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Background: Three CDK 4/6 inhibitors (inh) are FDA approved in combination with endocrine therapy (ET) for HR+/HER2- metastatic breast cancer (MBC), however, there are no validated predictive markers of response to this class of drugs. Approximately 30-40% of patients (pts) have little to no response to these agents, with disease progression occurring in weeks to a few months after therapy initiation. We conducted an open-label, multicenter clinical trial (NCT03195192) to utilize cutting edge proteomic technologies to map the functional activation of the signaling architecture of pre-treatment tumor tissue from HR+/HER2- MBC pts receiving first line CDK4/6 inh plus ET, and to correlate these functional phosphoprotein-based signaling patterns with 1-year progression-free survival (1-yr PFS). Methods: We enrolled 29 of 100 planned pts, then the study closed early due to slow accrual. All pts were followed up to 12 months from starting a CDK4/6 inhibitor or until disease progression if it occurred earlier. The primary objective of this trial is to assess the correlation between baseline phosphorylated Rb levels that indicate activated Rb in tumor tissue and 1-yr PFS. We hypothesized that high levels of activated Rb will identify pts who are more likely to respond to CDK4/6 inh. Secondary objective is to evaluate the correlation between 1-yr PFS and 8 pre-specified qualifying (protein/phosphoprotein CDK 4/6 kinase pathway biomarkers: total Rb, Rb (S780), total Cyclin D1, Cyclin D1 (S286), total p16INK, total p27, p27 (T187), FoxM1 (T600). Results: Pre-treatment diagnostic FFPE biopsy material available from 24 evaluable pts were analyzed by a Laser Capture Microdissection (LCM) Reverse Phase Protein Microarray (RPPA) workflow. Seventeen of 24 pts (71%) were White, 7 (29%) African American; median age 65 (range 36-79); 22 pts (92%) received an aromatase inh and 2 (8%) had fulvestrant. The primary analysis expressed the relationship between phospho-RB and 1-yr PFS as a 2x2 table of frequencies summarized either above or below the median values observed in all pt values using a Pearson chi-squared test. Similarly, we analyzed the qualified protein/phosphoprotein markers quantified by RPPA by median dichotomization. Univariate analysis showed that 1-yr PFS correlated with below median levels of phospho and total RB (Chi-sq = 8.71, p-value = 0.003); phospho-FoxM1 (Chi-sq = 4.44, p-value = 0.035); Cyclin D1 S286 (Chi-sq = 4.44, p-value = 0.035); total and phospho-p27 and Ki67 (Chi-sq = 4.44, p-value = 0.035). None of these biomarkers were individually significant as continuous variables on multivariate analysis by logistic regression. Conclusions: Functional CDK 4/6 drug target pathway mapping analysis is possible from the pre-treatment diagnostic FFPE material. Our results indicate that pts whose tumors had low inherent proliferative and CDK 4/6 kinase activity were more likely to respond to first line CDK4/6 inh plus ET indicating possible prognostic determinants of the biomarkers. Citation Format: Maysa M Abu-Khalaf, Christos Hatzis, K. Alex Hodge, Frances Valdes, William Sikov, Monica Mita, Neelima Denduluri, Kris Awerkamp, Rita Murphy, Daniel Zelterman, Bryant Dunetz, Emanuel Petricoin, Mariaelena Pierobon. Multiomic advanced diagnostics for CDK 4/6 drug target activation mapping of HR+/HER2- metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-28.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), became a pandemic in early 2020. Lateral flow immunoassays for antibody testing have been viewed as a cheap and rapidly deployable method for determining previous infection with SARS-CoV-2; however, these assays have shown unacceptably low sensitivity. We report on nine lateral flow immunoassays currently available and compare their titer sensitivity in serum to a best-practice enzyme-linked immunosorbent assay (ELISA) and viral neutralization assay. For a small group of PCR-positive, we found two lateral flow immunoassay devices with titer sensitivity roughly equal to the ELISA; these devices were positive for all PCR-positive patients harboring SARS-CoV-2 neutralizing antibodies. One of these devices was deployed in Northern Italy to test its sensitivity and specificity in a real-world clinical setting. Using the device with fingerstick blood on a cohort of 27 hospitalized PCR-positive patients and seven hospitalized controls, ROC curve analysis gave AUC values of 0.7646 for IgG. For comparison, this assay was also tested with saliva from the same patient population and showed reduced discrimination between cases and controls with AUC values of 0.6841 for IgG. Furthermore, during viral neutralization testing, one patient was discovered to harbor autoantibodies to ACE2, with implications for how immune responses are profiled. We show here through a proof-of-concept study that these lateral flow devices can be as analytically sensitive as ELISAs and adopted into hospital protocols; however, additional improvements to these devices remain necessary before their clinical deployment.

In this work high throughput technology and computational analysis were used to study two stage IV lung adenocarcinoma patients treated with standard chemotherapy with markedly different survival (128 months vs 6 months, respectively) and whose tumor samples exhibit a dissimilar protein activation pattern of the signal transduction. Tumor samples of the two patients were subjected to Reverse Phase Protein Microarray (RPPA) analysis to explore the expression/activation levels of 51 signaling proteins. We selected the most divergent proteins based on the ratio of their RPPA values in the two patients with short (s-OS) and long (l-OS) overall survival (OS) and tested them against a EGFR-IGF1R mathematical model. The model with RPPA data showed that the activation levels of 19 proteins were different in the two patients. The four proteins that most distinguished the two patients were BADS155/136 and c-KITY703/719 having a higher activation level in the patient with short survival and p70S6S371/T389 and b-RAFS445 that had a lower activation level in the s-OS patient. The final model describes the interactions between the MAPK and PI3K-mTOR pathways, including 21 nodes. According to our model mTOR and ERK activation levels were predicted to be lower in the s-OS patient than the l-OS patient, while the AMPK activation level was higher in the s-OS patient. Moreover, KRAS activation was predicted to be higher in the l-OS KRAS-mutated patient. In accordance with their different biological properties, the Moment Independent Robustness Indicator in s-OS and l-OS predicted the interaction of MAPK and mTOR and the crosstalk AKT with p90RSK as candidates to be prognostic factors and drug targets.

Background: The biological mechanisms underlying early- and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin–paclitaxel treatment. Methods: Tumour epithelia were isolated from two independent sets of primary EOC (n=72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1–AMPK and AKT–mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin–paclitaxel-sensitive and -resistant tumours. Results: Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK–AKT–mTOR axis compared to early EOC (P

Immune-checkpoint blockade has emerged as a powerful therapeutic strategy in lung cancer. However, the molecular mechanisms associated with response are still poorly understood. Using lung cancer as a model system, we employed protein pathway activation mapping and multiplex immunofluorescence to explore pathway-centered signaling events and tumor-immune spatial interactions associated with response to Nivolumab. Signaling data were collected from laser dissected tumor epithelia and the surrounding tumor-stroma interface (TSI) of 28 lung cancer patients treated with Nivolumab using the Reverse Phase Protein Microarray. As expected, quantitative PD-L1 measurements associated with response to treatment. Pathway-centered analysis revealed increased TLR4-based signaling events, pro-inflammatory molecules, and tumor-associated antigens in patients that benefitted from treatment. CD68+/PD-L1+ antigen-presenting cells spatial distribution was also associated with response to treatment. Taken together, these results suggest that a tumor ecology that favors immune activation may play a primary role in modulating response to compounds targeting the tumor-immune axis.

Background Non-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis. Methods Sixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed. Results Phosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p

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

Background: Although the efficacy of different immunotherapeutic agents varies greatly across tumor types, little is known about the role of different metastatic tumor microenvironments in modulating the expression of immune-checkpoints like PD-L1. Using metastatic breast cancer as a model, this work explored whether metastatic lesions derived from different patients, but colonizing the same host target organ, showed organ-specific immune signatures. Methods: Snap frozen metastatic lesions collected from 30 breast cancer patients enrolled in a prospective phase II multi-omic precision medicine clinical trial (“Side-Out II”) were used for this analysis. Sites of metastasis were: liver (LI) (n=10), skin/chest wall (SC) (n=10), and other lesions (OL) (n=10) [including lymph nodes (LN) (n=4), lung (LU) (n=3), and intra-abdominal lesions (IA) (n=3)]. Pure tumor epithelia were isolated from each specimen via Laser Capture Microdissection (LCM). PD-L1 expression (Antibody Clone E1L3N) was quantitatively measured using the Reverse Phase Protein Microarray (RPPA), a high-throughput, multiplex immunoassay that provides operator-independent quantitative data starting from a small amount of biological material. Proportion of patients with PD-L1 expression above the median and the 75th percentile of the population was compared across metastatic sites. Results: PD-L1 expression had a >5 fold dynamic range across breast cancer-derived metastatic lesions with different distribution across metastatic sites. Specifically, PD-L1 expression of 6/10 LIs, 5/10 OLs (including 2 LU and 3 LN positive lesions), and 4/10 SC metastases was greater than the population median of all metastases combined. LI metastases were the most represented group above the 75th percentile of the population (4/10), followed by OS (2/10) (including 1 LU and 1 LN positive lesions), and SC (1/10). Of the 15 lesions above the population median, 6 were LI (40%), 5 were OS (33%), and 4 were SC (27%); of the 7 metastases above the 75th percentile, 4 were LI (57%), 2 were OS (29%), and 1 was SC (14%). Conclusions: The LCM-RPPA workflow can capture PD-L1 protein expression on a continuous quantitative scale and provide a broader understanding of the dynamic range of expression. Our data suggest that organ specific microenvironments in which metastatic lesions develop may strongly influence overall PD-L1 tumor cell expression. If different metastatic host organs can modulate PD-L1 expression, location of the metastatic lesion should be kept in consideration when selecting patients that are candidates for immunotherapy. Such hypotheses should be further validated in prospective clinical trial where quantitative PD-L1 expression of different metastatic lesions is evaluated along with response to targeted immuno-oncology compounds. Citation Format: Mariaelena Pierobon, K Alex Hodge, Elisa Baldelli, Steven Anthony, Nicholas J. Robert, Donald W. Northfelt, Mohammad J. Jahanzeb, Linda Vocila, Lance A. Liotta, Bryant Dunetz, Emanuel F. Petricoin. PD-L1 expression differs across cancer metastatic sites from breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3129.

35 Background: FDA approved IHC-based companion/complementary assays are routinely used to measure PD-L1 for treatment selection. However, IHC cut-point values vary across platforms and are based on subjective analysis that requires antigen retrieval methods. Even in highly selected populations of PD-L1 “positive” patients, clinical benefits are seen only in subgroups of patients. We tested the feasibility of utilizing LCM and RPPA as a new methodology for quantitative, operator independent measurements of PD-L1 on tumor cells. Methods: PD-L1 quantification by RPPA was compared to IHC on 23 lung cancers (LC). Tumor cells were isolated from the surrounding microenvironment using LCM. The E1L3N clone from Cell Signaling was used to quantify PD-L1 expression by RPPA and IHC. Reproducibility across antibody clones was then assessed on LCM procured tumor epithelia from 10 FFPE LC and 71 snap-frozen ovarian cancers (OC). PD-L1 measurements were compared between the Cell Signaling E1L3N and the Ventana SP-142 clone. A quantitative PD-L1 calibrated assay was developed and assessed with the pilot population data. Results: Of the 23 LC, 5 were PD-L1 positive by IHC. The 5 IHC-positive patients had the greatest level of PD-L1 expression by RPPA indicating that the RPPA platform correlates with IHC. However, RPPA quantification showed a dynamic range ~ 4-fold across IHC negative samples. Correlation between PD-L1 detection with the E1L3N and the SP-142 clone in the OC was significant (R2 = 0.85), indicating great concordance across clones. Results were confirmed in the 10 LC samples (R2 = 0.87). Conclusions: The LCM-RPPA workflow captures PD-L1 expression on a continuous quantitative scale. This quantitative output correlated with IHC although it captures a much broader dynamic range in both IHC negative and positive populations. PD-L1 quantification by LCM-RPPA may be less dependent upon the clone used for the detection than IHC. Because the detection is unconstrained by antigen retrieval issues as well as subjectivity of IHC interpretation, this approach may generate a more accurate cut-point of therapeutic response prediction.

Adiponectin has antilipogenic and anti-inflammatory effects, while tumor necrosis factor alpha (TNF-alpha) reduces insulin sensitivity and has proinflammatory effects. We examined (1) the extent to which hypoadiponectinemia and TNF-alpha activation are features of nonalcoholic steatohepatitis (NASH) and (2) whether serum levels of these markers correlate with the severity of histological changes in 109 subjects with nonalcoholic fatty liver disease (NAFLD), including 80 with NASH and 29 with simple steatosis. By multivariate analysis, subjects with NASH had reduced adiponectin level and increased TNF-alpha and soluble TNF receptor 2 (sTNFR2)-but not leptin levels, compared with controls matched by age, sex, and body mass index; these differences were independent of the increased insulin resistance (by homeostasis model [HOMA-IR]) in NASH. When compared with simple steatosis, NASH was associated with lower adiponectin levels and higher HOMA-IR, but there were no significant differences in the levels of TNF-alpha and sTNFR2. The majority of subjects with steatohepatitis (77%) had adiponectin levels less than 10 microg/mL and HOMA-IR greater than 3 units, but only 33% of those with pure steatosis had these findings. HOMA-IR and low serum adiponectin were also independently associated with increased grades of hepatic necroinflammation. In conclusion, hypoadiponectinemia is a feature of NASH independent of insulin resistance. Reduced adiponectin level is associated with more extensive necroinflammation and may contribute to the development of necroinflammatory forms of NAFLD.