The ten differential proteins identified by SRM have biological significance in cancer progression. The final signature of 14 proteins identified by SRM could be used to identify AJCC stage III melanoma patients likely to have poor outcomes who may benefit from adjuvant systemic therapy.
Abstract There is increasing evidence that the aberrant expression of cancer-testis (CT) antigens - a family of ca. 150 proteins that are both auto-immunogenic and mainly restricted to tumors in various types of human cancers - makes them attractive immunotherapy targets, as well as possible cancer diagnostic markers. The underlying hypothesis of our study was that there were measurable differences in auto-antibody repertoires between pre- and post-treatment cancer patient samples, which would correlate with the likelihood, nature and extent of response of these individual patients to a given therapeutic treatment. We carried out a retrospective serological study of primary and secondary autoimmune responses of selected malignant melanoma cancer patients prior to and following kinase inhibitor treatment (either dabrafenib, vemurafenib, trametinib, or dabrafenib and trametinib in combination), using archived human serum samples. Kinase inhibitor treatment has been seen to be associated with increased immune cell infiltrates, raising the possibility that clinical benefit in some of these patients may have, in part, an immune basis, thus justifying the use of an immune monitoring platform for evaluating a treatment approach that is not primarily intended to target immune mechanisms. Therefore, our goals were to explore the utility and general applicability of our recently developed and validated novel cancer antigen protein microarray platform in the cancer immunology field. In addition, we sought to cross-correlate this protein microarray data with both patient clinical information and in vitro T-cell re-stimulation assays for a selected subset of patients, as a means of determining the biological significance of this data. We observed a large number (81% of the cohort) of autoantibody responses – statistically significant changes in antibody titers – to treatment, and successfully identified well-established therapeutic targets – NY-ESO-1 and MAGEB1 – which were the two most abundantly expressed proteins amongst those tested, present across 61% and 56% patients, respectively. Furthermore, our CT antigen array data suggested preliminary evidence of direct correlation between our observed autoantibody responses and the clinically reported ones across a subset of patients (approximately 35% of the cohort). However, it appears plausible that other factors, which we have yet to determine, may be contributing towards a lack of correlation amongst the remaining cohort. Such factors may include patient-specific differences such as tumor-induced systemic immune suppression or evasion mechanisms, the absence or presence of tumor infiltrating lymphocytes and the favorability of the tumor microenvironment. In conclusion, we showed that our novel protein microarray platform represents a sensitive, high-throughput and readily customizable means to detect and quantify the presence of large panels of cancer-specific human autoantibodies in serum, obtaining consistently robust, high quality and reproducible data, and demonstrating its potential feasibility and inferred biological significance. Applications of this tool include prospective use in identifying novel diagnostic, disease progression, prognostic, treatment resistant and even predictive biomarkers, which could aid in the detection and management of cancer. Most importantly, these could lead to the discovery of novel cancer therapeutic targets, patient stratification biomarkers prior to treatment, and a means to monitor cancer patient responses to treatment. Citation Format: Jessica Duarte, Janique Peyper, Katherine Woods, Simon Tsao, Georgina Long, Richard Kefford, Richard Scolyer, Jonathan Cebon, Jonathan Blackburn. Protein microarrays for the immunological profiling of melanoma. [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 A024.
9000 Background: Nicotinamide (vitamin B3) enhances DNA repair and prevents cutaneous immune suppression after ultraviolet (UV) radiation exposure. It reduces photocarcinogenesis in mice, and human non-melanoma skin cancers (NMSC) in Phase 2 clinical trials. We report the outcomes of the Phase 3 Oral Nicotinamide to Reduce Actinic Cancer (ONTRAC) Study. Methods: ONTRAC was a double-blind RCT conducted in two tertiary treatment centers in Sydney, Australia from 2012-2014. 386 immune competent participants with ≥ 2 histologically-confirmed NMSC in the past 5 years were randomized (1:1) to oral nicotinamide 500mg bd (NIC) or matched placebo (PBO) for 12 months. The primary endpoint was the number of new NMSCs to 12 months. Secondary endpoints included number of squamous cell carcinomas (SCCs), basal cell carcinomas (BCCs), and actinic keratoses (AKs) to 12 months. Skin reviews by dermatologists were performed 3 monthly. The sample size provided 90% power to detect a 33% difference in NMSC rates. Analysis was by intention-to-treat. Results: The mean age of study population was 66 years, the mean number of NMSC in the past 5 years was 8, and 63% were men. Treatment discontinuation rates were 9% for PBO versus 10% for NIC. 99% of patients underwent at least one post-baseline skin assessment. The average NMSC rate was significantly lower for NIC (1.77) than PBO (2.42). The estimated relative rate reduction (RRR) was 0.23 (95% CI: 0.04 to 0.38, p = 0.02) adjusting for center and NMSC history, and 0.27 (95% CI: 0.05 to 0.44; p = 0.02) with no adjustment. Treatment effects of comparable magnitude were found for both BCCs (RRR = 0.20, 95% CI: -0.06 to 0.39, p = 0.1) and SCCs (RRR = 0.30, 95% CI: 0 to 0.51, p = 0.05). AK counts were reduced for NIC compared to PBO by 11% at 3 months (p = 0.01), 14% at 6 months (p < 0.001), 20% at 9 months (p < 0.0001) and 13% at 12 months (p < 0.005). There were no clinically relevant differences in adverse event rates between the two arms. Conclusions: Nicotinamide reduces NMSC formation in high risk patients and is well tolerated. Furthermore, it is widely accessible as an inexpensive over-the-counter vitamin supplement and presents a new chemopreventive opportunity against NMSCs that is readily translatable into clinical practice. Clinical trial information: ACTRN12612000625875.
Read moreFigure S1. Distribution plots of the 65 cytokines targeted by the Discovery assay. Boxplot graphs showing relative fluorescence intensity (RFU; y axis) of the plasma samples (X) for (a), 60 of the 65 cytokines that were within the range of the external standards (from known low (S1) to high (S7) concentrations) while (b). 5 of the 65 cytokines were out of the standard curve range. Blank (B) values were also included in the assay. Figure S2. Distribution plots of five cytokines detected below the standard curve range in the Discovery assay. Histogram graphs showing distribution of the relative fluorescence units (RFU) of 47 plasma samples for Eotaxin-3, IL-21, IL-3, IL-9 and TSLP. Figure S3. Correlation of high and low abundance proteins. Median RFU of highly abundant proteins CTACK and Eotaxin-1, and low abundance proteins IL-7 and I-309 from the SOMAscan and Discovery assays were plotted; each point corresponds to a different patient sample (PRE and EDT plasmas). Proteins that are high and low abundance show poor correlation between the two assays. (DOCX 1640 kb)
Read moreAlternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4-8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of <i>BRAF</i> exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibitors, alternate BRAF exon 4-8 splicing is not associated with this intronic branch point mutation. We also confirm that melanoma cells expressing BRAF splicing variants retain exquisite sensitivity to existing FDA-approved MEK inhibitors.
Read moreLong-term survival occurs for a minority of patients receiving MAPK inhibitor treatment alone. Sex, serum LDH, BRAF genotype, and primary melanoma ulceration status are independent factors associated with treatment outcomes. Patients with a complete response to treatment have the best survival, but relapses still occur.
Read moreDermoscopy and RCM represent complementary/synergistic methods for diagnosis of amelanotic/light-coloured skin lesions.
Read moreAbstract Melanoma patients with clinically palpable regional lymph node metastases (AJCC Stage III) carry a risk of relapse and death that approaches 70% at 5 years. The NeoCombi study is a pilot phase II study of neoadjuvant dabrafenib plus trametinib in patients with resectable Stage IIIB-C BRAFV600 mutation positive melanoma. Patients receive 12 weeks of combined drug treatment, followed by surgical resection of all disease. Patients then continue combination drug treatment for a further 40 weeks. We aimed to identify host and tumour biomarkers that predict response to MAPK inhibition. Blood and tumour samples were obtained at baseline, early during treatment (3-7 days on therapy), at completion resection after 12 weeks on therapy, and, if relevant, on progression. Additional blood samples were also collected every 2 weeks up to 12 weeks of therapy. Samples were processed to generate plasma, peripheral blood mononuclear cells (PBMCs), fresh frozen tumour and where possible, tumour-dissociates and cell lines (tumour and immune cells). We have profiled tumour-specific and peripheral immune responses using a combination of flow cytometry, mass cytometry and multiplex cytokine/chemokine arrays. We also profiled circulating tumour-associated DNA (ctDNA) in plasma. To date, we have accrued 17 patients onto the NeoCombi study. Primary analysis of the first 12 patients has been completed. Decreases in ctDNA and tumour viability, in addition to immunophenotype changes, were seen within 1 week of starting treatment. Changes in immunophenotype continued throughout the first 12 weeks of treatment with conversion of CD8+ and CD4+ naive (CD45RA+) cells to effector cells (CD45RA-) in most patients. We also noted that activation of regulatory T cells (CD4+CD25+CD127-HLA-DR+) increased over the first 12 weeks of treatment in patients with partial pathological responses compared to those with complete pathological responses. Interestingly, the cytokine profile remained relatively stable across treatment within each patient, suggesting that factors already present at baseline may determine response to treatment. A pro-inflammatory cytokine signature has been identified as a potential biomarker of patients likely to relapse and in vitro validation studies are currently underway. Additionally, although ctDNA was only detectable in 42% of patients (compared to over 70% in patients with stage IV disease), high levels of ctDNA identified patients at highest risk of relapse and, if detectable, proved a useful marker to monitor treatment response. In conclusion, we have identified both host and tumour-specific biomarkers which may be useful to predict (and to monitor) response to MAPK inhibition in melanoma patients. Citation Format: Sarah J. Welsh, Suzanah Boyd, Helen McGuire, Maria Gonzales, Alexander M. Menzies, Hojabr Kakavand, Elisa James, Elizabeth Smith, Barbara Fazekas, Richard F. Kefford, Alex Guminski, Richard A. Scolyer, Georgina V. Long, Helen Rizos. In depth immune profiling of the response of melanoma to MAPK inhibition. [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 2650.
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