HLA-A*02 and its prognostic traits in cancer : an immunological biomarker as a tool towards individualised cancer therapy.

This thesis is based on the findings that HLA-A*02, a common allele in the Swedish population, has a decreasing frequency in the European continent in relation to geographic latitude. Furthermore, mortality of ovarian cancer correlates to the HLA-A*02 frequency and geographic latitude. The aims of the thesis were to study the HLA haplotypes and overrepresentation of HLA-A*02 in ovarian cancer patients, to determine the prognostic traits of HLA-A*02 in combination with MHC class I expression, as well as to analyse the role of HLA-A*02 in colon cancer patients together with MHC class I expression and CD8+ lymphocyte infiltration. In the first paper, HLA-A*02 is determined in 97 ovarian cancer patients and correlated with overall survival. HLA-A*02 was analysed from FFPE tumour samples. Patients with serous ovarian cancer and HLA-A*02 positivity had a poorer survival compared to those who were HLA-A*02 negative. In the second paper, blood samples were collected from 32 patients with advanced epithelial ovarian cancer (EOC) to determine HLA-A, -B, -Cw and -DRB1. The results showed an unusual representation of HLA alleles in EOC compared to healthy Swedish bone-marrow donors where HLA-A*02 was overrepresented, also HLA-A*02 homozygotes were two-fold higher among EOC patients. The frequency of HLA-A*01 was also increased whereas HLA-A*03 was decreased. Combinations of A*02 with B*05, B*15, DR1*03, DRB1*04, Cw*3 and DRB1*03 were also significantly increased, but only when considered as single, non-corrected analysis. These results indicated that the prognostic trait was restricted to HLA-A*02. In the third paper, blood samples and FFPE tumour samples were collected from 162 EOC patients and analysed for HLA-A*02 genotype as well as MHC class I and β2 microglobulin expression. Patients were with advanced stage, serous histology and HLA-A*02 positivity were grouped and named “worse prognosis group”. In this group there was an increased downregulation of MHC class I, which also correlated to a poorer prognosis. However the level of MHC expression was independent of prognosis when excluded from the worse prognosis group, suggesting that the prognostic trait of HLA-A*02 was not dependent on MHC class I expression. In the fourth paper, FFPE tumour samples were collected from 520 patients with colon cancer randomised to surgery and adjuvant chemotherapy or surgery alone. Tumour slides were analysed for HLA-A*02 genotype, MHC class I expression, HLA-G expression and CD8+ lymphocyte infiltration. Expression of HLA-G was a negative prognostic marker for the male patients. Also a high infiltration of CD8+ lymphocytes was important in the male patients, where a high frequency of infiltration correlated with a good prognosis. The HLA-A*02 genotype was, on the other hand, a superior negative prognostic marker for the female patients compared to CD8+ lymphocyte infiltration. In the fifth paper, blood samples and FFPE tumour samples were collected from 16 EOC patients. Blood samples were HLA typed at the clinical immunology laboratory. DNA was extracted from the FFPE samples and primers were designed to amplify exons 2, 3 and 4 of HLA-A. The amplification product was then sequenced and the sequence was aligned against all known HLAalleles. Complete HLA-A alleles could be assigned to 14/16 patients, in one case only one allele could be assigned and in one case no alleles could be assigned. The conclusion is thus that HLAA typing can be performed in fragmented FFPE-derived DNA using this approach

The need for a network to establish and validate predictive biomarkers in cancer immunotherapy.

Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, the entire medical oncology field has been revolutionized by the introduction of immune checkpoints inhibitors. Despite success in a variety of malignancies, responses typically only occur in a small percentage of patients for any given histology or treatment regimen. There are also concerns that immunotherapies are associated with immune-related toxicity as well as high costs. As such, identifying biomarkers to determine which patients are likely to derive clinical benefit from which immunotherapy and/or be susceptible to adverse side effects is a compelling clinical and social need. In addition, with several new immunotherapy agents in different phases of development, and approved therapeutics being tested in combination with a variety of different standard of care treatments, there is a requirement to stratify patients and select the most appropriate population in which to assess clinical efficacy. The opportunity to design parallel biomarkers studies that are integrated within key randomized clinical trials could be the ideal solution. Sample collection (fresh and/or archival tissue, PBMC, serum, plasma, stool, etc.) at specific points of treatment is important for evaluating possible biomarkers and studying the mechanisms of responsiveness, resistance, toxicity and relapse. This white paper proposes the creation of a network to facilitate the sharing and coordinating of samples from clinical trials to enable more in-depth analyses of correlative biomarkers than is currently possible and to assess the feasibilities, logistics, and collated interests. We propose a high standard of sample collection and storage as well as exchange of samples and knowledge through collaboration, and envisage how this could move forward using banked samples from completed studies together with prospective planning for ongoing and future clinical trials

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA

The aim of this study was to establish a novel approach for human leukocyte antigen (HLA)-typing from formalin-fixed paraffin-embedded-derived DNA. HLAs can be a prognostic factor in cancer and have an extensive polymorphism. This polymorphism is predominantly restricted to exons, which encode the peptide-binding domain of the protein. Formalin-fixed paraffin-embedded material is routinely collected in the clinic and therefore a great source of DNA for genetic analyses. However, its low quality due to fragmentation and nucleotide changes has often created obstacles in designing genetic assays. In this study, we amplified the most polymorphic exons of the HLA-A gene, exons 2, 3, and 4, in 16 formalin-fixed paraffin-embedded samples >10 years old. These tissue samples belonged to patients already HLA-typed by peripheral blood samples at the routine laboratory. Acquired amplification products were used for sequencing, which provided enough information to establish an HLA allele. The same method was applied to DNA extracted from peripheral blood from a healthy volunteer with known HLA type. Of the samples, 14/16 (88%) were successfully typed, in one sample only one of the alleles could be determined, and in one sample no allele could be determined. The amplification of the most polymorphic exons of HLA-A was a successful alternative when DNA quality prevented positive results with previously described methods. The method is usable when an HLA type is needed but the patients are deceased and/or no whole blood samples can be collected. It has thus potential to be used in several fields such as the clinic, research, and forensic science. © 2014 USCAP, Inc All rights reserved

Sex Differences in Overall Survival and the Effect of Radiotherapy in Merkel Cell Carcinoma—A Retrospective Analysis of A Swedish Cohort

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer where Merkel cell Polyomavirus (MCPyV) contributes to the pathogenesis. In an adjuvant setting, radiotherapy (RT) is believed to give a survival benefit. The prognostic impact of sex related to MCPyV-status and adjuvant RT were analyzed in patients referred to Karolinska University Hospital. Data were collected from 113 patients’ hospital records and MCPyV analyses were made in 54 patients (48%). We found a significantly better overall survival (OS) for women compared to men and a significant difference in OS in patients receiving adjuvant RT. Furthermore, we found that men with virus negative MCC have an increased risk for earlier death (HR 3.6). This indicates that MCPyV positive and negative MCC act as two different diseases, and it might be due to different mechanism in the immune response between male and female patients. This could have significance in tailoring treatment and follow-up in MCC patients in the future

Sex Differences in Overall Survival and the Effect of Radiotherapy in Merkel Cell Carcinoma—A Retrospective Analysis of A Swedish Cohort

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer where Merkel cell Polyomavirus (MCPyV) contributes to the pathogenesis. In an adjuvant setting, radiotherapy (RT) is believed to give a survival benefit. The prognostic impact of sex related to MCPyV-status and adjuvant RT were analyzed in patients referred to Karolinska University Hospital. Data were collected from 113 patients’ hospital records and MCPyV analyses were made in 54 patients (48%). We found a significantly better overall survival (OS) for women compared to men and a significant difference in OS in patients receiving adjuvant RT. Furthermore, we found that men with virus negative MCC have an increased risk for earlier death (HR 3.6). This indicates that MCPyV positive and negative MCC act as two different diseases, and it might be due to different mechanism in the immune response between male and female patients. This could have significance in tailoring treatment and follow-up in MCC patients in the future

Prognostic Interactions between FAP plus Fibroblasts and CD8a+T Cells in Colon Cancer

Simple Summary In addition to malignant cells, tumors are composed also of other cell types including immune cells and fibroblasts. These cell types interact with each other and with the malignant cells. Prognosis associations have previously been demonstrated for CD8-positive immune cells. Recent studies suggest that fibroblasts can affect the function of immune cells. The aim of this study was to investigate if the fibroblast composition of tumors affected the prognosis association of CD8 immune cells. This study demonstrated that in colon cancer, CD8 prognosis associations was restricted to the group of tumors with high expression the FAP fibroblast marker. Our findings suggest continued mechanistic studies regarding crosstalk between FAP-positive fibroblasts and the different immune cell types; and also support the investigation of fibroblast/T-cell interactions for therapeutic purposes. Inter-case variations in immune cell and fibroblast composition are associated with prognosis in solid tumors, including colon cancer. A series of experimental studies suggest immune-modulatory roles of marker-defined fibroblast populations, including FAP-positive fibroblasts. These studies imply that the fibroblast status of tumors might affect the prognostic significance of immune-related features. Analyses of a population-based colon cancer cohort demonstrated good prognosis associations of FAP intensity and CD8a density. Notably, a significant prognostic interaction was detected between these markers (p = 0.013 in nonadjusted analyses and p = 0.003 in analyses adjusted for cofounding factors) in a manner where the good prognosis association of CD8 density was restricted to the FAP intensity-high group. This prognostic interaction was also detected in an independent randomized trial-derived colon cancer cohort (p = 0.048 in nonadjusted analyses). In the CD8-high group, FAP intensity was significantly associated with a higher total tumor density of FoxP3-positive immune cells and a higher ratio of epithelial-to-stromal density of CD8a T cells. The study presents findings relevant for the ongoing efforts to improve the prognostic performance of CD8-related markers and should be followed by additional validation studies. Furthermore, findings support, in general, earlier model-derived studies implying fibroblast subsets as clinically relevant modulators of immune surveillance. Finally, the associations between FAP intensity and specific immune features suggest mechanisms of fibroblast-immune crosstalk with therapeutic potential