like (n=23). Compared to primary tumors (n=110), the proportion of HER2-enriched disease in metastatic tumors (n=63) was significantly higher (6% vs 20%; p=0.005). Protein expression analysis was performed in 146 samples (94 primary and 57 metastasis). In 19 cases (13%), the outlier expression of some targetable proteins (FGFR1 [n=4, 2.7%], IGF1R [n=4, 2.7%], EGFR [n=1, 0.7%], CEACAM5 [n=6, 4.1%], IDO1 [n=2, 1.37%], TROP2 [n=2, 1.37%]) were identified. Of note, HER2 overexpression (>740 amol/μg) was observed in 4 HER2- cases. Finally, among WT tumors, 21% presented a potential drug-matched protein target. Conclusions: Nationwide molecular screening in Spain is feasible. Nearly half of patients had tumors with mutation(s), mostly PIK3CA, that could be matched to a potential drug or drug class. PAM50 profile might be helpful to navigate towards a therapeutic decision making, although the MAB could not make any targeted-driven recommendation yet with this data. More clinical evidence is needed to use MASS-SPEC as a diagnostic tool. Citation Format: Pernas S, Villagrasa P, Nuciforo PG, Vivancos A, Scaltriti M, Rodón J, Burgués O, Canes J, Dueñas M, Cecchi F, Vidal M, Lluch A, Perelló A, Llombart A, Dorca J, Montaño A, Oliveira M, Ribas G, Rapado I, Paré L, Prat A, Ciruelos E. Primary and secondary results of the first nationwide molecular screening program in Spain for patients with advanced breast cancer (AGATA SOLTI-1301 study) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-02.

This abstract was not presented at the conference. Citation Format: Gasparini E, Bisagni A, Di Cicilia R, Kuhn E, Falco G, Ferrari G, Foroni M, Tamagnini I, Bassano C, Ragazzi M, Gardini G, Cecchi F, Hembrough T, Bisagni G. Not presented [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-24.

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.

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.

Across all stages of drug discovery and development, experimental assays are performed to understand the effect of a drug or drug candidate—at the molecular, cellular, organ, or organism level. Imaging is a key technology in this process, and an imaging assay consisting of sample preparation, image acquisition, and image analysis provides a quantitative readout of a system. Historically, chemical assays have been the workhorse of early discovery, screening millions of compounds for a simple endpoint, whereas imaging was primarily used in lower throughput mechanistic studies. However, with development of high-throughput high-content microscopy platforms, the throughput of imaging assays now rivals chemical screens. Similarly, innovations in image analysis mean that robust quantitative conclusions can be derived from complex and multimodal image data, driving informed decision making later in the drug development process. For these reasons, imaging is widely used throughout the pharmaceutical industry and throughout multiple stages of the drug discovery process.

International audience; 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 exomesequencing, 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.

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

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.

Background: Selecting chemotherapy based on tumor biology can improve response rates and avert toxicity. Studies of the relationship between tumor expression of TOP2A protein and response to anthracycline-based chemotherapy have yielded contradictory results. Here we used mass spectrometry (MS) to evaluate associations between tumor molecular profiles and pathological complete response (pCR) in breast cancer patients treated with neoadjuvant anthracycline-containing therapy. Methods: Patients were selected from the ERNEST-B (Erlangen Neoadjuvant Study Breast), which is a retrospective cohort study. Archived tumor samples from anthracycline-treated breast cancer patients (n=133) were microdissected and solubilized. In each tumor sample, TOP2A and other target proteins were quantitated with a mass spectrometry assay. Molecular profiling also included RNA sequencing of the tumor and whole genome sequencing of both tumor and matched normal tissue sections. The cohort was dichotomized into high and low expressors of TOP2A using a protein level cutoff of 515 amol/ug of tumor protein. The difference in pCR (ypT0ypN0) rates between high and low expressors of TOP2A was assessed using a z-test for differences in proportion. Results: TOP2A protein was detected in 84 of 133 (63%) tumor samples from anthracycline-treated patients (range: 178 to 3044 amol/ug). Patients whose tumor expressed TOP2A protein above the cutoff of 515 amol/ug had higher pCR rates than patients with lower TOP2A expression (35.5% vs. 12.1%; odds ratio (OR): 4.48 [95% CI: 1.53-13.28], Fisher' exact test p = 0.004). The difference retained statistical significance in a logistic regression model adjusting for HR and HER2 status (OR: 2.23 [95% CI: 1.15-4.30], p=0.017). In a separate cohort of 19 breast cancer patients who did not receive anthracycline, there was no association between TOP2A protein expression level and pCR rate. Results from a validation cohort as well as the genomic analysis will be presented at the meeting. Conclusions: In a retrospective analysis of anthracycline-treated breast tumors, TOP2A expression was associated with a higher rate of pCR. Targeted proteomics may predict the response of breast cancer patients to anthracycline-based therapy. Citation Format: Schwartz S, Cecchi F, Tian Y, Yau C, Szeto C, Rübner M, Erber R, Beckmann MW, Hartmann A, Benz S, Hembrough T, Fasching P. Identifying patients sensitive to anthracycline-containing therapy with quantitative proteomic and genomic profiling [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-10-06.

Protein expression in formalin-fixed, paraffin-embedded (FFPE) patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry(MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal Growth Factor Receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue selected reaction monitoring (LT-SRM) mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R2 =0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

Background A wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies. Patients and methods GC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics. Results Quantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy. Conclusions Proteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods.

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

Summary 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 β, 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.

Background T-DXd (Enhertu®) is an FDA-approved antibody-drug conjugate (ADC) targeting HER2. T-DXd has shown anti-tumor activity, not only in patients with HER2-overexpressing (IHC3+/2+ ISH+) breast cancer (BC) but also in patients with BC with low HER2 expression (IHC1+/2+ ISH−). Current HER2 protein expression assessment is based on manual pathologist scoring that classifies tumors by the percentage of tumor cells with highest intensity and completeness of staining. A critical need exists for more objective and quantitative methods to assess HER2 expression, specifically to better identify patients with low-level expression if T-DXd proves to be efficacious in this patient population. Methods We used deep learning (DL)-based image analysis (IA) to generate a novel HER2 Quantitative Continuous Score (QCS). Data analytic techniques determined optimal HER2 QCS for the J101 trial (NCT02564900) of 151 patients with varying HER2 expression levels (1+, 2+, 3+). HER2 QCS consists of DL models to detect membrane, cytoplasm, and nuclei of all tumor cells. QCS was extensively trained using pathologists’ annotations, and the performance was validated on unseen data to ensure its generalization and robustness. QCS was blindly applied to J101 data. The optical density (OD; level of brown stain intensity) was computed on detected membrane to derive features that could be linked to survival prediction. QCS features were selected to maximize ORR in positive group, minimize ORR in negative group maintaining while high prevalence in the positive group. Results Analytical validation showed high correlation between QCS from automatically detected membranes and QCS from those annotated by pathologists (R=0.993). This is in the same range as correlation between three pathologists (R=0.995). HER2 QCS was largely consistent with pathologist HER2 scoring as well but showed broad quantitative overlap between IHC and ISH categories. HER2 QCS showed a direct linear relationship between ORR and increased HER2 expression across the entire assay range. In the HER2-low population (n = 65), for whom HER2-targeting therapies are not currently approved, 42% of patients responded to T-DXd, with a median PFS (mPFS) of 11 mo. Using HER2 QCS, we were able to further stratify this population into a subgroup of QCS-high patients (above a staining intensity cut-off determined by IA), with response and mPFS increased to 53% (95% CI: 36%-68%) and 14.5 mo (95% CI: 10.9 mo-NR) respectively, while the QCS-low group only showed ORR of 24% (95% CI: 9%-45%) and mPFS of 8.6 mo (95% CI: 4.2 mo-NR). Generally, best-performing QCS cutoffs were driven by most tumor cells expressing a minimal amount of HER2, in contrast to current clinical guidelines that are driven by a minority of cells expressing higher levels of HER2. We also examined spatial heterogeneity by characterizing cells as either bearing membrane stain above a determined OD threshold (positive cell) or lying within certain distances from a positive cell. We observed similar efficacy with best performing-cutoffs, again, being found when a minimal level of HER2 expression (OD) was examined. Conclusions Taken together, these data establish a first proof-of-concept demonstrating that use of HER2 QCS can potentially enhance prediction of patient outcome with T-DXd by increasing sensitivity and specificity of response, especially in the HER2-low population. The ability to identify patients in the HER2-low group who could benefit from T-DXd is critical for its use in a patient population with a high unmet need that would otherwise not be treated with anti-HER2 therapy. Further clinical verification and validation is ongoing. Citation Format: Mark Gustavson, Susanne Haneder, Andreas Spitzmueller, Ansh Kapil, Katrin Schneider, Fabiola Cecchi, Sriram Sridhar, Guenter Schmidt, Sotirios Lakis, Regina Teichert, Anatoliy Shumilov, Ana Hidalgo-Sastre, Magdalena Wienken, Hadassah Sade, J. Carl Barrett, Danielle Carroll. Novel approach to HER2 quantification: Digital pathology coupled with AI-based image and data analysis delivers objective and quantitative HER2 expression analysis for enrichment of responders to trastuzumab deruxtecan (T-DXd; DS-8201), specifically in HER2-low patients [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 PD6-01.

Protein-targeted therapies are expected to selectively kill tumor cells that express the targeted protein biomarker. Although a tumor mass may initially respond to targeted therapies based on expression of the targeted protein, all cells within a tumor may not express the targeted protein above a critical threshold level; therefore, those cells that do not express, or that downregulate expression of, the targeted protein may not be responsive to therapy. The ability to monitor the dynamic expression of these protein biomarkers throughout the course of therapy may allow for treatment to be personalized in real-time in response to the evolving nature of the tumor. This report demonstrates, by monitoring a single patient through multiple therapies, how targeted mass spectrometry is an effective, quantitative method that provides real-time analysis of multiple therapeutically associated targeted proteins that can be used to personalize a patient's treatment strategy throughout the course of care.