A comparative study of gastric adenocarcinoma HER2 IHC phenotype and mass spectrometry-based quantification.

Introduction: Gastric cancer is a highly heterogeneous malignant tumor of the digestive system. Anti-HER2 treatment can inhibit downstream signaling pathways and improve clinical treatment and outcomes in patients with HER2 protein overexpression. Currently, two standard methods for evaluating HER2 expression status are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, these low-throughput assays often produce discordant or equivocal results. Methods: In this study, we presented a new HER2 protein detection method based on mass spectrometry selected reaction monitoring (MS-SRM) and validated the method. We conducted a retrospective study on 118 formalin-fixed paraffin-embedded (FFPE) tissues from patients with advanced gastric adenocarcinoma in northern China, and we compared the MS-SRM results with those from IHC and correlated them with FISH. Results: We established and validated the upper and lower detection limits (300-700 amol/µg) for abnormal HER2 protein expression in advanced gastric cancer. We also found that, among samples with mixed Lauren subtypes, those with a high level of HER2 expression had typical intestinal type features in pathology. Discussion: This study demonstrated that the MS-SRM method can overcome the limitations and deficiencies of IHC, directly quantify the expression of HER2 protein in tumor cells and be used as a supplement to IHC. It has the potential to be used as a companion diagnosis for new drugs used to treat advanced gastric cancer. Large-scale clinical validation is required.

Early-pregnancy prediction of risk for pre-eclampsia using maternal blood leptin/ceramide ratio: discovery and confirmation.

OBJECTIVE: This study aimed to develop a blood test for the prediction of pre-eclampsia (PE) early in gestation. We hypothesised that the longitudinal measurements of circulating adipokines and sphingolipids in maternal serum over the course of pregnancy could identify novel prognostic biomarkers that are predictive of impending event of PE early in gestation. STUDY DESIGN: Retrospective discovery and longitudinal confirmation. SETTING: Maternity units from two US hospitals. PARTICIPANTS: Six previously published studies of placental tissue (78 PE and 95 non-PE) were compiled for genomic discovery, maternal sera from 15 women (7 non-PE and 8 PE) enrolled at ProMedDx were used for sphingolipidomic discovery, and maternal sera from 40 women (20 non-PE and 20 PE) enrolled at Stanford University were used for longitudinal observation. OUTCOME MEASURES: Biomarker candidates from discovery were longitudinally confirmed and compared in parallel to the ratio of placental growth factor (PlGF) and soluble fms-like tyrosine kinase (sFlt-1) using the same cohort. The datasets were generated by enzyme-linked immunosorbent and liquid chromatography-tandem mass spectrometric assays. RESULTS: Our discovery integrating genomic and sphingolipidomic analysis identified leptin (Lep) and ceramide (Cer) (d18:1/25:0) as novel biomarkers for early gestational assessment of PE. Our longitudinal observation revealed a marked elevation of Lep/Cer (d18:1/25:0) ratio in maternal serum at a median of 23 weeks' gestation among women with impending PE as compared with women with uncomplicated pregnancy. The Lep/Cer (d18:1/25:0) ratio significantly outperformed the established sFlt-1/PlGF ratio in predicting impending event of PE with superior sensitivity (85% vs 20%) and area under curve (0.92 vs 0.52) from 5 to 25 weeks of gestation. CONCLUSIONS: Our study demonstrated the longitudinal measurement of maternal Lep/Cer (d18:1/25:0) ratio allows the non-invasive assessment of PE to identify pregnancy at high risk in early gestation, outperforming the established sFlt-1/PlGF ratio test.

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.

EGFR Blockade Reverts Resistance to KRASG12C Inhibition in Colorectal Cancer.

Most patients with KRAS G12C-mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRAS G12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRAS G12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRAS G12C colorectal cancer. SIGNIFICANCE: The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079.

HER2-Mediated Internalization of Cytotoxic Agents in ERBB2 Amplified or Mutant Lung Cancers.

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.

Risk factors for postdischarge growth retardation among very-low-birth-weight infants: A nationwide registry study in Taiwan.

BACKGROUND: Very-low-birth-weight (VLBW) infants have a high risk of postdischarge growth retardation (GR). Continued GR might exert negative long-term effects on their health. This study examined the prevalence and the risk factors for postdischarge GR among VLBW infants in Taiwan. METHODS: Nationwide data from the Taiwan Premature Infant Follow-up Network between 2007 and 2011 were analyzed. Infants with a gestational age (GA) < 37 weeks and birth body weight (BBW) < 1500 g were enrolled. They were followed up after discharge at the corrected ages of 6, 12, and 24 months. Associations between postdischarge GR at the follow-ups and possible risk factors were analyzed. RESULTS: The prevalence of postdischarge GR among 2124 VLBW infants was 17.3%, 19.4%, and 13.8% at the corrected age (CA) of 6, 12, and 24 months, respectively. The significant perinatal factors of postdischarge GR were being small for gestational age (SGA) and extremely low birth weight (ELBW). ELBW infant with extra-uterine growth retardation (EUGR) at discharge or longer length of hospital stay (LOS) had poorer growth outcomes. Among non-ELBW infants, EUGR at discharge and surgical necrotizing enterocolitis (NEC) were the main influencing factors of unfavorable growth outcomes. RDS with surfactant therapy had a positive effect of postdischarge growth outcomes in ELBW infants. CONCLUSION: Postdischarge GR is still a serious problem in Taiwan. Being SGA and ELBW and EUGR were significant risk factors for postdischarge GR throughout the first two years of life in VLBW infants. An integrated and organized team for postdischarge care as well as scheduled follow-ups, detailed nutritional education, and thorough inspection are necessary.

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.

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.

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.

A micro-computed tomographic evaluation of dentinal microcrack alterations during root canal preparation using single-file Ni-Ti systems.

The aim of the present study was to evaluate the length of dentinal microcracks observed prior to and following root canal preparation with different single-file nickel-titanium (Ni-Ti) systems using micro-computed tomography (micro-CT) analysis. A total of 80 mesial roots of mandibular first molars presenting with type II Vertucci canal configurations were scanned at an isotropic resolution of 7.4 µm. The samples were randomly assigned into four groups (n=20 per group) according to the system used for root canal preparation, including the WaveOne (WO), OneShape (OS), Reciproc (RE) and control groups. A second micro-CT scan was conducted after the root canals were prepared with size 25 instruments. Pre- and postoperative cross-section images of the roots (n=237,760) were then screened to identify the lengths of the microcracks. The results indicated that the microcrack lengths were notably increased following root canal preparation (P<0.05). The alterations in microcrack length in the OS group were more significant compared with those in the WO, RE and control groups (P<0.05). In conclusion, the formation and development of dentinal microcracks may be associated with the movement caused by preparation rather than the taper of the files. Among the single-file Ni-Ti systems, WO and RE were not observed to cause notable microcracks, while the OS system resulted in evident microcracks.

MET tyrosine kinase receptor expression and amplification as prognostic biomarkers of survival in gastroesophageal adenocarcinoma.

BACKGROUND: MET gene amplification and Met protein overexpression may be associated with a poor prognosis. The MET/Met status is typically determined with fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Targeted proteomics uses mass spectrometry-based selected reaction monitoring (SRM) to accurately quantitate Met expression. FISH, IHC, and SRM analyses were compared to characterize the prognostic value of MET/Met in gastroesophageal adenocarcinoma (GEC). METHODS: Samples from 447 GEC patients were analyzed for MET gene amplification (FISH) and Met protein expression (IHC and SRM). Cox proportional hazards models and Kaplan-Meier estimates were applied to explore relations between Met, overall survival (OS), and clinical/pathological characteristics. Spearman's rank coefficient was used to assess the correlation between parameters. RESULTS: Patients with MET-amplified tumors had worse OS when: the MET/centromere enumeration probe for chromosome 7 FISH ratio was ≥ 2 (hazard ratio [HR], 3.13; 95% confidence interval [CI], 1.84-5.33), the MET gene copy number was ≥5 (HR, 2.51; 95% CI, 1.45-4.34), or ≥ 10% of the cells had ≥15 copies (HR, 4.28; 95% CI, 2.18-8.39). Similar observations were made with Met protein overexpression by IHC (≥1 + intensity in ≥ 25% of the tumor cell membrane: HR, 1.39; 95% CI, 1.04-1.86) or SRM (≥400 amol/µg: HR, 1.76; 95% CI, 1.06-2.90). A significant correlation was observed between MET FISH/Met IHC, MET FISH/Met SRM, and Met IHC/Met SRM; only MET FISH and Met SRM were independent negative prognostic biomarkers in multivariate analyses. CONCLUSIONS: MET amplification and overexpression, assessed by multiple methods, were associated with a worse prognosis in univariate analyses. However, only MET amplification by FISH and Met expression by SRM were independent prognostic biomarkers. Compared with IHC, SRM may provide an added benefit for informed decisions about Met-targeted therapy. Cancer 2017;123:1061-70. © 2016 American Cancer Society.

Therapeutically Induced Changes in HER2, HER3, and EGFR Protein Expression for Treatment Guidance.

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.

Mass-spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: comparison to IHC and FISH.

BACKGROUND: Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results. METHODS: We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/µg) of Her2-SRM protein in cell lines (n = 27) and GEC tissues (n = 139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cutoffs in order to identify HER2 gene amplification. RESULTS: After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with the HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM expression of the covariates Met, Egfr, Her3, and HER2 heterogeneity, as well as their interactions (cell lines r (2) = 0.9842; FFPE r (2) = 0.7643). In GEC tissues, Her2-SRM protein was detected at any level in 71.2 % of cases. ROC curves demonstrated that Her2-SRM protein levels have a high specificity (100 %) at an upper-level cutoff of >750 amol/µg and sensitivity of 75 % at a lower-level cutoff of <450 amol/µg for identifying HER2 FISH-amplified tumors. An "equivocal zone" of 450-750 amol/µg of Her2-SRM protein was analogous to IHC2+ but represented fewer cases (9-16 % of cases versus 36-41 %). CONCLUSIONS: Compared to IHC, targeted SRM-Her2 proteomics provided more objective and quantitative Her2 expression with excellent HER2/CEP17 FISH correlation and fewer equivocal cases. Along with its multiplex capability for other relevant oncoproteins, these results demonstrate a refined HER2 protein expression assay for clinical application.

Quantification of Anaplastic Lymphoma Kinase Protein Expression in Non-Small Cell Lung Cancer Tissues from Patients Treated with Crizotinib.

BACKGROUND: Crizotinib has antitumor activity in ALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non-small cell lung cancer (NSCLC). The current diagnostic test for ALK rearrangement is breakapart fluorescence in situ hybridization (FISH), but FISH has low throughput and is not always reflective of protein concentrations. The emergence of multiple clinically relevant biomarkers in NSCLC necessitates efficient testing of scarce tissue samples. We developed an anaplastic lymphoma kinase (ALK) protein assay that uses multiplexed selected reaction monitoring (SRM) to quantify absolute amounts of ALK in formalin-fixed paraffin-embedded (FFPE) tumor tissue. METHODS: After validation in formalin-fixed cell lines, the SRM assay was used to quantify concentrations of ALK in 18 FFPE NSCLC samples that had been tested for ALK by FISH and immunohistochemistry. Results were correlated with patient response to crizotinib. RESULTS: We detected ALK in 11 of 14 NSCLC samples with known ALK rearrangements by FISH. Absolute ALK concentrations correlated with clinical response in 5 of 8 patients treated with crizotinib. The SRM assay did not detect ALK in 3 FISH-positive patients who had not responded to crizotinib. In 1 of these cases, DNA sequencing revealed a point mutation that predicts a nonfunctional ALK fusion protein. The SRM assay did not detect ALK in any tumor tissue with a negative ALK status by FISH or immunohistochemistry. CONCLUSIONS: ALK concentrations measured by SRM correlate with crizotinib response in NSCLC patients. The ALK SRM proteomic assay, which may be multiplexed with other clinically relevant proteins, allows for rapid identification of patients potentially eligible for targeted therapies.

Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

BACKGROUND: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. METHODS: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). RESULTS: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification. CONCLUSIONS: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.

Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue.

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.