Individual Patient Tumors Research Articles

Multimodal Assessment of Estrogen Receptor mRNA Profiles to Quantify Estrogen Pathway Activity in Breast Tumors

BackgroundMolecular markers have transformed our understanding of the heterogeneity of breast cancer and have allowed the identification of genomic profiles of estrogen receptor (ER)-α signaling. However, our understanding of the transcriptional profiles of ER signaling remains inadequate. Therefore, we sought to identify the genomic indicators of ER pathway activity that could supplement traditional immunohistochemical (IHC) assessments of ER status to better understand ER signaling in the breast tumors of individual patients. Materials and MethodsWe reduced ESR1 (gene encoding the ER-α protein) mRNA levels using small interfering RNA in ER+ MCF7 breast cancer cells and assayed for transcriptional changes using Affymetrix HG U133 Plus 2.0 arrays. We also compared 1034 ER+ and ER− breast tumors from publicly available microarray data. The principal components of ER activity generated from these analyses and from other published estrogen signatures were compared with ESR1 expression, ER-α IHC, and patient survival. ResultsGenes differentially expressed in both analyses were associated with ER-α IHC and ESR1 mRNA expression. They were also significantly enriched for estrogen-driven molecular pathways associated with ESR1, cyclin D1 (CCND1), MYC (v-myc avian myelocytomatosis viral oncogene homolog), and NFKB (nuclear factor kappa B). Despite their differing constituent genes, the principal components generated from these new analyses and from previously published ER-associated gene lists were all associated with each other and with the survival of patients with breast cancer treated with endocrine therapies. ConclusionA biomarker of ER-α pathway activity, generated using ESR1-responsive mRNAs in MCF7 cells, when used alongside ER-α IHC and ESR1 mRNA expression, could provide a method for further stratification of patients and add insight into ER pathway activity in these patients.

Abstract A15: Establishing an ex-vivo, tumor spheroid culture system to assess molecular-targeted therapies for personalized treatment of non-small cell lung cancer

Abstract Molecularly-targeted therapeutics are being developed for personalized cancer treatment based on the ideas that treatments should be determined by specific characteristics of an individual patient's tumor and that treatments should target tumor cells without damaging normal cells. Accumulating evidence indicates tumor heterogeneity can lead to acquired resistance to molecularly-targeted therapies. Currently, preclinical investigations rely heavily on the use of tumor cell lines and mouse xenograft models. The high failure rate in oncology drug development suggests that such test systems do not adequately model tumor heterogeneity. Therefore, more predictive preclinical models are needed to ensure that only the most promising molecularly-targeted, combination therapies are assessed in clinical studies. Here we established a novel 3D lung adenocarcinoma tissue-originating spheroid model, which may be used to investigate the efficacy of molecularly-targeted cancer therapeutics. Fresh lung adenocarcinomas were minced and either digested with Liberase DH enzyme (5.6 units) or homogenized to generate cancer tissue-originating spheroids. Spheroids were plated in Matrigel and cultured in stem cell media, with 0.01, 0.1, 1 or 10 μM erlotinib or the DMSO vehicle. Erlotinib is an epidermal growth receptor inhibitor commonly used to treat non-small cell lung cancer (NSCLC). Using an ImageXpress Micro Widefield High Content Screening System (Molecular devices), z-stack bright field images were collected 1, 7, and 14 days after plating. Composite z-stack images were analyzed using LASX (Leica Microsystems) software. Total spheroid area (in pixels) was measured for replicate cultures exposed to different erlotinib concentrations at each time point. The total area of spheroids for 7 and 14 days were normalized to day 1 measurements and cell death was evaluated as the decrease in total area of the spheroids in culture. Our results indicate that direct homogenization of lung adenocarcinomas generated at least ten times greater yield of spheroids than was achieved using digestion with the Liberase DH. The yield and appearance of spheroids varied significantly. Spheroids prepared from some lung adenocarcinomas were more spherical and aggregated than those prepared from other tumors. There was time-dependent decrease in total area of spheroids following 7 and 14 days in culture. Also spheroids prepared from different lung adenocarcinomas responded differently to different concentrations of erlotinib. High-sensitivity quantification of specific mutations (EGFR, KRAS PIK3CA and BRAF) will be conducted before and after erlotinib treatments to determine whether this model can be used to assess the potential outgrowth of drug-resistant tumor subpopulations. Citation Format: Malathi Banda, Karen L. McKim, Barbara Parsons. Establishing an ex-vivo, tumor spheroid culture system to assess molecular-targeted therapies for personalized treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A15.

Abstract A20: Personalized models to guide precision medicine

Abstract Background: Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient's cancer. Available cell line models alone often do not accurately recapitulate the genetic profile of individual patient tumors and therefore limit preclinical evaluation of newly targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to the use of monolayer cultures as the initial screening method that is associated with highly variable responses and does not predict clinically observed chemoresistance. In our Englander Institute for Precision Medicine we developed a program utilizing patient derived tumor organoids, in combination with individualized genomic sequencing, targeted and/or high throughput drug screenings to nominate drug candidates in a precision patient care setting. Drug candidates are further validated with personalized in vivo models. Utilizing these various genomic and biological platforms for pharmacological screenings, we can more closely recapitulate the in vivo tumor of individual patients and can more accurately model personalized therapeutic response and resistance in vitro and in vivo. Design: Fresh tissue samples were collected, washed and mechanically or enzymatically dissociated and then plated in a Matrigel (BD) scaffold with primary culture media. Primary spheres were characterized according to our cytology, histology and genomic platforms. Established and characterized tumor organoids were expanded, cryopreserved for banking, used for in vitro studies and implanted in nude mice for patient derived xenografts (PDXs) to further validate potential drug candidates. Results: Our success rate in generating patient derived pan-cancer tumor organoids is 30%, depending on specimen quality and tumor type (e.g. endometrial cancer 70%, metastatic prostate cancer 15%). Morphology and molecular profiles show good concordance among tumor organoids and native tumor tissues. The success rate in establishing PDXs from organoid cultures is currently at 70-80%. In vitro and in vivo drug screenings show tumor specific drug sensitivity. Conclusion: We have developed protocols for the generation and characterization of individual patient-derived tumor organoids. Cytopathology, histopathology and molecularpathology represent important platforms in our Precisicon Medicine Program. Tumor organoid characterization, pharmacological screenings and drug validation in PDX models are effective models which can be used to tailor standard of care treatment, study drug resistance, and nominate novel therapeutic targets unique to the individual genomic landscape and biology of each tumor. Citation Format: Chantal Pauli, Loredana Puca, Benjamin Hopkins, Brooke M. Emerling, Andrea Sboner, Olivier Elemento, Terra J. McNary, Yelena Churakova, Himisha Beltran, Mark A. Rubin. Personalized models to guide precision medicine. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A20.

Abstract CT020: MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial

Abstract The majority of metastatic cancers remain incurable since the current methods of treatment often fail to target the heterogeneous nature of each individual patient's tumor. Personalized approaches targeting each individual patient's tumor may therefore bring significant improvements. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically validate a pioneering RNA-based immunotherapy concept for the treatment of triple negative breast cancer (TNBC) by targeting shared tumor antigens and individual neo-antigens in TNBC patients. MERIT combines two personalized treatment concepts: (i) treatment with vaccines containing “off-the-shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode shared breast cancer tumor antigens expressed in the respective patient's tumor; (ii) treatment with mRNAs engineered on-demand that encode patient-specific mutated neo-antigens identified by next generation sequencing (NGS) and ranked according to the predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation, which specifically targets APCs and consequently induces antigen-specific T cell responses. MERIT is a multi-center phase I trial (NCT02316457) conducted in four European countries to assess the feasibility, safety and biological efficacy of this personalized immunotherapy. TNBC patients (pT1cN0M0 - anyTanyNM0) after surgery and adjuvant chemotherapy will be allocated to one of two study arms. Patients in ARM1 will receive eight vaccination cycles with a personalized set of shared tumor antigens selected from the WAREHOUSE that correspond to the patient tumor's antigen-expression profile. Patients in ARM2 will be first treated with the personalized WAREHOUSE vaccine approach followed by six vaccination cycles of on-demand manufactured MUTANOME vaccine targeting the unique mutation signature of the individual patient. During the clinical trial, patients will receive the individualized combination of the RNAs in parallel to standard radiotherapy. The clinical trial is approved and the study start is planned for Q1 2016. The consortium has built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept, which covers the whole individualized drug development cycle from target discovery, validation to GMP manufacturing and drug release for each individual patient. We will present the therapeutic concept and study protocol as well as the methodologies required for this highly innovative phase I trial. The personalized immunotherapy overcomes the current limitations of fixed, off-the-shelf therapeutics and thus might increase the clinical benefit for TNBC patients. This project is a collaborative effort of five partners from academia and industry funded by the European Commission's FP7 and led by BioNTech AG. Citation Format: Sandra Heesch, Valesca Bukur, Janina Buck, Jan Diekmann, Mustafa Diken, Kerstin Ewen, Heinrich Haas, Alexandra Kemmer-Brueck, Björn-Philipp Kloke, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Anna Paruzynski, Kathrin Schultheiß, Doreen Schwarck, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Özlem Türeci, Ugur Sahin. MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT020.

Abstract 1405: Vesigenurtacel-L stimulates tumor infiltration of unique polyclonal T cell clones in non-muscle invasive bladder cancer patients

Abstract Non-muscle invasive bladder cancer affects approximately 50,000 Americans each year, requires chronic medical management with a combination of surgical resection and intravesical BCG instillations for treatment. While BCG improves recurrence-free survival in this population, there remains a significant unmet medical need for therapeutics in patients who have a particularly high risk of recurrence and/or progression to muscle-invasive disease. Vesigenurtacel-L is an allogeneic, cell based, vaccine engineered to secrete the heat shock protein, gp96-Ig, which facilitates cross-presentation of cell-derived antigens on MHC I to activate CD8+ T cell responses across MHC barriers. We recently reported that in patients treated with Vesigenurtacel-L in a Phase I clinical trial, a minimum of 15 antigens were shared between the vaccine and individual patient tumors. Although no patient received a complete course of maintenance BCG, 70% of patients remained recurrence free beyond 12 months. Baseline biopsies were collected from all patients, and serial biopsies for several patients when clinically indicated. Analysis of these tissues by RNA Sequencing revealed that recurring patient tumors expressed higher levels of B7-H4, GITR and KIR3 at baseline, while non-recurring patient tumors expressed an immune profile consistent with an interferon defense signature. In addition, patients with progressive elimination of residual disease in repeat biopsies showed increased levels of CD3, CD8, IL-23A and OX40. Deep sequencing of peripheral blood T cells and total tumor DNA for T cell receptors was performed at baseline and at several post-treatment time points. These data demonstrated polyclonal expansion of TCR clones that were either absent, or present at low frequencies at baseline in the peripheral blood. Remarkably, approximately 40% of these clones could also be found expanded within the tumor. These data correlate positively with the absolute increase in tumor infiltrating lymphocyte percentages observed by immunohistochemistry. ELISPOT assays were performed to demonstrate the specificity of expanded T cell clones toward shared tumor antigens, since the antigens in both the patients tumors and Vesigenurtacel-L were known. These data provide exciting clinical evidence of patient benefit derived from an allogeneic immunotherapy vaccine and compelling evidence for a vaccine therapy converting ‘cold’ tumor to (predominantly CD8+) TIL-rich tumors, with a corresponding analysis of the antigenic determinants of the response. Citation Format: Taylor H. Schreiber, George Fromm, Xin Xu, Eric Yusko, Brandon Early, Melissa Price. Vesigenurtacel-L stimulates tumor infiltration of unique polyclonal T cell clones in non-muscle invasive bladder cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1405.

Abstract 3346: Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates

Abstract We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of <1 CSC. The HS provides an analog of the CSC niche, enabling the CSC to proliferate (with some daughters differentiating into Amplifying Transit Cells (ATCs)) and undergo 10-15 symmetric divisions before exhausting the nutrients. This satisfies the McCulloch and Till (spleen colony assay) requirements for a stem cell. Applying Koch's postulates: (1) Does the patient's tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions. The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments. Conclusion: The CSC hypothesis is validated in the HSA. Citation Format: Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3346.

Abstract 619: Personalized models to guide precision medicine

Abstract BACKGROUND Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient's cancer. Available cell line models alone often do not accurately recapitulate the genetic profile of individual patient tumors and therefore limit preclinical evaluation of newly targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to the use of monolayer cultures as the initial screening method that is associated with highly variable responses and does not predict clinically observed chemoresistance. In our Englander Institute for Precision Medicine we developed a program utilizing patient derived tumor organoids, in combination with individualized genomic sequencing, targeted and/or high throughput drug screenings to nominate drug candidates in a precision patient care setting. Drug candidates are further validated with personalized in vivo models. Utilizing these various genomic and biological platforms for pharmacological screenings, we can more closely recapitulate the in vivo tumor of individual patients and can more accurately model personalized therapeutic response and resistance in vitro and in vivo. DESIGN Fresh tissue samples were collected, washed and mechanically or enzymatically dissociated and then plated in a Matrigel (BD) scaffold with primary culture media. Primary spheres were characterized according to our cytology, histology and genomic platforms. Established and characterized tumor organoids were expanded, cryopreserved for banking, used for in vitro studies and implanted in nude mice for patient derived xenografts (PDXs) to further validate potential drug candidates. RESULTS Our success rate in generating patient derived pan-cancer tumor organoids is 30%, depending on specimen quality and tumor type (e.g. endometrial cancer 70%, metastatic prostate cancer 15%). Morphology and molecular profiles show good concordance among tumor organoids and native tumor tissues. The success rate in establishing PDXs from organoid cultures is currently at 70-80%. In vitro and in vivo drug screenings show tumor specific drug sensitivity. CONCLUSION We have developed protocols for the generation and characterization of individual patient-derived tumor organoids. Cytopathology, histopathology and molecular pathology represent important platforms in our Precisicon Medicine Program. Tumor organoid characterization, pharmacological screenings and drug validation in PDX models are effective models which can be used to tailor standard of care treatment, study drug resistance, and nominate novel therapeutic targets unique to the individual genomic landscape and biology of each tumor. Citation Format: Chantal Pauli, Loredana Puca, Brooke M. Emerling, Benjamin Hopkins, Andrea Sboner, Olivier Elemento, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. Personalized models to guide precision medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 619.

Abstract LB-030: Building personalized genetic models of human cancer in zebrafish to predict response to therapy and characterize disease mechanisms

Abstract A key challenge posed by personalized medicine is the ability to functionally characterize specific mutations identified in human tumors in order to guide treatment decisions. Transgenic approaches in zebrafish offer an opportunity to model individual patient tumors in order to develop personalized models. To test this concept, we performed a genetic characterization of a patient with thyroid carcinoma and created an in vivo personalized thyroid cancer model in zebrafish. Our studies suggest that this approach can be used to build preclinical models suitable for chemical-genetic studies. Chromosomal rearrangements in RET often lead to chimeric in-frame fusions of its kinase domain with one of several partners, resulting in ectopic, ligand-independent activation of RET. RET/PTC1 encodes a CCDC6-RET fusion and results in ectopic transcriptional activation of RET in thyrocytes and is associated with papillary thyroid cancer (PTC). Cabozantinib, a selective RET inhibitor, restricts growth of RET/PTC1 cell lines and shows marked activity in patients with RET-mutant medullary thyroid cancer. However, its efficacy is limited due unknown mechanisms of resistance. Genetic analysis of the index patient's tumor revealed a RET/PTC1 rearrangement, alterations in the TERT promoter and a focal loss of TP53. We performed molecular analyses of the index patient at distinct points throughout disease to generate a genomic timeline of disease and identify putative landmarks and molecular subtypes of disease. To functionally assess genetic alterations in the index patient, we transiently expressed RET/PTC1 in zebrafish thyroid follicular cells using a zebrafish thyroglobulin promoter. We observed tumor growth by 14 days post fertilization (dpf) in transgenic larvae, with histopathologic characteristics consistent with PTC. A transcriptional profile of RET/PTC1 transgenic zebrafish tumors revealed a set of genes associated with thyrocyte differentiation, as well as transcriptional signatures associated with cell proliferation and cAMP signaling in human thyrocytes. Ex vivo treatment of zebrafish RET/PTC1 tumors with cabozantinib, blocks RET activation and downstream signaling. As a whole, these studies provide proof-of-concept data to develop a transgenic zebrafish platform for patient specific models of thyroid carcinoma and other solid tumors. Citation Format: Jacques A. Villefranc, Chantal Pauli, Viviana Anelli, Sagar Chhangawala, Rohan Bareja, Theresa Scognamiglio, Andrea Sboner, Mark A. Rubin, Yariv Houvras. Building personalized genetic models of human cancer in zebrafish to predict response to therapy and characterize disease mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-030.

Abstract 243: Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells

Abstract Advanced prostate cancer (PC) is treated primarily by means of chemotherapy with one of the clinically approved microtubule (MT)-targeting agents (MTAs): paclitaxel, docetaxel and cabazitaxel. The mechanism of action of the MTAs on the cellular level is to bind to MTs and inhibit MT-dependent intracellular pathways, ultimately leading to cell death. In PC, MTAs are the only chemotherapy class shown to improve survival, however, there is high variability of response to MTA chemotherapy among patients as well as emergence of resistance to drug action which hampers the clinical efficacy. Clinicians need additional information to match individual patient tumors to the most effective drugs or refrain from treating patients that will not respond. However, the precise mechanism of action or resistance development to MTAs is not understood, which represents a critical gap in our knowledge to select the best treatment. Our hypothesis is that there are inherent differences in tumor MT dynamics among individual patients which dictates their response to treatment with specific MTAs. To this end, we envision that a systematic characterization of PC MT dynamics and their response to MTAs will allow chemotherapy customization and prolong survival. Specifically, our approach consists of the analysis of metastatic castrate resistant PC (mCRPC) organoids (Gao D. et al, 2014) derived from bone (acetabulum, vertebrae), soft tissue (salvage prostatectomy, lymph nodes), pleural effusion and circulating tumor cells biopsy samples obtained from metastatic patients who have received hormonal therapy and the MTA docetaxel or hormonal therapy alone or no treatment. We apply a quantitative single-cell image analysis algorithm (ClusterTrack) to measure a panel of twelve distinct MT dynamics parameters (a MT dynamics signature). Preliminary analysis suggests differences between different mCRPC organoids. MT tracking results indicate a correlation of decreased MT growth rate with increased Op18/stathmin expression. In this context, we hypothesize that differential expression of MT regulating genes affects specific parameters of MT dynamics, thus determining the sensitivity to MTA treatment. Citation Format: Alexandre Matov, Johan de Rooij, Jay Gatlin, Julia Rohrberg, Nikta Ahmad, Rahul Aggarwal, Jeff Simko, Andrei Goga, Charles Ryan, Torsten Wittmann. Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 243.

Intra‐tumor heterogeneity in head and neck cancer and its clinical implications

The presence of heritable differences among cancer cells within a tumor, called intra-tumor genetic heterogeneity, has long been suspected of playing a role in poor responses to therapy. Research over the past decade has documented the existence of such heterogeneity within tumors of individual patients and documented its potential clinical significance. The research methods for identifying this heterogeneity were not, however, readily adaptable to widespread clinical application. After a brief review of this background, we describe the development of a measure of intra-tumor genetic heterogeneity, based on whole-exome sequencing of individual tumor samples, that could be applied to biopsy specimens in a clinical setting. This measure has now been used in head and neck squamous cell carcinoma (HNSCC) to document, for the first time, a relation of high intra-tumor genetic heterogeneity to shorter overall survival in a large, multi-institutional study. The implications of heterogeneity for research and clinical care thus now need to be addressed.

Systems medicine in colorectal cancer: from a mathematical model toward a new type of clinical trial.

Current colorectal cancer (CRC) treatment guidelines are primarily based on clinical features, such as cancer stage and grade. However, outcomes may be improved using molecular treatment guidelines. Potentially useful biomarkers include driver mutations and somatically inherited alterations, signaling proteins (their expression levels and (post) translational modifications), mRNAs, micro‐RNAs and long noncoding RNAs. Moving to an integrated system is potentially very relevant. To implement such an integrated system: we focus on an important region of the signaling network, immediately above the G1‐S restriction point, and discuss the reconstruction of a Molecular Interaction Map and interrogating it with a dynamic mathematical model. Extensive model pretraining achieved satisfactory, validated, performance. The model helps to propose future target combination priorities, and restricts drastically the number of drugs to be finally tested at a cellular, in vivo, and clinical‐trial level. Our model allows for the inclusion of the unique molecular profiles of each individual patient's tumor. While existing clinical guidelines are well established, dynamic modeling may be used for future targeted combination therapies, which may progressively become part of clinical practice within the near future. WIREs Syst Biol Med 2016, 8:314–336. doi: 10.1002/wsbm.1342This article is categorized under: 1Biological Mechanisms > Cell Signaling2Analytical and Computational Methods > Computational Methods3Translational, Genomic, and Systems Medicine > Translational Medicine

A genetic basis for the variation in the vulnerability of cancer to DNA damage.

Radiotherapy is not currently informed by the genetic composition of an individual patient's tumour. To identify genetic features regulating survival after DNA damage, here we conduct large-scale profiling of cellular survival after exposure to radiation in a diverse collection of 533 genetically annotated human tumour cell lines. We show that sensitivity to radiation is characterized by significant variation across and within lineages. We combine results from our platform with genomic features to identify parameters that predict radiation sensitivity. We identify somatic copy number alterations, gene mutations and the basal expression of individual genes and gene sets that correlate with the radiation survival, revealing new insights into the genetic basis of tumour cellular response to DNA damage. These results demonstrate the diversity of tumour cellular response to ionizing radiation and establish multiple lines of evidence that new genetic features regulating cellular response after DNA damage can be identified.

Cytologic Differential Diagnosis of Malignant Mesothelioma and Reactive Mesothelial Cells With FISH Analysis of p16.

Mesothelioma patients often present with serosal effusions, which are ideal for cytopathological diagnoses. However, the morphological overlap between malignant and benign mesothelial proliferation can make a conclusive cytological diagnosis of mesothelioma elusive because immunohistochemical staining does not discriminate definitively between the two in this setting. p16 is deleted in up to 80% of pleural mesotheliomas. The aim of this study was to establish the correlation between the p16 deletion status of the cell block with that of its corresponding tumor using fluorescence in situ hybridization (FISH) analysis for individual patient tumors. Twenty-two biopsies and 24 corresponding cell blocks, containing serosal effusions with atypical mesothelial cells from 22 patients with histologically confirmed pleural mesotheliomas, were analyzed with p16 FISH. Seventeen cell blocks consisting of serosal effusions with reactive mesothelial cells from nonmesothelioma cases were also analyzed. Combined immunofluorescence and FISH were also performed. Seventeen of the 22 mesothelioma patients (77.3%) showed homozygous deletions of p16 in the tumor tissue and in the atypical mesothelial cells from the cell blocks. p16 FISH followed by immunofluorescence with EMA was helpful towards identifying the mesothelioma cells in the cell blocks. We confirmed that the p16 FISH results obtained from the cell blocks are as reliable as those from the tissue sections. Cell block analysis is recommended for patients with serosal effusions of unknown origins with the following methods: immunohistochemistry should be performed to validate the mesothelial origin, and p16 FISH should be performed to confirm malignancy. Diagn. Cytopathol. 2016;44:591-598. © 2016 Wiley Periodicals, Inc.

Progress in the treatment of ovarian cancer—lessons from homologous recombination deficiency—the first 10 years

For several years, a major obstacle in the systemic treatment of ovarian cancer has been the lack of a therapeutic strategy tailored to specific biomarkers present in the individual patient's tumour. However, considerable progress has been made recently through the development of drugs targeting cells deficient in the key mechanism of double-strand DNA repair, known as homologous recombination (HRD). These drugs, inhibitors of the enzyme poly (ADP) ribose polymerase (PARP), selectively kill HRD cells through a process known as tumour-selective synthetic lethality. Olaparib is the first such agent, now approved for the treatment of ovarian cancer associated with mutations in the BRCA 1/2 genes, since these are characterised by cells with HRD. Importantly, another group of patients with tumours bearing a similar repair deficiency but without BRCA mutations may also be susceptible to PARP inhibition and efforts to develop an HRD assay are therefore a priority so that these patients can be identified as PARPi candidates. In addition, combination strategies are an area of intense research; these include combinations with antiangiogenic agents and with inhibitors of the P13K/AKT pathway and others are likely to merit assessment since resistance to PARP inhibitors will certainly emerge as the next challenge. While olaparib is the first PARP inhibitor to receive approval for ovarian cancer treatment, others including rucaparib and niraparib are clearly effective in this disease and, within the next year or two, the results of ongoing randomised trials will clarify their respective roles. PARP inhibitors are generally well tolerated; regulatory approval at present supports their use as a maintenance therapy (in Europe) and as treatment for advanced recurrent disease (in the United States), but it is likely that these indications will extend as the results of ongoing trials become available. Ten years have elapsed between the first pre-clinical publications and the regulatory approval of PARP inhibitors and the next 10 years promise to be even more productive.

Mucinous epithelial ovarian carcinoma

Mucinous tumours involving the ovary may be benign, borderline, or malignant. Malignant tumours may be primary or metastatic. Differentiation between primary and metastatic involvement of the ovary is critical for optimal patient management. Even among skilled pathologists, this distinction can be problematic, as can the distinction between borderline ovarian tumour of intestinal type and well-differentiated invasive primary mucinous ovarian carcinoma. Primary invasive mucinous ovarian carcinoma and mucinous carcinoma metastatic to the ovary do have distinct patterns of macroscopic and microscopic involvement which will reveal the correct diagnosis in many cases. There are also well-recognized patterns of immunohistochemical staining that can further assist in this differentiation. As a result of the application of these histopathological techniques, the incidence of primary invasive mucinous epithelial carcinoma has fallen over recent years from ∼12% to ∼3%. However, even in recent multicentre clinical trials such as GOG 182, expert pathological review suggests that ∼60% of tumours originally classified as primary invasive mucinous carcinomas were in fact metastatic tumours to the ovary. Review of outcome data for patients with mucinous carcinoma entered into multicentre trials suggests that this subtype of disease has a particularly poor prognosis in comparison with other subtypes of ovarian carcinoma. Historically, patients with mucinous epithelial ovarian carcinoma (mEOC) have been treated in the same way as other subtypes of ovarian carcinoma. While there is undoubtedly a response rate to platinum-based chemotherapy, retrospective reviews of individual centre experience suggest that this is substantially lower than for high-grade papillary serous carcinoma and in the order of only 30%-40%. The mEOC trial was established to investigate the possibility that the combination of capecitabine and oxaliplatin (chemotherapy drugs more commonly used in colorectal carcinoma) may be superior to conventional carboplatin and paclitaxel chemotherapy. In a 2 × 2 factorial design, there was also a randomization to bevacizumab. Unfortunately, this trial closed early, 5 years after initiation having recruited just 50 of a proposed 322 patients. mEOC is now characterized as a type I tumour with an identifiable stepwise progression from a premalignant lesion, through non-invasive, to invasive malignancy. Molecular characterization of mEOC reveals it to be distinct from other subtypes of the disease with a KRAS mutation occurring in 40%-50% of patients. Other gene abnormalities including HER2 amplification in ∼19% also occur. This raises the possibility of the use of targeted molecular therapies which with molecular analysis of individual patient tumours could form the basis of a future clinical trial. It is, however, clear that if trials are to be conducted in this rare subtype of disease, they will need to be truly international in nature and carefully designed, possibly using an adaptive stepwise approach and will require an appropriate level of funding with a realistic assessment of likely recruitment. Associated translational research will clearly be essential.

Correlating Surrogate Endpoints with Overall Survival at the Individual Patient Level in BRAFV600E-Mutated Metastatic Melanoma Patients Treated with Vemurafenib.

Surrogate endpoints are needed that correlate with overall survival (OS). We analyzed individual patient tumor data from a phase III trial of vemurafenib versus dacarbazine (BRIM3) to identify criteria for tumor measures that correlated with OS. Correlates were validated using a separate data set from a phase II trial of vemurafenib (BRIM2). Deidentified tumor measurements and OS data from BRIM3 and from BRIM2 were analyzed. Target tumor measurement data and nontarget tumor data were available from pretreatment, weeks 6,12, and every 9 weeks thereafter. In the BRIM3 data set, associations of OS with both early tumor response (first 12 weeks) and time to progression (TTP) were assessed. Different definitions of response and progression were explored. Findings were validated using the BRIM2 data set. Thresholds of early response were explored ranging from any degree of tumor shrinkage to 100% tumor shrinkage. Correlation was weak at all thresholds tested. TTP, however, was more strongly correlated with OS. The strongest correlation was seen when progression was defined as ≥50% increase in the sum of tumor diameters or appearance of new tumors. This was confirmed by similar analyses in the BRIM2 cohort. TTP defined as ≥50% increase in the sum of tumor diameters or appearance of new tumors was more strongly associated with OS than early tumor shrinkage in melanoma patients treated with RAF inhibitor. In future trials, consideration should be given to replacing response rate with TTP or PFS as preferable clinical endpoints in early-phase studies.

Highlights of This Issue

Legat and colleagues report a clinical trial in 16 advanced-stage melanoma patients vaccinated with five tumor peptides, Montanide and IMP321/LAG-3Ig, a non-TLR agonist with interesting adjuvant properties including dendritic cell activation. The treatment was well tolerated. Combinatorial immune monitoring revealed multiple tumor antigen-specific CD8 and CD4 T-cell responses in most patients, supporting the further use of these vaccine components for the development of combination immunotherapies.Zabor and colleagues analyzed individual patient tumor data from a phase III clinical trial of vemurafenib versus dacarbazine in melanoma patients to identify criteria for tumor measures that correlated with OS. Correlation between OS and early response was weak no matter how the response was defined; however, a strong correlation was seen between OS and progression when defined as ≥50% increase in sum of tumor diameters or appearance of new tumors. These data suggest that tumor progression is a better surrogate endpoint for OS than tumor response, such as RECIST.Zhuang and colleagues studied whether targeting IL-1a inhibits NF-κB activity and suppresses PDAC cell growth. Previously, studies show that KRASG12V induces expression of IL-1a in genetically engineered mouse models that express mutant KRAS and p53 and in cell lines expressing KRASG12V with p16 knockdown. The authors also observed a correlation between mutant KRAS and IL-1a expression in PDAC patient samples. The experimental evidence suggests a combination of gemcitabine and IL-1 receptor antagonist can reduce PDAC cell growth by inhibiting IL-1a-induced NF-κB activity, thus suggesting a new therapeutic approach for PDAC.Poulain and colleagues analyzed the genomic landscape of CXCR4 somatic mutation in Waldenström macroglobulinemia (WM) combining deep sequencing and SNP array. The study showed existence of intraclonal (variation in coexpression of MYD88 and CXCR4 mutations) and interclonal (mutually exclusive BCR and CXCR4 mutations in MYD88L265P WM) heterogeneity of the molecular spectrum of CXCR4 mutation. Furthermore, CXCR4 mutated WM displayed a specific clinicobiologic and genomic and transcriptomic signature. This dataset further suggests the genomic heterogeneity of WM beyond the current indolent versus symptomatic classification.

Abstract P4-08-04: Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine

Abstract Background: Treatment of BC is conventionally based on the presence/absence of ER/PR or HER2 status of the primary tumor. We have enriched this approach by including major genetic and proteomic changes in tumors of individual patients in order to develop a better treatment-rationale based on an alteration driven signaling algorithm. Methods: Genomic and proteomic data from 75 BC patients seen in our center were retrospectively analyzed. Patients were re-biopsied after consultation and samples were characterized (IHC for ER, PR, and HER2; FFPE samples for genomic [Foundation Medicine] and proteomic analyses [Theranostics]). In vivo studies were conducted using xenograft models. Results: Although alterations of PIK3CA, PIK3R1, AKT, PTEN, MDM2, MDM4, TSC1, mTOR and RICTOR are most frequently observed in our patients, there is a distinct pattern of alteration(s) of the PI3K pathway genes in different subtypes of BC. A total of 76 genes were altered in 48 ER+BC patients. In 79% of ER+BC patients the above mentioned PI3K pathway genes were altered. Analyzing the set of alterations of genes in individual patients, we observed that within these 48 patients 25% exhibited alterations in more than one node of the pathway; the most common combination (alterations) being the amplification/mutation of PIK3CA with the amplification of MDM2/4 genes. The percentage of patients belonging to HER2+ & TNBC exhibiting similar alterations in the PI3K pathway genes were significantly lower (∼40%). Our previous in vivo studies demonstrated that GDC-0980 and BEZ235 enhanced the antitumor activity of ABT888 plus carboplatin in TNBC or trastuzumab in HER2+ BC respectively and blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, pS6RP and CD31. Mechanistically the action of the PI3K-mTOR pathway targeted drug(s) was tested using cell line based models of BC subtypes pertaining to their respective genomic alterations. A combination of a pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) blocked proliferative signals and enhanced apoptosis (cleaved caspase3) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells respectively as demonstrated by WB, flow cytometry, cell proliferation, viability and cytotoxicity assays. A recent study demonstrated that exposure to chemotherapy induced a phenotypic shift or cell state transition towards a transient CD44Hi/CD24Hi chemotherapy-tolerant state, leading to the activation of downstream non-receptor tyrosine kinase signaling towards an emerging adaptive resistance (Goldman et al., Nature Comm. 2015). Hence drug combination(s) are being tested for their effect on CD44/CD24 expression levels, results of which will be presented in the meeting. Conclusion: Plotting the genetic alterations from the patient on the signaling landscape will be useful in cracking the code leading to improved treatment options. Patient specific in-depth plotting of genetic alterations of the PI3K-mTOR pathway and the relevance of these alterations in the context of (1) mechanisms of PI3K-mTOR pathway targeted drugs and (2) cell signaling are critical in determining choice of drugs in BC subtypes. Citation Format: Carlson JH, Krie A, Williams C, Sun Y, Lin X, Williams K, Klein J, Friedman L, De P, Dey N, Leyland-Jones B. Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-04.

Abstract OT2-03-02: DI study: Decision impact of the NanoString® Technologies Prosigna™ in early breast cancers

Abstract Backgrounds: More than a decade of research, clinical studies, and peer reviewed publications support the value of molecular subtyping based on gene expression analyses to assess prognosis and treatment options for patients with early-stage breast cancer. Therefore, genomic assays are now being introduced to supplement the conventional diagnostic tools. Prosigna is a standardized test that measures the expression levels of 50 classifier genes in formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue samples and provide a subtype classification based on the fundamental biology of individual patient's tumor (referred to as molecular subtyping), as well as a prognostic score (referred to as risk of recurrence (ROR) score) that predicts the probability of cancer recurrence over 10 years. The primary objective of this study is to assess the extent to which Prosigna affects the medical oncologist's treatment recommendations regarding adjuvant chemotherapy and actual treatments received for patients with early-stage breast cancer. Changes will include hormonal therapy alone, hormonal therapy plus chemotherapy, and changes in types of chemotherapy if chemotherapy was recommended before and after the test. Secondary objectives will be to elicit information on investigators' confidence in the recommendations before and after the test, and by cancer recurrence risk groups, rate of chemotherapy related adverse events stratified by administration of chemotherapy, and patients' decisional conflict status, anxiety levels, and functional status before and after Prosigna results. Multicentric prospective study. Prosigna will be performed on operative piece for all consecutively postmenopausal women matching the inclusion criteria and having signed an informed consent. Data on patient demographics, disease status, intended cancer-specific postoperative management before and after the test, tests results, investigators and patients' confidence in the treatment and in the test, will be recorded in the inclusion visit, after the tests results and 6 month post-assay. Inclusion criteria: Postmenopausal patients with resected node-negative, estrogen-receptor-positive, HER2-negative (by the local laboratory) early-stage invasive breast cancer (T1-T2, N0, pN0 (i+), pN1 (micrometastatic), M0), able to give consent, eligible for treatment of breast cancer with adjuvant chemotherapy and with ECOG performance status of 0 or 1. Statistical methods: The clinical and demographic characteristics of the study sample will be described using mean, median, standard deviation, and range for continuous/ordinal variables and frequency and proportion for categorical variables. Bivariate plots and crosstabs will be performed to inspect bivariate associations between variables. The proportion of patients for whom the physicians' choice of treatment changed from baseline to follow-up will be calculated along with the 95% confidence interval. The change in investigator confidence in treatment recommendations before and after Prosigna results were known will be analyzed by calculating the mean and 95% CI for the question regarding whether a physician is more confidence in treatment recommendation after ordering Prosigna. 47 patients have been included on 200 scheduled. Citation Format: Lerebours F, Hequet D, Guinebretière J-M, Roulot A, Callens C, Gentien D, Penault-LLorca F, Zilberman S, Salmon R, Foa C, Berseneff H, Huchon C, Katz G, MacDonald M, Morel P, Bieche I, Dubot C, Rouzier R. DI study: Decision impact of the NanoString® Technologies Prosigna™ in early breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT2-03-02.

Abstract A113: iVacALL: A personalized peptide-vaccination design platform for pediatric acute lymphoblastic leukemia patients based on patient-individual tumor-specific variants

Abstract We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Standard chemotherapy is a successful treatment in 80% of patients, only about 20% develop a relapse, however these patients have a dismal prognosis. Prevention of relapse after first-line chemotherapy or stem cell transplantation (SCT) is therefore mandatory. Accumulation of somatic mutations is one characteristic feature of malignant cells. These single nucleotide variants (SNVs) can lead to altered amino acid sequences of the translated proteins, which in turn can be presented as antigenic peptides on HLA molecules of the malignant cells. Mutated peptides represent ideal T cell targets as they are true neoantigens, specific for the tumor, and should not have been subject to central tolerance selection mechanisms. A peptide vaccination composed of mutated T cell epitopes specific for individual patient tumors is therefore a promising approach to prevent relapse in high-risk patients. For this purpose we detect nonsynonymous mutations by whole exome and transcriptome sequencing of patient leukemic blasts and normal reference tissue. HLA binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan for the patients' individual HLA type. Whole exome sequencing was performed for 17 c-ALL, 2 cortical T-ALL and 1 pro-B-ALL sample pairs. ALL-specific SNVs, as well as insertions and deletions (InDels) were identified using a comparative bioinformatics pipeline. The determined variants were further validated by deep sequencing in 9/20 patients so far, with an average of 12 (+/- 8) validated mutations per patient. For all patients with validated variants, MHC class I and MHC class II epitopes could be predicted successfully. We applied the platform for 3 patients based on compassionate need and designed individual peptide vaccines. One patient underwent haploidentical SCT with relapsed c-ALL, a second patient received autologous SCT with ependymoma and the third patient got allogeneic SCT with pro-B ALL. In all cases validated mutations could be identified and epitope prediction was performed for MHC I & II binders. The predicted peptides were synthesized and vaccination cocktails were formulated. The vaccination schedule provides 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvant. The vaccination was generally well tolerated. Response to the vaccination was monitored by detection of T cells recognizing the vaccinated peptides occurring over time in peripheral blood of the patients. Monitoring was performed for each vaccination time point by prestimulation with the peptides and subsequent intracellular cytokine staining (ICS) of T cells and FACS analysis. In all 3 patients we could detect a developing CD4+ T cell response against the vaccinated mutated MHC II binding peptides. Whole exome sequencing of pediatric ALL patients is feasible and yields a small amount of validated mutations per patients. However, these few mutations seem sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T cell responses in patients. Moreover, the platform is not limited to ALL/leukemia but can also be applied for solid tumor patients. Citation Format: Armin Rabsteyn, Christina Kyzirakos, Christopher Schröder, Marc Sturm, Christopher Mohr, Mathias Walzer, Ulrike Pflückhahn, Michael Walter, Magdalena Feldhahn, Karoline Laske, Michael Bonin, Martin Ebinger, Stefan Stevanovic, Peter Bauer, Oliver Kohlbacher, Cecile Gouttefangeas, Hans-Georg Rammensee, Rupert Handgretinger, Peter Lang. iVacALL: A personalized peptide-vaccination design platform for pediatric acute lymphoblastic leukemia patients based on patient-individual tumor-specific variants. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A113.