Zuanel Diaz

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor’s biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

Resistance to cancer treatment: the role of somatic genetic events and the challenges for targeted therapies

Therapeutic resistance remains a major cause of cancer-related deaths. Resistance can occur from the outset of treatment or as an acquired phenomenon after an initial clinical response. Therapeutic resistance is an almost universal phenomenon in the treatment of metastatic cancers. The advent of molecularly targeted treatments brought greater efficacy in patients whose tumors express a particular target or molecular signature. However, resistance remains a predictable challenge. This article provides an overview of somatic genomic events that confer resistance to cancer therapies. Some examples, including BCR–Abl, EML4–ALK, and the androgen receptor, contain mutations in the target itself, which hamper binding and inhibitory functions of therapeutic agents. There are also examples of somatic genetic changes in other genes or pathways that result in resistance by circumventing the inhibitor, as in resistance to trastuzumab and BRAF inhibitors. Yet other examples results in activation of cytoprotective genes. The fact that all of these mechanisms of resistance are due to somatic changes in the tumor’s genome makes targeting them selectively a feasible goal. To identify and validate these changes, it is important to obtain biopsies of clinically resistant tumors. A rational consequence of this evolving knowledge is the growing appreciation that combinations of inhibitors will be needed to anticipate and overcome therapeutic resistance.

Recruiting Terminally Ill Patients into Non-Therapeutic Oncology Studies: views of Health Professionals

BackgroundNon-therapeutic trials in which terminally ill cancer patients are asked to undergo procedures such as biopsies or venipunctures for research purposes, have become increasingly important to learn more about how cancer cells work and to realize the full potential of clinical research. Considering that implementing non-therapeutic studies is not likely to result in direct benefits for the patient, some authors are concerned that involving patients in such research may be exploitive of vulnerable patients and should not occur at all, or should be greatly restricted, while some proponents doubt whether such restrictions are appropriate. Our objective was to explore clinician-researcher attitudes and concerns when recruiting patients who are in advanced stages of cancer into non-therapeutic research.MethodsWe conducted a qualitative exploratory study by carrying out open-ended interviews with health professionals, including physicians, research nurses, and study coordinators. Interviews were audio-recorded and transcribed. Analysis was carried out using grounded theory.ResultsThe analysis of the interviews unveiled three prominent themes: 1) ethical considerations; 2) patient-centered issues; 3) health professional issues. Respondents identified ethical issues surrounding autonomy, respect for persons, beneficence, non-maleficence, discrimination, and confidentiality; bringing to light that patients contribute to science because of a sense of altruism and that they want reassurance before consenting. Several patient-centered and health professional issues are having an impact on the recruitment of patients for non-therapeutic research. Facilitators were most commonly associated with patient-centered issues enhancing communication, whereas barriers in non-therapeutic research were most often professionally based, including the doctor-patient relationship, time constraints, and a lack of education and training in research.ConclusionsThis paper aims to contribute to debates on the overall challenges of recruiting patients to non-therapeutic research. This exploratory study identified general awareness of key ethical issues, as well as key facilitators and barriers to the recruitment of patients to non-therapeutic studies. Due to the important role played by clinicians and clinician-researchers in the recruitment of patients, it is essential to facilitate a greater understanding of the challenges faced; to promote effective communication; and to encourage educational research training programs.

Methods for Sample Acquisition and Processing of Serial Blood and Tumor Biopsies for Multicenter Diffuse Large B-cell Lymphoma Clinical Trials

Increasingly, targeted therapies are being developed to treat malignancies. To define targets, determine mechanisms of response and resistance, and develop biomarkers for the successful investigation of novel therapeutics, high-quality tumor biospecimens are critical. We have developed standard operating procedures (SOPs) to acquire and process serial blood and tumor biopsies from patients with diffuse large B-cell lymphoma enrolled in multicenter clinical trials. These SOPs allow for collection and processing of materials suitable for multiple downstream applications, including immunohistochemistry, cDNA microarrays, exome sequencing, and metabolomics. By standardizing these methods, we control preanalytic variables that ensure high reproducibility of results and facilitate the integration of datasets from such trials. This will facilitate translational research, better treatment selection, and more rapid and efficient development of new drugs. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2688–93. ©2014 AACR.

Physician Recruitment of Patients to Non-Therapeutic Oncology Clinical Trials: Ethics Revisited

Tailoring medical treatment to individual patients requires a strong foundation in research to provide the data necessary to understand the relationship between the disease, the patient, and the type of treatment advocated for. Non-therapeutic oncology clinical trials studying therapeutic resistance require the participation of patients, yet only a small percentage enroll. Treating physicians are often relied on to recruit patients, but they have a number of ethical obligations that might be perceived as barriers to recruiting. Concepts such as voluntariness of consent and conflicts of interest can have an impact on whether physicians will discuss clinical trials with their patients and how patients perceive the information. However, these ethical obligations should not be prohibitive to physician recruitment of patients – precautions can be taken to ensure that patients’ consent to research participation is fully voluntary and devoid of conflict, such as the use of other members of the research team than the treating physician to discuss the trial and obtain consent, and better communication between researchers, clinicians, and patients. These can ensure that research benefits are maximized for the good of patients and society.

Abstract 3888: Molecular profiling of sequential biopsies in patients with metastatic colorectal cancer identifies genomic alterations that evolve during first-line therapy and could have therapeutic implications: A prospective study to identify molecular mechanisms of clinical resistance (QCROC-01: NCT00984048).

Therapeutic resistance remains a major obstacle in metastatic colorectal cancer (mCRC) and biomarkers to guide treatment are essential to improving survival and quality of life in mCRC patients. A biopsy-driven prospective study was designed to identify biomarkers and mechanisms of resistance to a standard first-line therapy in patients with mCRC which could be useful in guiding treatment selection (QCROC-01; NCT00984048). We also hoped to recognize molecular changes over time, or resulting from the selection pressure of treatment, which could have implications for subsequent therapy. This study is ongoing and approved at thirteen sites with one-hundred patients enrolled so far. Patients with mCRC receiving FOLFOX (5-fluorouracil, leucovorin and oxaliplatin) with bevacizumab consented to three needle core tumour biopsies at pre-treatment and at the time of resistance. The rate of both patient and physician acceptance of biopsies has steadily risen with time and experience. Serial bloods were also collected for proteomic analysis and circulating tumor DNA. Twenty-five biopsy samples were profiled using exome sequencing (tumor and germ line), RNAseq, low pass genome sequencing and miRNA analysis. Differential gene expression analysis revealed signatures associated with clinical response and resistance when comparing tumours obtained pre- and post-treatment. We detect changes in variant allele fraction including both depletion and enrichment of individual somatic mutations over the course of treatment, the latter of which may indicate subclonal and acquired “driver” mutations that confer therapeutic resistance. A small number of genes show recurrent evidence for changes in clonal enrichment at the time of relapse across multiple patients. These could also represent therapeutic targets for subsequent therapy for these patients, and as such, represent new treatment opportunities. Our findings provide insights into tumor evolution during first-line chemotherapy of mCRC that may hold clues to optimize current first-line therapeutic decision making and identifies potential target pathways for second-line stratification of patients. This study is part of the Canadian Colorectal Cancer Consortium which is a multi-site collaboration funded by the Terry Fox Research Institute and le fonds de recherche du quebec - sante. Citation Format: Suzan McNamara, Ryan Morin, Mathilde Couetoux du Tertre, Rosemary McCloskey, Rebecca Johnston, Daniel Fornika, Benoit Samson, Bernard Lesperance, Thierry Alcindor, Yoo-Joung Ko, Richard Dalfen, Eve St-Hilaire, Lucas Sideris, Felix Couture, Hans Prenen, Sabine Tejpar, Ronald Burkes, Andre Constantin, Errol Camlioglu, Adriana Aguilar, Adrian Gologan, Benoit Tetu, Celia M. Greenwood, Cyrla Hoffert, Samia Qureshi, Zuanel Diaz, Maud Marques, Micheal Witcher, Therese Gagnon-Kugler, Petr Kavan, Gerald Batist. Molecular profiling of sequential biopsies in patients with metastatic colorectal cancer identifies genomic alterations that evolve during first-line therapy and could have therapeutic implications: A prospective study to identify molecular mechanisms of clinical resistance (QCROC-01: NCT00984048). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2015-3888

Abstract 64: Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials.

Introduction: Personalized medicine strategies targeting specific aberrations unique to an individual tumor, are promising to become a new paradigm in the fight against cancer. Such tailored approaches rely on the thorough profiling of malignant tissues by high-throughput technologies followed by selection of appropriate treatment targeting tumor-specific aberrations. One fundamental challenge, whose resolution is necessary for any successful personalized medicine endeavor, is isolation of useful amounts of good quality molecular material for reliable tumor characterization in downstream assays. Here, we describe our standardized protocol for biopsy collection and results from a multi-center phase II clinical trial investigating the use of panobinostat with or without rituximab in relapsed diffuse large B-cell lymphoma (NCT01238692). Results: Biopsies are performed with patient consent, before treatment initiation and after 15 days of treatment, in patients with safely accessible lesions. Four needle core biopsies are collected at each time point using standard operating procedures to limit pre-analytical variability. Of these, one is preserved in formalin for immunohistochemical (IHC) analyses, while the remaining three are pooled together in RPMI 1640 media. These samples are sent to a central laboratory for further processing. The needle cores in RPMI 1640 undergo B-cell purification using a negative selection kit (StemCell Technologies). Isolation of tumor RNA and DNA is performed using a commercially available kit (Qiagen). Of the 23 patients currently enrolled, 16 (70%) have had a pre-treatment biopsy performed and of these, 7 (44% of those biopsied, 30% of all patients) have also had a post-treatment biopsy, demonstrating the feasibility of tissue collection at participating sites. We assess the effect of transport on the quality and yield of RNA and DNA as well as differences in quality and yield between pre-treatment biopsy vs. biopsies at day 15. The material isolated from these biopsies is evaluated for suitability in downstream applications, namely IHC, gene expression microarray and DNA exome sequencing. Conclusions: The development of standard operating procedures for the collection and processing of biospecimens is essential to control for pre-analytical variability inherent to multicenter trials. Our experience shows that this is both feasible and crucial to understanding of tumor molecular profile changes associated with treatment. Our protocol allows extraction of RNA and DNA of sufficient quality and quantity to permit downstream multi-dimensional analysis. Patient acceptance of research biopsies has been high although the rate of pre-treatment biopsies is greater than that of post-treatment biopsies. Finally, our work demonstrates that the timing of post-treatment biopsies is critical. Citation Format: Torsten H. Nielsen, Zuanel Diaz, Rosa Christodoulopoulos, Lu Yao, Samia Qureshi, Naciba Benlimame, Errol Camlioglu, Michael Crump, Ryan D. Morin, Nathalie Johnson, Tina P. Haliotis, Wilson H. Miller, Sarit Assouline, Koren K. Mann. Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 64. doi:10.1158/1538-7445.AM2013-64

Abstract B24: De novo and acquired resistance to first-line standard therapy in colorectal cancer: from cell lines to metastatic tumors

Introduction: Personalized medicine (PM) is a concept that has raised high expectations amongst scientists, clinicians, and patients. An emerging approach is to examine tumor biopsy material for genomic changes that are known targets of currently available therapeutic agents, with the assumption that a clinical benefit will be observed if the target is inhibited. While striking anecdotal reports are predictable from this approach, the clinical impact of these agents is limited by the inevitable development of therapeutic resistance. Our focus is on the design of parallel research programs using both in vitro and in vivo strategies, in an effort to delay or inhibit resistance. We present here preliminary data for a signature of resistance to standard first-line treatment - fluorouracil, folinic acid, oxaliplatin and bevacizumab (FOLFOX/B) using cell line models of resistance to this regimen. In parallel, we are conducting a prospective study to identify biomarkers of clinical resistance to first-line therapy in patients with metastatic colorectal cancer (CRC) (NCT00984048). Methods: Ten established CRC cell lines were treated with FOLFOX/B and categorized as resistant or sensitive based on IC50 values. In parallel, patients who consented to an initial biopsy and one at disease progression following an initial response were identified as intrinsically resistant or as having acquired resistance during treatment. CRC cell lines that were initially sensitive were rendered resistant to mimic the acquired resistance in patients, by serial passages with gradual increases in concentration of the combination regimen. We compared microarray data from three sensitive and three resistant cell lines. Results: We found a different expression pattern from microarray data comparing sensitive and resistant cell lines, thereby indicating a potential signature of resistance to FOLFOX/B. Interestingly, we found that the Src family kinase Lyn was overexpressed in resistant cells lines. Treating cells with non-cytotoxic concentration of dasatinib, a dual Src family kinase and Abl inhibitor, sensitized both the parental sensitive cells and the cells with acquired resistance to FOLFOX/B, thereby suggesting that combination treatment with dasatinib may be effective in delaying or inhibiting resistance. We have thus far collected needle core biopsies from liver metastases from forty patients who agreed to partake in this multi-center trial. Eligible patients have confirmed metastatic CRC, measurable disease, and consent to three needle-core biopsies (NCBs) of a non-resectable liver metastasis before treatment and at resistance, as well as serial blood collection throughout the study. Using standard operating procedures developed for this trial, we were able to both preserve morphology and obtain high-quality genomic material from biopsy tissue. We will determine if the resistance signature and overexpression of Lyn observed in the resistant CRC cell lines are similarly demonstrated in patients that were intrinsically resistant to FOLFOX/B. Conclusions: We have designed parallel in vitro and in vivo experiments to study resistance to standard first-line treatment for mCRC. These studies provide insight on metastatic signatures of resistance and suggest combination therapies to delay or inhibit therapeutic resistance in patients.

Abstract 5534: Building the organization framework for biopsy-driven translational research: The Quebec Clinical Research Organization in Cancer (Q-CROC) experience

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The success of personalized medicine in oncology relies on translational research efforts to identify biomarkers that will influence clinical management. The discovery and validation of biomarkers is a concerted effort requiring an organizational framework that is often underestimated. The Quebec Clinical Research Organization in Cancer (Q-CROC) consortium is a multi-disciplinary and multi-institutional group of scientists and clinicians devoted to integrating and enhancing translational and clinical research capacity in Quebec. We describe here the organizational framework driving a multicenter, prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer ([NCT00984048][1], Q-CROC-01). Results: The Q-CROC consortium has put in place an organizational infrastructure to support the activities and operations of its translational projects. We identified and addressed several critical issues during the course of the Q-CROC-01 translational project that were also common to our subsequent biomarker-driven trial in lymphoma (Q-CROC-02, [NCT01238692][2]) and breast cancer (Q-CROC-03, [NCT01276899][3]). Examples of these issues include: (i) feasibility and burden of tissue collection at participating sites, (ii) limiting pre-analytical variability in blood and tissue specimens for functional downstream applications, (iii) verification of tumor content on biopsy specimens, (iv) tracking sample flow, (v) integration of clinical data with discovery platforms, and (vi) engaging participation throughout all steps of the project. In part to address the above issues, we established five operational Cores: clinical, biobank, biospecimen processing, bioanalytical and bioinformatic. A further challenge was the integration between these Cores, who for the most part operated in silos. We observed that a critical element to unify all components of the consortium was a scientific project management team, consisting of dedicated individuals regularly interacting with each Core to ensure that objectives were aligned and deliverables were met. This academic framework for translational research may be comparable to that of multicenter clinical trials undertaken by industry, but some challenges, including financial and time constraints, data sharing and IP agreements, and engagement of its members, may be more palpable in the academic setting. Conclusion: Infrastructure science is underestimated and under-reported in translational cancer research and is crucial to the success of any large-scale biomarker discovery effort. Our experience with three multi-institutional biomarker-driven trials is that progress hinges upon the availability of an infrastructure that is not only the sum of its parts but that provides a concrete link between each component. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5534. doi:1538-7445.AM2012-5534 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00984048&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01238692&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01276899&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom

Abstract 3389: Determining optimal conditions for collection and processing of metastatic liver biopsies collected for a multicenter, prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The biomarker discovery process requires patient tissue samples from which histology is verified and high-quality genomic material is isolated. Several methods have been developed to either preserve tissue morphology or extract sufficient quality and quantity of RNA and DNA for downstream discovery efforts. As clinical trials incorporate patient biopsies for biomarker discovery or validation, it is becoming increasingly important to ensure quality material and identify methods that allow for preservation of morphology and stabilization of molecular content concurrently. We assessed, in liver needle-core biopsies, different sampling, fixation, and genomic isolation methods to maintain morphology and obtain high-quality genomic material for a multi-center prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer. Four sampling methods (snap freezing, RNAlater, frozen RNAlater, formalin), two different fixation protocols for histological studies (10% formalin, RNAlater followed by OCT embedding and freezing) and two RNA isolation procedures (Triazol and AllprepDNA/RNA isolation) were evaluated. Results: Keeping in mind site feasibility, we report that the ideal condition to both preserve morphology and obtain high-quality genomic material of patient liver biopsy samples is to collect biopsies in RNAlater for shipping to a Central Pathology core, followed by washing with cold PBS (on dry ice) to permit proper RNA preservation during OCT embedding and cryostat sectioning for histological verification. Simultaneous extraction of DNA and RNA from the same biopsy core yields nucleic acids of optimal concentration and quality for downstream genomic applications. Conclusion: The collection of biospecimens using pre-determined protocol-specific standard operating procedures (SOPs) is essential to control for pre-analytical variability inherent to multicenter trials. Furthermore, histological control of percent tumor cells in each biopsy is absolutely necessary to ensure optimal representation of tumor (>70%) in the specimen. The above conditions were used in the multicenter Q-CROC-01 study ([NCT00984048][1]), where three needle core biopsies are collected from liver metastases of patients with colorectal cancer. One biopsy is collected in formalin and is set aside for downstream immunohistochemistry experiments. Two biopsies are collected in RNAlater and verified for histology. If they pass quality control, both DNA and RNA are isolated concurrently and sent to discovery platforms (DNA: array comparative genomic hybridization (aCGH), methylation profiles, RNA: gene expression profiles, RT-PCR, microRNA profiles, alternative splicing profiles). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3389. doi:1538-7445.AM2012-3389 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00984048&atom=%2Fcanres%2F72%2F8_Supplement%2F3389.atom