Alan Spatz

The validity of circulating microRNAs in oncology: Five years of challenges and contradictions

MicroRNAs (miRNAs) in circulation have received an increasing amount of interest as potential minimal invasive diagnostic tools in oncology. Several diagnostic, prognostic and predictive signatures have been proposed for a variety of cancers at different stages of disease, but these have not been subjected to a critical review regarding their validity: reproducible identification in comparable studies and/or with different platforms of miRNA detection. In this review, we will critically address the results of circulating miRNA research in oncology that have been published between January 2008 and June 2013 (5.5 years), and discuss pre‐analytical challenges, technological pitfalls and limitations that may contribute to the non‐reproducibility of circulating miRNA research.

Loss of microRNA-200a and c, and microRNA-203 expression at the invasive front of primary cutaneous melanoma is associated with increased thickness and disease progression

Loss of E-cadherin expression in melanoma correlates with increased tumor thickness and reduced disease-free survival. The molecular mechanisms underpinning its differential expression in melanoma tissue remain elusive. MicroRNAs (miRNAs) have been implicated in tumor progression and regulation of E-cadherin expression. Here, we demonstrate a significant correlation between tumor thickness and loss of expression of miR-200a, miR-200c, and miR-203 in a series of 23 frozen primary melanomas, where it was confirmed in two subsequent validation series (series 1: six nevi, 15 primary melanomas, and 16 metastases; series 2: 11 matched pairs of primary melanomas and metastases). Decreased levels of miR-200a, miR-200c, and miR-203 correlated with increasing thickness in the combined validation series (P = 0.024, 0.033, and 0.031, respectively). In addition, progressive loss of miR-200a expression with disease progression was observed in series 1 (P < 0.001) and in series 2 (P = 0.029). MiR-200 in situ hybridization and E-cadherin immunohistochemistry demonstrated reduced expression of both at the deep invasive margin of the tumor. Furthermore, a functional validation study using an anti-miR200 strategy demonstrated that loss of miR-200 expression in melanoma cell lines reduced E-cadherin expression. Collectively, our data point towards an important role for miR-200 and miR203 expression in regulating E-cadherin during melanoma progression.

Barriers and facilitators of adherence to medical advice on skin self-examination during melanoma follow-up care

BackgroundMelanoma is the fastest growing tumor of the skin, which disproportionately affects younger and middle-aged adults. As melanomas are visible, recognizable, and highly curable while in early stages, early diagnosis is one of the most effective measures to decrease melanoma-related mortality. Skin self-examination results in earlier detection and removal of the melanoma. Due to the elevated risk of survivors for developing subsequent melanomas, monthly self-exams are strongly recommended as part of follow-up care. Yet, only a minority of high-risk individuals practices systematic and regular self-exams. This can be improved through patient education. However, dermatological education is effective only in about 50% of the cases and little is known about those who do not respond. In the current literature, psychosocial variables like distress, coping with cancer, as well as partner and physician support are widely neglected in relation to the practice of skin self-examination, despite the fact that they have been shown to be essential for other health behaviors and for adherence to medical advice. Moreover, the current body of knowledge is compromised by the inconsistent conceptualization of SSE. The main objective of the current project is to examine psychosocial predictors of skin self-examination using on a rigorous and clinically sound methodology.Methods/DesignThe longitudinal, mixed-method study examines key psychosocial variables related to the acquisition and to the long-term maintenance of skin self-examination in 200 patients with melanoma. Practice of self-exam behaviors is assessed at 3 and 12 months after receiving an educational intervention designed based on best-practice standards. Examined predictors of skin self-exam behaviors include biological sex, perceived self-exam efficacy, distress, partner and physician support, and coping strategies. Qualitative analyses of semi-structured interviews will complement and enlighten the quantitative findings.DiscussionThe identification of short and long-term predictors of skin self-examination and an increased understanding of barriers will allow health care professionals to better address patient difficulties in adhering to this life-saving health behavior. Furthermore, the findings will enable the development and evaluation of evidence-based, comprehensive intervention strategies. Ultimately, these findings could impact a wide range of outreach programs and secondary prevention initiatives for other populations with increased melanoma risk.

Cytology cell blocks are suitable for immunohistochemical testing for PD-L1 in lung cancer

Background PD-L1 immunohistochemistry (IHC) testing is usually carried out on tissue blocks from core needle biopsy or surgical resections. In this study, we assessed the feasibility of using cytology cell blocks for PD-L1 IHC assay. Methods A total of 1419 consecutive cases of non-small-cell lung cancer (NSCLC), including 371 cytology cell blocks, 809 small biopsies, and 239 surgical specimens, were included in the study. The cytology cell blocks were prepared with formalin only, methanol/alcohol only or both. PD-L1 expression was examined by staining with Dako PD-L1 IHC 22C3 pharmDx kit. A Tumor Proportion Score (TPS) was categorized as <1%, 1%-49% and ≥50% tumor cells. A total of 100 viable tumor cells were required for adequacy. Results Of the cytology cell blocks, 92% of the specimens had an adequate number of tumor cells, not significantly different from small biopsies. The rate of TPS ≥50% differed between sample types and was observed in 42% of cytology cell blocks versus 36% of small biopsies (P = 0.04), and 29% of surgical resections (P = 0.001). The fixative methods did not affect the immunostaining, with overall PD-L1 high expression (TPS ≥50%) rates of 42% in formalin-fixed specimens versus 40% in specimens with combined fixation by methanol/alcohol and formalin (NS). The PD-L1 high expression rate was not associated with EGFR, ALK or KRAS molecular alterations. Higher stage (IV) was associated with higher PD-L1 TPS (P= 0.001). Conclusion Our results show that when the TPS ≥50% is used as the end point, PD-L1 IHC performs well with cytology cell blocks. Cell blocks should be considered as a valuable resource for PD-L1 testing in advanced NSCLC. The clinical significance of higher PD-L1 IHC scores in cytology specimens needs to be evaluated prospectively.

Congenital Melanocytic Nevi, Associated Neoplasms, and Pediatric Melanoma

By definition, the term congenital nevus connotes a melanocytic nevus present at birth or appearing within the first year of life (Table 6.1) [1–11]. However, congenital melanocytic nevi (CMN) are also defined by striking differences from common acquired nevi and the implications of these differences. The presence of a CMN on an individual often raises important medical, cosmetic, and psychosocial issues. The extent to which the latter issues become important is in general directly related to overall size or surface area or “nevus burden” of a CMN. Consequently, larger varieties of CMN, i.e., large and giant CMN, pose the greatest threat to patients in terms of risk for melanoma and potentially fatal central nervous system involvement by these nevi (neurocutaneous melanosis).

Abstract 1977: The protein phosphatase 2 subunit PR48 is a novel melanoma tumor suppressor gene.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cutaneous melanoma cell proliferation is a strong prognostic factor for distant metastasis free survival (DMFS). In the current study we tested if aberrant expression of a regulator both DNA replication and cell cycle progression, i.e. the protein phosphatase 2A subunit PR48, could be a driver of melanoma proliferation and progression. Quantitative analysis of PR48 mRNA expression revealed a strong correlation between low levels of expression and poor DMFS in a multivariate analysis (n=49, p=0.0007). In line with this, no or low PR48 protein expression was associated with poor overall survival in three independent samples sets of melanoma (n=339, P<0.001), and reduced PR48 expression correlated with increased proliferation (P=0.0023). Functional analysis using over expression and knock down strategies in melanoma cell lines demonstrated that PR48 alters cdc6-cdt1 complex formation, regulates pRb phosphorylation status, and entry into the S-phase of the cycle progression. Ectopic PR48 expression decreased tumorigenicity of melanoma cells, and shRNA-mediated reduced PR48 expression increased tumor take and growth in nude mice. Our data demonstrate that PPP2R3B is a novel tumor suppressor gene in melanoma, whose loss of expression contributes increased proliferation and poor survival. Citation Format: Leon CL van Kempen, Jonathan Jarry, Mounib Elchebly, Margaret Redpath, Per-Henrik Edqvist, Fredrik Ponten, Richard Scolyer, Dirk Schadendorf, Celia M.T. Greenwood, Joost van den Oord, Alan Spatz. The protein phosphatase 2 subunit PR48 is a novel melanoma tumor suppressor gene. [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 1977. doi:10.1158/1538-7445.AM2013-1977

Abstract CT098: Phase 1 first-in-human study of anti-clusterin antibody AB-16B5 in patients with advanced solid malignancies

Background. Secreted clusterin (sCLU) promotes survival and stimulates epithelial to mesenchymal transition (EMT) in cancer cells, leading to tumor invasion, metastasis and chemoresistance. AB-16B5 is a humanized IgG2 mAb specific for the EMT-inducing region in some isoforms of sCLU produced by tumor cells, with much less affinity for sCLU found in healthy individuals. Pre-clinical models showed that AB-16B5 inhibits EMT and displays synergy with chemotherapeutic agents. We performed a first-in-human phase I trial to assess safety and tolerability of AB-16B5 and to determine a recommended dose for phase II studies. Methods. Patients (pts) with evaluable and refractory advanced solid malignancies were eligible with adequate organ and bone marrow functions and an ECOG of 0-2. AB-16B5 was administered as a 60-min IV weekly infusion (21-day cycles). Dose escalation followed an accelerated scheme for the 1.5 and 3 mg/kg dose levels and a standard 3 + 3 design for subsequent levels (6, 9 and 12 mg/kg). All pts underwent collection of blood samples for PK analysis. Pre-treatment and post-treatment (end of cycle 2) tumor biopsies were collected if feasible in pts dosed at 9 and 12 mg/kg. Biopsies were used to evaluate EMT status and to investigate the presence of AB-16B5. Radiological imaging was performed every 6 weeks for the first 8 cycles, then every 9 weeks thereafter. Results. 15 pts (13 carcinomas of various origins, 1 melanoma and 1 sarcoma) were enrolled from May 2015 to October 2016. Pts received between 1 and 53 weekly doses (median: 9 doses). The most frequently reported adverse events (AEs, all causalities) were nausea, abdominal pain, constipation, vomiting, back pain, pruritus and dyspnea. Most of the AEs were of Grade 1 or 2. Among the AEs ≥ Grade 3, only 2 (Grade 3 infusion related reaction and rash) were judged related to AB-16B5. No dose-limiting toxicities were identified during the first cycle of treatment for any pt. 5 serious AEs were reported (sepsis, pyrexia, dyspnea, intra-abdominal hemorrhage and main stem bronchus obstruction), none of which were judged related to the study treatment. PK analysis across all dose levels confirmed that systemic exposure to AB-16B5 increased in a dose proportional manner. The presence of AB-16B5 at the tumor site was confirmed in all 5 pts where a post-treatment tumor lysate could be generated. Biomarker analysis in paired tumor biopsies provided some evidence for EMT inhibition as seen by increased E-cadherin expression after treatment with AB-16B5 in 2 pts. In one of these 2 pts with advanced gastric cancer, this was also accompanied by a loss of vimentin expression. This pt had stable disease (SD) with clinical benefit and remained on treatment for 24 weeks. Another pt with follicular thyroid cancer had SD for almost 1 year. Conclusion. Weekly infusions of AB-16B5 are well tolerated up to 12 mg/kg. This dose level is the recommended phase II dose for future trials. Early correlative studies on tumor tissue provide evidence of molecular modulation of the tumor environment in humans. Clinical trial registry number: NCT02412462 Citation Format: Cristiano Ferrario, Julie Laurin, Leon Van Kempen, Caroline Lambert, Alan Spatz, Oksana Markova, Gerald Batist, Adrian Langleben, Mario Filion, Jacques Jolivet. Phase 1 first-in-human study of anti-clusterin antibody AB-16B5 in patients with advanced solid malignancies [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 CT098. doi:10.1158/1538-7445.AM2017-CT098

Molecular testing in Cutaneous Melanoma

Cutaneous melanoma is associated with strong prognostic phenotypic features such as gender, Breslow’s thickness, and ulceration although the biological significance of these variables is largely unknown. It is likely that these features are surrogates of important biological events rather than promoting directly melanoma progression. High-throughput expression studies have helped deciphering the mechanism and role of melanoma cells replication. Another important phenotypic variable in melanoma is the presence of histopathological features of chronic sun exposure damage. The presence or absence of solar elastosis correlates with the rate and the type of BRAF mutations. Genetic defects found in cutaneous melanoma involve most often receptor tyrosine kinase and downstream kinase pathways. Thus, the mitogen-activated protein kinase pathway and the PI3K pathway are activated in most melanomas. BRAF has a recurrent gain-of-function mutation V600E in about 7 % of all cancers and 43–50 % of melanomas. Anti-BRAF therapy has been the first targeted therapy of metastatic melanomas and completely changed the therapeutic landscape. One of the most important unmet needs in the melanoma field is to shift from prognostication based on artificial segmentation of continuous variables to continuous likelihood scores for diagnosis, prognosis, and response to treatment predictions. This implies to use shared biomarkers and clinical databases.

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