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Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non–small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry.Experimental Design: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing.Results: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry–based DNA sequencing.Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.

Supplementary Methods, Results and Figure Legends from Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer

Supplementary Data from Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer

Purpose: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited.Experimental Design: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples.Results and Conclusions: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.

Supplementary Figures from Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer

Background Long-acting injectable (LAI) antiretroviral therapy (ART) has the potential to improve medication adherence, reduce human immunodeficiency virus (HIV) stigma, and promote equity in care outcomes among people with HIV (PWH). We describe our early experience implementing LAI-cabotegravir/rilpivirine (CAB/RPV) for maintenance HIV-1 treatment. Methods We launched a pilot LAI-ART program at a large Ryan White-funded clinic in the Southeast, which accept provider-initiated referrals from April 14, 2021 to December 1, 2021. Our interdisciplinary program team (Clinician-Pharmacy-Nursing) verified clinical eligibility and pursued medication access for eligible patients. We describe (1) demographic and clinical variables of PWH referred and enrolled and (2) early outcomes among those accessing LAI-CAB/RPV. Results Among 58 referrals, characteristics were median age 39 (Q1–Q3, 30.25–50) years, 74% male, and 81% Black, and payor source distribution was 26% Private, 21% Medicare, 19% Medicaid, and 34% AIDS Drugs Assistance Program. Forty-five patients (78%) met clinical eligibility for LAI-CAB/RPV; ineligibility concerns included evidence of confirmed or possible RPV resistance (n = 8), HIV nonsuppression (n = 3), possible RPV hypersensitivity (n = 1), and pregnancy (n = 1). Among 45 eligible PWH, 39 (87%) enrolled and 15 (38%) initiated LAI-CAB/RPV after a median of 47 (Q1–Q3, 31–95) days since enrollment. Conclusions Implementing LAI-ART at a Southern US Ryan White-funded clinic has been challenged by the following: substantial human resource capital to attain drug, administer injections, and support enrolled patients; delayed therapy initiation due to insurance denials; patient ineligibility primarily due to possible RPV resistance; and inability to provide drug regardless of payor source. These barriers may perpetuate disparities in ART access and outcomes among PWH and should be urgently addressed so that LAI-ART can be offered equitably.

Background Pre-exposure prophylaxis (PrEP) is a highly effective biomedical strategy to decrease Human Immunodeficiency Virus (HIV) acquisition. Effectiveness of oral PrEP is linked to medication adherence. In 2018, Grady Health System (GHS) launched a PrEP program to increase PrEP access among un- and underinsured individuals living in metro Atlanta, Georgia. The purpose of this study is to determine PrEP medication adherence, PrEP discontinuation rates, and associated individual factors of patients enrolled during the first 18 months of the program’s implementation. Methods A single-center, retrospective chart review was conducted on patients enrolled in the GHS PrEP program between June 1, 2018 and February 29, 2020 who received more than one monthly PrEP prescription. Adherence was estimated using the medication possession ratio (MPR). The primary outcome was mean adherence to PrEP. Secondary outcomes include rate of high percent adherence (MPR > 80%), median time of engagement in care, PrEP discontinuation rates, rates of PrEP re-engagement, and individual factors associated with PrEP discontinuation and low adherence. Results This study included 154 patients, 70.8% of them were Black, 62.3% were cisgender men, 59.1% were uninsured, and the mean age was 34. The majority of patients identified as men who have sex with men (51.9%). Mean PrEP adherence was 89.2%; 77.3% of patients demonstrated a high rate of adherence. No individual or social factors were associated with low adherence, but younger age was associated with higher rates of PrEP discontinuation (p< 0.0061). At the end of the follow up period on October 30, 2020, 53.8% of patients were active in the program and 12.7% of those who discontinued had re-engaged with the program. The average length of program engagement was 9.8 months. Table 1. Baseline socio-demographic characteristics (N=154) Table 2. PrEP Adherence and Discontinuation at the GHS PrEP Program from 2018 to 2020 (N=154) Table 4. Multivariate analysis of individual factors associated with PrEP discontinuation and low adherence Conclusion Mean PrEP adherence at a safety net PrEP program in Atlanta was high and PrEP discontinuation rates were comparable to other PrEP clinics nationwide. We found no association with individual factors previously linked to lower adherence, including Black race, younger age, and insurance status. Program-related factors that may have impacted these findings need to be investigated. Other future areas of research include strategies to optimize engagement in care in younger patients. Disclosures Bradley L. Smith, Pharm.D., AAHIVP, Gilead Sciences, Inc (Advisor or Review Panel member)

Background HIV disproportionally affects transgender women (TGW) of color, with a prevalence of 26% and 44% among Latinx and Black TGW, respectively. Low medication adherence likely contributed to suboptimal pre-exposure prophylaxis (PrEP) efficacy among TGW in clinical trials, but real-world PrEP outcome data for TGW is limited. In this study, we developed the PrEP care continuum for TGW referred to a PrEP program at a large, safety-net urban hospital in the Southeast. Figure 1. PrEP care continuum of TGW referred a PrEP program. Referrals include all TGW referred to PrEP clinic, eligibility includes all those referred who were deemed eligible for PrEP, linkage refers to those eligible who had ongoing care at the PrEP clinic, prescription refers to those who received their first prescription of PrEP, initiation includes those who started taking the PrEP they were prescribed, and persistence includes those who had a visit within 6 months of study end. Methods We analyzed data for those referred to the PrEP program from 3/2018 to 2/2020. We determined the proportion of TGW who were linked to the program, provided a PrEP prescription, started PrEP, and persisted in PrEP care, defined as having at least one follow-up visit within 6 months. Using a multivariate regression model, including age, race, ethnicity, mental health co-morbidities, and substance use, we determined factors associated with persistence in PrEP care. We calculated rates of sexually transmitted infections (STIs) and HIV incidence. Results Of the 321 total referrals for PrEP, 42 (13%) were TGW. 81% of TGW were referred from a co-located gender clinic. Median age was 28.5 years (IQR 23-34), 62% were Black, 21% had mental health co-morbidities, 45% used substances, and 35% engaged in transactional sex. Of all TGW who were referred, 37 (88%) were eligible for PrEP and linked to care, 36 (85.7%) were prescribed and initiated PrEP, and 22 (52.4%) persisted in care at the end of the study period. There were no factors associated with persistence in PrEP care. The most common STIs during the first visit were pharyngeal gonorrhea (22.7%) and syphilis (16.7%). STI incidence was highest for rectal chlamydia (12.5%) and pharyngeal gonorrhea (6.5%). There was one HIV seroconversion during the study period. Conclusion In a public hospital-based PrEP clinic in Atlanta with a co-located gender clinic, TGW had high rates of linkage to care and PrEP prescription and initiation, despite high rates of mental health diagnoses and substance use. However, there was a significant drop-off in persistence. STI prevalence and incidence were high, but there was only one HIV seroconversion, highlighting the potential benefits of PrEP. Future studies are needed to assess interventions to optimize persistence in PrEP care among TGW. Disclosures Bradley L. Smith, PharMD, AAHIVP, Gilead Sciences, Inc (Advisor or Review Panel member)

Purpose Hospital emergency medication kits (HEMKs) are used to provide certain critical medications in emergent situations, despite many technological advancements for patient safety and medication distribution. We sought to evaluate HEMK usage and analyze associated costs to identify and recommend process improvements. Methods Mayo Clinic in Rochester, MN, is a large multisite academic medical center with 2 hospital campuses and many ambulatory clinics. All documentation of the approximately 250 HEMKs in circulation was analyzed from January to November 2017. The primary outcome was HEMK use. Secondary outcomes included individual medication usage and associated costs. These data were then used to recommend process improvements. Results Of 880 HEMKs evaluated, 675 (76.7%) were used, resulting in expiration 23.3% of the time. A total of 1,024 emergency medications were used, most commonly for hypoglycemia. Many of these medications are also available in automated dispensing machines for patient care use. Cost analysis revealed an average annual cost of nearly $200,000 associated with HEMKs. The results of our analysis indicated little added benefit of HEMKs in the setting of automated dispensing machine optimization. Steps for HEMK retirement are described. Conclusion HEMKs offered little added benefit considering technological advancements that have been made in patient safety and medication distribution since their inception. Retirement of HEMKs is anticipated to increase pharmacy operational efficiency by using automated dispensing machine technology and appropriate emergency response protocols to ensure optimal patient care.

Background Atlanta, GA ranks third in the nation for highest rates of new HIV diagnoses, disproportionally affecting Black men and women. Pre-exposure prophylaxis (PrEP) is underutilized in this population due to multiple barriers to uptake, including limited access to PrEP delivery programs. The advantages of a primary pharmacy-led PrEP program include: relatively low service fees, perform and assess point-of-care testing, and provide adherence counseling. Similar programs across the United States have been shown to effectively increase PrEP uptake and optimize retention in care. Grady Health System (GHS), the fifth largest public hospital system in the United States, is located at the epicenter of the HIV epidemic: downtown Atlanta. It encompasses 11 different primary care clinics, accounting for 850,000 outpatient visits per year. In August 2018, we launched a developmental pilot of a GHS pharmacy-based tele-PrEP program, aiming to optimize PrEP access for vulnerable populations who would otherwise not be able to obtain it. PrEP services are provided directly to the community and through a consultative support program for all clinical sites within the GHS system. The key pilot interventions included developing a user-friendly electronic medical record (EMR)-based PrEP order sets and brief provider education interventions in 6 GHS primary care clinics, to increase PrEP awareness among non-HIV clinicians. Methods We conducted a retrospective process evaluation of the pilot PrEP program based on the PrEP continuum of care. Results Over 9 months, 95 referrals were received from providers within the GHS clinics. Of the 95 patients referred, 56 (59%) started PrEP. Two patients were started on post-exposure prophylaxis prior to initiation of PrEP. Forty-five patients (81%) remain on PrEP as of April 2019. Six clients were diagnosed with 9 STIs on screening (4 syphilis, 2 gonorrhea, 2 chlamydia, 1 lymphogranuloma venereum). There have been no HIV seroconversions in patients on PrEP. Conclusion Utilizing a pharmacy-based PrEP program to train and support clinical providers in a large, hospital system can facilitate PrEP uptake and retention for patients in primary care. Disclosures All authors: No reported disclosures.

This article summarizes a presentation made at the joint BIA/MHRA conference, “Challenges of Development of Targeted Agents and Companion Diagnostics,” in London in March 2011. It focuses on the challenges the pharmaceutical industry is facing with the development of targeted agents in the field of oncology, in parallel with the challenges associated with the identification and development of the necessary diagnostic tools to support patient selection for personalized therapy. This article will encompass clinical, technical, and regulatory aspects.

Background Limited data exist to evaluate safety-related outcomes in Outpatient Parenteral Antimicrobial Therapy (OPAT) patients treated with antimicrobial agents for Gram-positive infections. Methods This retrospective, single-center study enrolled Mayo Clinic OPAT patients between 2013 and 2017. The primary objective of the study compared rates of therapy modification due to drug-related toxicity for staphylococcal infections treated with ceftriaxone, cefazolin, nafcillin, oxacillin, vancomycin, daptomycin, ceftaroline, linezolid, or ertapenem. Secondary objectives included determination of the frequency and type of adverse drug events (ADEs) attributed to OPAT and rate of readmission due to ADEs attributed to OPAT. Results One hundred seventy-two patients were identified (cefazolin n = 54, ceftriaxone n = 49, vancomycin n = 30, daptomycin n = 16, nafcillin n = 9, ertapenem n = 6, ceftaroline n = 4, oxacillin n = 3, linezolid n = 1). The overall treatment completion rates were high (153/172, 89.0%). Patients completed an average of 35.3 days (7 to 95) of therapy with their original antibiotic. Fourteen patients required change to a different antibiotic due to antimicrobial toxicity (ceftriaxone=5; vancomycin=2; cefazolin = 2; daptomycin = 2; ceftaroline = 1; nafcillin = 1; oxacillin = 1) and five patients experienced treatment failure required an additional agent (ceftriaxone = 2; nafcillin = 2; linezolid = 1). Adverse drug events (ADEs) were the most common reason for antimicrobial adjustment (14/19, 73.7%). The most common ADEs were hypokalemia (28/172, 16.3%) and diarrhea (25/172, 14.5%). There were only two cases of Clostridium difficile. Thirty-day readmissions due to antimicrobial therapy were low with 11 patients. Conclusion OPAT with Gram-positive agents used for staphylococcal infections is effective, but antimicrobial modifications still occur. Clinicians should be aware of the risk of ADEs and readmissions in OPAT patients. A multidisciplinary approach may enhance management of ADEs and possibly preventing readmissions Disclosures All authors: No reported disclosures.

Our understanding of the mechanisms by which BCR-ABL drives CML is based, in part, on the use of model cell lines such as the K562 cell line. However, the BCR-ABL translocation may occur via a number of different junction points. In addition, CML is a disease of hematopoietic stem cells and, as a result, can give rise to multiple lineages of tumor cells. In this study, we examined the cellular signaling profiles following imatinib mesylate treatment of eight model CML and ALL cell lines that encompass three BCR-ABL junction points and multiple lineages. We used phosphorylation-specific antibodies and flow cytometry to determine the kinase and pathway activation states with each of the cell lines before and after imatinib mesylate exposure. The comparisons of signaling response profiles, junction points and lineages indicate that cell line lineage rather than BCR-ABL junction point may determine cellular response to imatinib mesylate. The large amount of variation observed among the cell lines suggests that further analysis is required to understand the complex signaling profiles present in CML patients.

Background Stat51 (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition. Methods Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4′,6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies. Results Optimal fixation and staining parameters were established for pSTAT5 antibodies with K562 cells. These cells displayed high levels of immunoreactivity with pSTAT5 probes that could be inhibited uniformly with imatinib mesylate in a dose-response and time-dependent manner. The IC50 for downregulation of pSTAT5 immunoreactivity for K562 cells by cytometry was ∼70 nM. The inhibition half-time was approximately 1 min. At micromolar doses this reactivity remained minimal for up to 7 days. Cultured cells also displayed a population of negative cells that increased under conditions related to cessation of cell growth (media nutrient depletion). This study also showed quantitatively that a rabbit polyclonal antibody that cross-reacted with an additional epitope could be used successfully as a measure of Bcr/Abl activity. Conclusion We have developed a sensitive cytometric assay for Bcr/Abl kinase activity in human hematopoietic cell lines. Cytometry Part A 54A:75–88, 2003. © 2003 Wiley-Liss, Inc.

Expression of the human immunodeficiency virus (HIV) Nef protein has been linked to both decreased cell surface expression of CD4 and an impairment of signal transduction. The recently reported association of Nef with an unidentified serine kinase provides a clue as to how Nef might exert its effects. Considering the key role of protein kinase C (PKC) in T cell activation, we investigated the possibility that Nef interacts with PKC. Our results, using two approaches for detecting interactions between Nef and PKC isozymes in Jurkat cells, show that Nef interacts preferentially with thetaPKC. The interaction of Nef and thetaPKC is independent of calcium, enhanced by phospholipid activators of PKC and not affected by a PKC pseudosubstrate peptide. Phorbol 12-myristate 13-acetate and phytohemagglutinin stimulation of Jurkat cells expressing Nef fails to produce the usual translocation of thetaPKC from the cytosol to the particulate fraction; translocation of betaPKC and epsilonPKC was unaffected. Indeed, there appears to be a net loss of thetaPKC in Nef-expressing cells following stimulation. The loss of thetaPKC, which may be a result of inhibition of its binding to RACKs due to Nef binding, could contribute to the various impairments of T cell function associated with HIV infection and Nef expression.

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non–small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. Experimental Design: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. Results: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry–based DNA sequencing. Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.

Eukaryotic initiation factor (eIF) 4E, the mRNA 5′-cap-binding protein, mediates the association of eIF4F with the mRNA 5′-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (∼30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras–expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

SummaryDespite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRα and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.

Purpose: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited. Experimental Design: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples. Results and Conclusions: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.

Deregulated signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is common in many types of cancer, including glioblastoma. Dissecting the molecular events associated with activation of this pathway in glioblastoma patients in vivo presents an important challenge that has implications for the development and clinical testing of PI3K pathway inhibitors. Using an immunohistochemical analysis applied to a tissue microarray, we performed hierarchical clustering and multidimensional scaling, as well as univariate and multivariate analyses, to dissect the PI3K pathway in vivo. We demonstrate that loss of the tumor suppressor protein PTEN, which antagonizes PI3K pathway activation, is highly correlated with activation of the main PI3K effector Akt in vivo. We also show that Akt activation is significantly correlated with phosphorylation of mammalian target of rapamycin (mTOR), the family of forkhead transcription factors (FOXO1, FOXO3a, and FOXO4), and S6, which are thought to promote its effects. Expression of the mutant epidermal growth factor receptor vIII is also tightly correlated with phosphorylation of these effectors, demonstrating an additional route to PI3K pathway activation in glioblastomas in vivo. These results provide the first dissection of the PI3K pathway in glioblastoma in vivo and suggest an approach to stratifying patients for targeted kinase inhibitor therapy.

Although a high frequency of tumors contain constitutively activated signal transducers and activators of transcription 3 (Stat3), its relationship to breast cancer and patient survival has not been determined in a large retrospective study of node-negative tumors. To further elucidate the role of Stat3 in breast cancers, the expression patterns of Stat3 and Phospho-tyrosine residue 705 (Tyr705) Stat3 were correlated with survival outcome and clinicopathological parameters in a large cohort of node-negative breast cancer tumors.Immunohistochemical analysis of Stat3 and Phospho-Stat3 was performed on a breast cancer tissue microarray of 346 node-negative breast cancer specimens. These results were correlated with overall survival and other clinicopathological data.Positive Stat3 cytoplasmic expression was seen in 69.2% of tumors, and positive Phospho-Stat3 (Tyr705) cytoplasmic expression was seen in 19.6% of tumors. Neither cytoplasmic expression showed significant association with survival or other clinical parameters. However, 23.1% of tumors had positive Stat3 nuclear expression, and those patients had a significantly improved short-term survival (P = 0.0332) at 5 years of follow-up. Upon analysis of positive Phospho-Stat3 (Tyr705) nuclear expression, seen in 43.5% of tumors, positive tumors had a significantly improved survival at both short-term 5-year survival (P = 0.0054) and long-term 20-year (P = 0.0376) survival analysis. Additionally, positive Phospho-Stat3 (Tyr705) nuclear expression is an independent prognostic marker of better overall survival node-negative breast cancer by multivariate analyses that included the variables of nuclear grade, Ki-67, estrogen receptor staining, progesterone receptor staining, Her2 staining, age, and tumor size.These findings support a role for Stat3 and Phospho-Stat3 (Tyr705) overexpression in node-negative breast cancer and provide initial evidence that Phospho-Stat3 (Tyr705) may be a marker for improved overall survival independent of other prognostic markers.

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the beta-catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of beta-catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated beta-catenin, as detected by a phospho-specific antibody, may be useful to predict outcome in melanoma. We use immuno-histochemical analysis of beta-catenin and phospho-beta-catenin, first to verify the specificity of the phospho-beta-catenin antibody and then to assay expression in a tissue microarray-based study. The subcellular localization of beta-catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho-beta-catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho-beta-catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho-beta-catenin was more common in metastatic lesions. High levels of nuclear phospho-beta-catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho-specific antibodies to beta-catenin define a unique subset of cases and that monitoring of phospho-beta-catenin expression may be useful for assessing prognosis in malignant melanoma.

Background: The adoption of NGS platforms and development of targeted oncology drugs have enabled matching of patients and drugs. The authors undertook an observational, clinical study to explore the feasibility and potential clinical benefits of an upfront approach to the genomic profiling of tumors from metastatic colorectal cancer (mCRC) patients. The study sought to determine the number of drug targetable genomic changes, which occur within mCRC patients including a comparison of patients who progress early versus late. Methods: The study targeted enrollment of 50 mCRC patients within the US Oncology Network followed by collection of archival FFPE samples and genomic testing. Sample collection and processing was performed at Quintiles Central Laboratories followed by testing and bioinformatic analysis at the Quintiles Genomic Laboratory. Genomic profiling was performed on the Ion Torrent PGM following enrichment of tumor DNA via the AmpliSeq Cancer Hotspot Panel v2 assay, enriching for hotspots within 50 cancer-related genes. Clinical annotation and reporting to the doctors was provided by NofOne. Basic demographic and clinical information was collected but formal disease monitoring and follow-up was not performed. Clinicians were asked to report the impact of the genomic test report on patient recommendations. Results: The study enrolled and profiled 51 stage IV mCRC patients from July 2013 to October 2013 from 18 sites in the US. Subjects were stratified by time to progression prior to entering the study. The study population was evenly distributed across early (< 1yr) and late progressors (> 1yr) with a median age of 62. Test turn-around time averaged 15 days. 98% of the bases sequenced in the genomic analysis reached the target coverage necessary to identify 5% variant frequency in the sample. Genomic variants associated with approved therapies in mCRC were observed in 7.8% of patients while 64.7% of patients had variants associated with approved therapies in other indications. 84.3% of patients had variant associated with open clinical trials. Of these 43 patients, 32 had multiple biomarkers with associated trials. Overall, more than 100 mutations were identified including alterations in KRAS, BRAF, EGFR, PIK3CA, GNAS, TP53, APC and other genes. The number of actionable mutations was not associated with progressor status. Doctors recommended clinical trials following profiling and reporting of genomic alterations in 15 out of the 51 patients (29%), Conclusions: The outcome of this observational study demonstrates the feasibility of rapid screening and reporting of NGS genomic results targeting actionable mutations in mCRC. The lack of an association between early and late progressors, suggests that a greater sample size will be required for future studies. The reported impact on clinician recommendations indicates the value of the results to inform treatment and clinical trial decisions. Citation Format: Bradley L. Smith, Philip Breitfeld, Jennifer Cubino, Victor Weigman, Donald P. Richards, Ki Y. Chung. Feasibility study of genomic biomarker profiling for patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5187. doi:10.1158/1538-7445.AM2014-5187

We tested the hypothesis that the translocation and function of protein kinase C (PKC) requires the binding of PKC to its intracellular receptors (RACKs), using insulin-induced maturation of Xenopus oocytes. We show that after exposure of oocytes to insulin, PKC translocated from the cytosol to the particulate fraction. PKC is also required for insulin-induced oocyte maturation: microinjection of a PKC inhibitory peptide delayed maturation. To determine whether translocation of PKC was a result of the binding of PKC to the RACKS in the particulate fraction, we microinjected purified rat brain RACKS into oocytes before insulin exposure. Microinjection of RACKS, but not inactive phosphorylated RACKS, inhibited PKC translocation and delayed oocyte maturation. These results suggest an in vivo role for RACKS in a function mediated by PKC.

Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.