Kirsten H. Edmiston

A Portrait of Tissue Phosphoprotein Stability in the Clinical Tissue Procurement Process

Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (±20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase β Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3αβ Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.

Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival.

Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patients breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patients original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. Conclusions Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

Tissue is alive: New technologies are needed to address the problems of protein biomarker pre-analytical variability.

Instability of tissue protein biomarkers is a critical issue for molecular profiling. Pre‐analytical variables during tissue procurement, such as time delays during which the tissue remains stored at room temperature, can cause significant variability and bias in downstream molecular analysis. Living tissue, ex vivo, goes through a defined stage of reactive changes that begin with oxidative, hypoxic and metabolic stress, and culminate in apoptosis. Depending on the delay time ex vivo, and reactive stage, protein biomarkers, such as signal pathway phosphoproteins will be elevated or suppressed in a manner which does not represent the biomarker levels at the time of excision. Proteomic data documenting reactive tissue protein changes post collection indicate the need to recognize and address tissue stability, preservation of post‐translational modifications, and preservation of morphologic features for molecular analysis. Based on the analysis of phosphoproteins, one of the most labile tissue protein biomarkers, we set forth tissue procurement guidelines for clinical research. We propose technical solutions for (i) assessing the state of protein analyte preservation and specimen quality via identification of a panel of natural proteins (surrogate stability markers), and (ii) using multi‐purpose fixative solution designed to stabilize, preserve and maintain proteins, nucleic acids, and tissue architecture.

One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens.

Background There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. Results Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79. Conclusion In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.

The Neoadjuvant Model is Still the Future for Drug Development in Breast Cancer

The many improvements in breast cancer therapy in recent years have so lowered rates of recurrence that it is now difficult or impossible to conduct adequately powered adjuvant clinical trials. Given the many new drugs and potential synergistic combinations, the neoadjuvant approach has been used to test benefit of drug combinations in clinical trials of primary breast cancer. A recent FDA-led meta-analysis showed that pathologic complete response (pCR) predicts disease-free survival (DFS) within patients who have specific breast cancer subtypes. This meta-analysis motivated the FDAs draft guidance for using pCR as a surrogate endpoint in accelerated drug approval. Using pCR as a registration endpoint was challenged at ASCO 2014 Annual Meeting with the presentation of ALTTO, an adjuvant trial in HER2-positive breast cancer that showed a nonsignificant reduction in DFS hazard rate for adding lapatinib, a HER-family tyrosine kinase inhibitor, to trastuzumab and chemotherapy. This conclusion seemed to be inconsistent with the results of NeoALTTO, a neoadjuvant trial that found a statistical improvement in pCR rate for the identical lapatinib-containing regimen. We address differences in the two trials that may account for discordant conclusions. However, we use the FDA meta-analysis to show that there is no discordance at all between the observed pCR difference in NeoALTTO and the observed HR in ALTTO. This underscores the importance of appropriately modeling the two endpoints when designing clinical trials. The I-SPY 2/3 neoadjuvant trials exemplify this approach. Clin Cancer Res; 21(13); 2911–5. ©2015 AACR.

Carcinoembryonic antigen induction of IL-10 and IL-6 inhibits hepatic ischemic/reperfusion injury to colorectal carcinoma cells

Tumor cells cause ischemia/reperfusion (I/R) injury as they arrest within the hepatic microvasculature with the production of nitric oxide (NO) and reactive oxygen species (ROS) that kill both host liver and implanting tumor cells. Carcinoembryonic antigen (CEA) both facilitates the survival of experimental metastasis to nude mouse liver by weakly metastatic human colorectal carcinomas (CRCs) and induces the release of the proinflammatory cytokine IL‐6. We hypothesized that CEA also stimulates the release of the antiinflammatory cytokine IL‐10 causing inhibition of the toxicity of hepatic I/R injury and indirect stimulation of tumor cell colonization of the liver. Intravenous injection of CEA produced more than 1 ng/ml of IL‐10 in the systemic circulation within 1 hr which subsided by 8 hr. The IL‐10 response is specific to CEA since the pentapeptide sequence in CEA that binds to the CEA receptor stimulated isolated Kupffer cells to produce IL‐10. IL‐10, but not IL‐6, increased the survival of weakly metastatic CRC cocultured with ischemic‐reoxygenated liver fragments but did not affect the survival of CRC exposed to oxidative stress in the absence of any host cells. CEA, IL‐6 and IL‐10 pretreatment reduced expression of iNOS but only CEA and IL‐10 strongly inhibited NO and total reactive species production by ischemic‐rexoygenated liver. IL‐6 was toxic to CRC exposed to oxidative stress while IL‐10 did not have a direct effect on CRC. Thus, CEA stimulates production of IL‐10 that may enhance metastasis by promoting the ability of circulating CRC cells to survive the I/R injury of implantation.

Molecular network analysis using reverse phase protein microarrays for patient tailored therapy.

The practice of medicine has always aimed at individualized treatment of disease. The relationship between patient and physician has always been a personal one, and the physicians choice of treatment has been intended to be the best fit for the patients needs. The necessary pooling/grouping of disease families and their assignment to a number of drugs or treatment methods has, consequently, led to an increase in the number of effective therapies. However, given the heterogeneity of most human diseases, and cancer specifically, it is currently impossible for the treating clinician to effectively predict a patients response and outcome based on current technologies, much less the idiosyncratic resistances and adverse effects associated with the limited therapeutic options.

Innovations in Clinical Trial Design in the Era of Molecular Profiling

Historically, cancer has been studied, and therapeutic agents have been evaluated based on organ site, clinical staging, and histology. The science of molecular profiling has expanded our knowledge of cancer at the cellular and molecular level such that numerous subtypes are being described based on biomarker expression and genetic mutations rather than traditional classifications of the disease. Drug development has experienced a concomitant revolution in response to this knowledge with many new targeted therapeutic agents becoming available, and this has necessitated an evolution in clinical trial design. The traditional, large phase II and phase III adjuvant trial models need to be replaced with smaller, shorter, and more focused trials. These trials need to be more efficient and adaptive in order to quickly assess the efficacy of new agents and develop new companion diagnostics. We are now seeing a substantial shift from the traditional multiphase trial model to an increase in phase II adjuvant and neoadjuvant trials in earlier-stage disease incorporating surrogate endpoints for long-term survival to assess efficacy of therapeutic agents in shorter time frames. New trial designs have emerged with capabilities to assess more efficiently multiple disease types, multiple molecular subtypes, and multiple agents simultaneously, and regulatory agencies have responded by outlining new pathways for accelerated drug approval that can help bring effective targeted therapeutic agents to the clinic more quickly for patients in need.

Abstract CT140: PINC trial: Preventing invasive breast neoplasia with chloroquine

The PINC trial (NCT01023477) examined the dosage efficacy of oral chloroquine (CQ), an autophagy inhibitor, as a neoadjuvant therapy to reduce the volume, cause regression and decrease the recurrence of breast ductal carcinoma in situ (DCIS), for any grade or ER/PR/Her2 status. Study Objectives: Establish the safety of preventive doses of chloroquine in patients receiving external beam radiation. Elucidate functional molecular and cellular impacts of in vivo autophagy pathway treatment for DCIS. Study the impact of autophagy inhibitors on the MRI characteristics of DCIS lesions. Study the molecular cytogenetic profile of DCIS lesions before and after therapy. This trial implemented a general strategy to accelerate the pace of community-based translational research. Technology for providing immediate feedback on the therapeutic efficacy at the molecular level can be broadly extended to other trials. Methodology: 12 patients diagnosed with DCIS (any grade or ER/PR/Her2 status) were enrolled and randomly assigned to receive CQ at 250mg/week (n=5) or 500mg/week (n=7) for 4 weeks, followed by standard of care surgical therapy. MRI was performed before/after CQ treatment. DCIS spheroid forming cells were isolated and propagated from fresh human DCIS lesions. DCIS cells were characterized by organ culture, xenograft transplantation, molecular cytogenetics, and 59 cell signaling kinases were quantified by Reverse Phase Protein Microarrays. Results: 12 patients completed 4 weeks of CQ treatment prior to surgical excision of their DCIS lesion, with 1 yr follow-up information. CQ treatment reduced PCNA proliferation index in DCIS lesions compared to untreated controls (p=0.001) and inhibited autophagic flux (LC3B positive puncta by IHC). CQ reduced the number of mammospheres in organoid culture without altering copy number variation. Xenograft transplants in NOD/SCID mouse mammary fat pads failed to generate tumors (n=4). 7/12 patients exhibited a reduction in lesion diameter by MRI, 3/12 patients exhibited no measurable change, and 2/12 had a slight increase. Calcium export channel protein (PMCA2) co-localized with 3+ HER2 positive DCIS lesions. Tumor infiltrating macrophages migrated into DCIS ducts following CQ therapy compared to controls (p=0.006). Conclusion: Oral chloroquine, as anti-autophagy therapy, generates a measurable reduction in proliferation of DCIS lesions and enhances immune cell migration into the duct. Citation Format: Virginia A. Espina, Lance Liotta, Svetlana Rassulova, Holly Gallimore, Thalia Grant-Wisdom, Geetha Menezes, Hassan Nayer, Kirsten Edmiston. PINC trial: Preventing invasive breast neoplasia with chloroquine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT140. doi:10.1158/1538-7445.AM2017-CT140

Non-enzymatic, serum-free tissue culture of pre-invasive breast lesions for spontaneous generation of mammospheres.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue. Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO₂ incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.