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Atezolizumab in combination with nab-paclitaxel has been introduced for the treatment of locally advanced or recurrent triple negative breast cancer (TNBC). Patient selection relies on the use of immunohistochemistry using a specific monoclonal PD-L1 antibody (clone SP142) in a tightly controlled companion diagnostic test (CDx) with a defined interpretative algorithm. Currently there are no standardized recommendations for selecting the optimal tissue to be tested and there is limited data to support decision making, raising the possibility that tissue selection may bias test results. We compared PD-L1 SP142 assessment in a collection of 73 TNBC cases with matched core biopsies and excision samples. There was good correlation between PD-L1-positive core biopsy and subsequent excision, but we found considerable discrepancy between PD-L1 negative core biopsy and matched excision, with a third of cases found negative on core biopsies converting to positive upon examination of the excision tissue. In view of these findings, we developed a workflow for the clinical testing of TNBC for PD-L1 and implemented it in a central referral laboratory. We present audit data from the clinical PD-L1 testing relating to 2 years of activities, indicating that implementation of this workflow results in positivity rates in our population of TNBC similar to those of IMpassion130 clinical trial. We also developed an online atlas with a precise numerical annotation to aid pathologists in the interpretation of PD-L1 scoring in TNBC.

This study identifies the various functions of open questions in whole-class teaching in language arts classrooms in primary school, and it explores how these questions may work as a mediating tool. Open questions are considered a valuable tool in classroom discourse, enhancing dialogue and students’ learning by giving the students an opportunity to elaborate on their thoughts. The analysis draws on two data sets and includes observations from four schools and an examination of whole-class teaching in 32 language arts lessons. The results show that the teacher practice of asking open questions has one core function – classroom management. The teachers also ask open questions that are more subject specific, and the material covers writing activities and orthography and grammar instruction. The functions of open questions are quite similar in the two data sets. This may indicate that the teachers’ open questions mediate an understanding of school culture and the values of the subject to the students, here how writing activities and grammar instruction should be interpreted. This article argues that we should go further than merely differentiating between open and closed questions and investigate the functions of open questions in the classroom.

This study aims to investigate textbook tasks in the Norwegian school subject Natural sciences and the views of science that the tasks mediate to the pupils. Natural science is an interdisciplinary subject taught in Years 1–11 that includes chemistry, physics, biology and parts of the geosciences. We conducted a qualitative content analysis of 2,927 tasks from three Year-11 textbooks. The results showed that most of the tasks in all three textbooks were closed and asked the pupils to reproduce facts from the textbook or other information sources. However, findings also indicated that there are differences between and within the different chapters of the textbooks. In the topics that include socio-scientific issues, the tasks are more open for the pupils to both explore and evaluate evidence. Furthermore, all the three books have an introductory chapter focusing on scientific methodology, and the tasks in this chapter are much more diverse than in the remaining chapters. It therefore seems that the aim of the textbooks is to shed light on the nature of science, but the tasks embedded in the textbooks do not support scientific inquiry and perpetuate the traditional approach of asking learners to provide already determined answers to scientific issues.

Under Covid-19 utbruddet i mars 2020 ble alle norske barneskoler ble stengt, og både elever og lærere måtte flytte skolen hjem. Dette utfordret den etablerte lærerrollen og førte til endringer i lærernes undervisningspraksis. Formålet med denne artikkelen er å diskutere lærerens rolle, konkretisert som lærerens undervisningspraksis, i tiden med hjemmeskole. For å belyse lærernes undervisningspraksis tar vi utgangspunkt i intervjuer med lærere og tekster fra elevene på barnetrinnet samlet inn i perioden med hjemmeskole. I lys av dette materialet, samt Gert Biestas to posisjoner undervises av og lære av, drøfter vi hvordan lærerens rolle og undervisningspraksis i hjemmeskolen kan forstås.

BackgroundCarcinoma associated fibroblasts (CAFs) play important roles in modulating tumor development and prognosis through biochemical and biomechanical signals, but also through their immuno-modulatory characteristics. Fibroblast activation protein alpha (FAP), a serine protease with selectively high expression on CAFs, may be an ideal target for therapeutic intervention, including cancer immunotherapy. Therefore, a thorough understanding of FAP expression, but also immune cell composition and especially their interaction is key to optimally inform drug development and patient enrichment strategies.MethodsFormalin-fixed paraffin embedded tissue specimens comprising 253 primary tumors and 277 metastatic lesions were included in this study. Tumor sections were analyzed by digital immunohistochemistry (IHC) to assess tumor-stroma composition, FAP content and immune cell infiltration, complemented by transcriptomic analyses.ResultsAcross different types of epithelial tumors, FAP was detected by digital IHC in the tumor-associated stroma at a low to moderate proportion and with heterogeneous distribution patterns. Primary tumors in breast and lung cancer demonstrated a higher median FAP content (6.5% and 6.6% area coverage, respectively) compared to renal cell carcinoma (0.2% area coverage), which was confirmed on mRNA expression level. Median FAP levels were similar between primary and metastatic lesions in most tumor types except for renal cancer, for which FAP levels were significantly increased in metastasis lesions (3.3% area coverage). FAP content positively correlated with the density of FoxP3 positive regulatory T cells, but indication and tissue type specific differences were observed. Transcriptomic analysis revealed that both stroma-richness as well as higher FAP content were positively correlated with macrophage and dendritic cell gene signatures. However, while a higher stromal content was associated with signatures related to endothelial cells and preadipocytes, higher FAP content showed a stronger correlation with regulatory T cells. These findings are suggestive of a distinct biological role of FAP positive stroma in human tumors.ConclusionsFAP-targeted therapy is a promising strategy to optimize accumulation and action of anti-cancer drugs in the tumor microenvironment, potentially leading to more specific and effective therapies. Our study further elucidates the role of FAP by providing a comprehensive and granular landscape of FAP content in primary and metastatic tumor lesions derived from the same patient population and its association with immune cell composition. Future studies aim to elucidate the complex and dynamic interplay between malignant, stromal and immune cell populations in both temporal and spatial contexts and how that contributes to outcome in cancer immunotherapy.

PURPOSE The purpose of this study was to evaluate the prognostic value of Immunoscore in patients with stage III colon cancer (CC) and to analyze its association with the effect of chemotherapy on time to recurrence (TTR). METHODS An international study led by the Society for Immunotherapy of Cancer evaluated the predefined consensus Immunoscore in 763 patients with American Joint Committee on Cancer/Union for International Cancer Control TNM stage III CC from cohort 1 (Canada/United States) and cohort 2 (Europe/Asia). CD3+ and cytotoxic CD8+ T lymphocyte densities were quantified in the tumor and invasive margin by digital pathology. The primary end point was TTR. Secondary end points were overall survival (OS), disease-free survival (DFS), prognosis in microsatellite stable (MSS) status, and predictive value of efficacy of chemotherapy. RESULTS Patients with a high Immunoscore presented with the lowest risk of recurrence, in both cohorts. Recurrence-free rates at 3 years were 56.9% (95% CI, 50.3% to 64.4%), 65.9% (95% CI, 60.8% to 71.4%), and 76.4% (95% CI, 69.3% to 84.3%) in patients with low, intermediate, and high immunoscores, respectively (hazard ratio [HR; high v low], 0.48; 95% CI, 0.32 to 0.71; P = .0003). Patients with high Immunoscore showed significant association with prolonged TTR, OS, and DFS (all P < .001). In Cox multivariable analysis stratified by participating center, Immunoscore association with TTR was independent (HR [high v low], 0.41; 95% CI, 0.25 to 0.67; P = .0003) of patient’s sex, T stage, N stage, sidedness, and microsatellite instability status. Significant association of a high Immunoscore with prolonged TTR was also found among MSS patients (HR [high v low], 0.36; 95% CI, 0.21 to 0.62; P = .0003). Immunoscore had the strongest contribution χ2 proportion for influencing survival (TTR and OS). Chemotherapy was significantly associated with survival in the high-Immunoscore group for both low-risk (HR [chemotherapy v no chemotherapy], 0.42; 95% CI, 0.25 to 0.71; P = .0011) and high-risk (HR [chemotherapy v no chemotherapy], 0.5; 95% CI, 0.33 to 0.77; P = .0015) patients, in contrast to the low-Immunoscore group ( P > .12). CONCLUSION This study shows that a high Immunoscore significantly associated with prolonged survival in stage III CC. Our findings suggest that patients with a high Immunoscore will benefit the most from chemotherapy in terms of recurrence risk.

Background: The tumor microenvironment is an important factor in cancer immunotherapy response. To further understand how a tumor affects the local immune system, we analyzed immune gene expression differences between matching normal and tumor tissue. Methods: We analyzed public and new gene expression data from solid cancers and isolated immune cell populations. We also determined the correlation between CD8, FoxP3 IHC, and our gene signatures. Results: We observed that regulatory T cells (Tregs) were one of the main drivers of immune gene expression differences between normal and tumor tissue. A tumor-specific CD8 signature was slightly lower in tumor tissue compared with normal of most (12 of 16) cancers, whereas a Treg signature was higher in tumor tissue of all cancers except liver. Clustering by Treg signature found two groups in colorectal cancer datasets. The high Treg cluster had more samples that were consensus molecular subtype 1/4, right-sided, and microsatellite-instable, compared with the low Treg cluster. Finally, we found that the correlation between signature and IHC was low in our small dataset, but samples in the high Treg cluster had significantly more CD8+ and FoxP3+ cells compared with the low Treg cluster. Conclusions: Treg gene expression is highly indicative of the overall tumor immune environment. Impact: In comparison with the consensus molecular subtype and microsatellite status, the Treg signature identifies more colorectal tumors with high immune activation that may benefit from cancer immunotherapy. Cancer Epidemiol Biomarkers Prev; 27(1); 103–12. ©2017 AACR.

BackgroundFibroblast activation protein alpha (FAP) is frequently over-expressed in the tumor microenvironment (TME) while exhibiting limited expression in normal tissues. FAP expression was reported to be immunosuppressive in tumor mouse models and generally associated with worse prognosis in clinical studies. Therefore, it is important to understand the context in which FAP both exhibits immunosuppressive characteristics and be a useful target for immunotherapy.MethodsComprehensive immunohistochemistry (IHC) analyses on formalin-fixed paraffin-embedded tissue specimens with emphasis on lymph nodes and primary and metastatic tumor lesions spanning a wide range of indications were undertaken in this study. FAP staining of tumor tissues was performed with an optimized IHC robust-prototype-assay (RPA) and manually scored. The area (normal stroma & neoplastic) staining positively relative to the total tumor area at each intensity level was recorded and an H-score calculated (FAP-intensity score).These were supplemented by gene expression analysis using public as well as Roche phase 1, 2 and 3 cancer immunotherapy (CIT) clinical trial data sets.ResultsAnalysing FAP expression on normal tissue confirmed the general absence of FAP apart from a subset of pancreatic islet cells. Unlike the more homogenous expression of typical protein targets on tumor cells, FAP expression in the TME is heterogeneous in both pattern and intensity, requiring the analysis of a large sample set. Therefore, we evaluated 1216 samples from 23 tumor indications and 70 sub-indications. FAP expression exhibited a significant spread ranging from indications with highly abundant expression to those with low coverage.Using data from matching IHC and gene expression samples we confirmed FAP mRNA expression to significantly correlate with RPA H-scores (Spearman correlation: 0.62) (N=289, P=1.2E-31). Gene expression data from 12 atezolizumab clinical studies, including standard of care (SOC) randomized studies, with more than 6000 samples from 4 major indications were interrogated for the association between FAP expression and clinical response as evaluated by overall and progression free survival. This analysis suggests that FAP expression is generally associated with higher hazard ratios across all atezolizumab-treated samples (OS: 95% CI 1.04–1.09; PFS: 1.04–1.08), with the highest effect observed in Renal Cell Carcinoma (OS: 95% CI 1.08–1.31; PFS: 1.05–1.21), indicating a potential role of FAP in limiting CIT.ConclusionsData from these analyses can tailor indication and patient enrichment strategies for achieving optimal FAP-targeting. We propose to select indications with FAP-levels that are high enough to enable drug accumulation, yet low enough to reduce immunosuppressive effects that can hamper successful immunotherapy.

The female genital tract (FGT) mucosa is a critically important site for immune defense against microbes. Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population that recognizes microbial riboflavin metabolite antigens in an MR1-dependent manner. The role of MAIT cells in the FGT mucosa is unknown. Here, we found that MAIT cells and MR1+ antigen-presenting cells were present in the upper and lower FGT, with distinct tissue localization of MAIT cells in endometrium vs. cervix. The MAIT cells from the FGT and blood displayed a distinct phenotype with expression of interleukin (IL)-18Rα, CD127, α4β7, PD-1, as well as the transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, Eomes, and T-bet. Their expression levels of PLZF and Eomes were lower in the FGT compared with blood. When stimulated with Escherichia coli, MAIT cells from the FGT displayed a bias towards IL-17 and IL-22 expression, whereas blood MAIT cells produced primarily IFN-γ, TNF, and Granzyme B. Furthermore, both FGT- and blood-derived MAIT cells were polyfunctional and contributed to the T-cell-mediated response to E. coli. Thus, MAIT cells in the genital mucosa have a distinct IL-17/IL-22 profile and may have an important role in the immunological homeostasis and control of microbes at this site.

Background: Features characterizing the immune contexture (IC) in the tumor microenvironment can be prognostic and predictive biomarkers. Identifying novel biomarkers can be challenging due to complex interactions between immune and tumor cells and the abundance of possible features. Methods: We describe an approach for the data-driven identification of IC biomarkers. For this purpose, we provide mathematical definitions of different feature classes, based on cell densities, cell-to-cell distances, and spatial heterogeneity thereof. Candidate biomarkers are ranked according to their potential for the predictive stratification of patients. Results: We evaluated the approach on a dataset of colorectal cancer patients with variable amounts of microsatellite instability. The most promising features that can be explored as biomarkers were based on cell-to-cell distances and spatial heterogeneity. Both the tumor and non-tumor compartments yielded features that were potentially predictive for therapy response and point in direction of further exploration. Conclusion: The data-driven approach simplifies the identification of promising IC biomarker candidates. Researchers can take guidance from the described approach to accelerate their biomarker research.

HPV-DNA positive (HPVDNA+) oropharyngeal squamous cell carcinoma (OSCC) has better clinical outcome than HPV-DNA negative (HPVDNA-) OSCC. Current treatment may be unnecessarily extensive for most HPV+ OSCC, but before de-escalation, additional markers are needed together with HPV status to better predict treatment response. Here the influence of HLA class I/HLA class II expression was explored. Pre-treatment biopsies, from 439/484 OSCC patients diagnosed 2000-2009 and treated curatively, were analyzed for HLA I and II expression, p16(INK4a) and HPV DNA. Absent/weak as compared to high HLA class I intensity correlated to a very favorable disease-free survival (DFS), disease-specific survival (DSS) and overall survival (OS) in HPVDNA+ OSCC, both in univariate and multivariate analysis, while HLA class II had no impact. Notably, HPVDNA+ OSCC with absent/weak HLA class I responded equally well when treated with induction-chemo-radiotherapy (CRT) or radiotherapy (RT) alone. In patients with HPVDNA- OSCC, high HLA class I/class II expression correlated in general to a better clinical outcome. p16(INK4a) overexpression correlated to a better clinical outcome in HPVDNA+ OSCC. Absence of HLA class I intensity in HPVDNA+ OSCC suggests a very high survival independent of treatment and could possibly be used clinically to select patients for randomized trials de-escalating therapy.

For histochemical staining, to highlight multiple biomarkers within a sample, multiple stains with different light spectral absorption characteristics are deployed (i.e. multiplexing). To reconstruct the single stain contrast from a multiplexed sample, the conventional color deconvolution method assumes that light extinction follows Lambert-Beer’s law during imaging process and the optical density (OD) measured from the image is linearly related to the stain amount. However, this assumption does not hold well for commonly used diaminobenzidine (DAB) stain due to its precipitate-forming reaction during sample processing. Besides absorption, scattering also contributes to the light extinction process which causes the non-linear relation between the OD value and the stain amount. Therefore, using the conventional method may not have sufficient accuracy for quantified stain analysis, especially when DAB presents at high concentration levels. In this paper, our study shows that DAB presents different chromatic properties at different concentration levels. Therefore, we propose a new color deconvolution method to address the issue by employing a set of reference colors vectors, each of which characterizes a DAB concentration level. Then, the reference color vector that best represents the true DAB concentration level in the mixture is automatically selected for color deconvolution. Both visual and quantified assessments are provided to show that the method enables detection for a broader dynamic range of DAB concentration and therefore should be preferred by the user for bright field image analysis.

Contains fulltext : 193579.pdf (Publisher’s version ) (Closed access) BACKGROUND: The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS: An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance. FINDINGS: Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0.97 for colon tumour; r=0.97 for invasive margin; p

Norsk ungdom vokser opp i et av verdens rikeste, lykkeligste og mest egalitære land. Samtidig vet de at mange i verden lider. Hvordan skal de føle, tenke og handle i møte med verdens urettferdige fordeling av goder? I denne boken analyseres fortellinger om privilegier og skyld som unge mennesker møter i skolen og på fritiden. Forfatterne går også inn i klasserommene og undersøker hvordan elevene forstår og diskuterer skyldfortellinger og spørsmål om sitt eget ansvar i en global sammenheng. Fortellingene er hentet fra mange forskjellige medier og sjangere: en av kongens taler, undervisningsmateriell (norsklærebøker og Operasjon Dagsverks kampanjemateriell), ungdomsromaner (av Simon Stranger og Kristín A. Sandberg), tv-serier (Skam), dikt (av Øyvind Rimbereid og Cathrine Grøndahl), film (av Margreth Olin), krim (av Jo Nesbø), noveller (av Laila Stien) og romaner (av Gert Nygårdshaug). To artikler analyserer klasseromspraksiser (helklassesamtaler og prosjektarbeid). Samlet sett gir denne boken et første forskningsgrunnlag for en didaktikk om skyldfortellinger i norsk skole. Den begrunner hvorfor man bør ha en slik undervisning, peker på hva den kan inneholde, og hvordan den kan drives.

Multiplex-brightfield immunohistochemistry (IHC) staining and quantitative measurement of multiple biomarkers can support therapeutic targeting of carcinoma-associated fibroblasts (CAF). This paper presents an automated digitalpathology solution to simultaneously analyze multiple biomarker expressions within a single tissue section stained with an IHC duplex assay. Our method was verified against ground truth provided by expert pathologists. In the first stage, the automated method quantified epithelial-carcinoma cells expressing cytokeratin (CK) using robust nucleus detection and supervised cell-by-cell classification algorithms with a combination of nucleus and contextual features. Using fibroblast activation protein (FAP) as biomarker for CAFs, the algorithm was trained, based on ground truth obtained from pathologists, to automatically identify tumor-associated stroma using a supervised-generation rule. The algorithm reported distance to nearest neighbor in the populations of tumor cells and activated-stromal fibroblasts as a wholeslide measure of spatial relationships. A total of 45 slides from six indications (breast, pancreatic, colorectal, lung, ovarian, and head-and-neck cancers) were included for training and verification. CK-positive cells detected by the algorithm were verified by a pathologist with good agreement (R2=0.98) to ground-truth count. For the area occupied by FAP-positive cells, the inter-observer agreement between two sets of ground-truth measurements was R2=0.93 whereas the algorithm reproduced the pathologists’ areas with R2=0.96. The proposed methodology enables automated image analysis to measure spatial relationships of cells stained in an IHC-multiplex assay. Our proof-of-concept results show an automated algorithm can be trained to reproduce the expert assessment and provide quantitative readouts that potentially support a cutoff determination in hypothesis testing related to CAF-targeting-therapy decisions.

Artikkelen viser hvordan forfatterne av to norsklærebøker for videregående skole har valgt ulike strategier for å inkludere et flerkulturelt perspektiv i norskfaget. Grip teksten henvender seg til elever med en tradisjonell norsk kulturbakgrunn og forsøker å gjøre dem bevisste på de stereotype og etnosentriske oppfatningene de har av mennesker fra andre kulturer. Moment henvender seg til alle elever i dagens flerkulturelle norske skole og minner dem om at den grunnlovfestede ytringsfriheten er en nødvendighet for dialog og fredelig sameksistens mellom kulturer.

Denne artikkelen utforsker hvilke forstaelser av skjonnlitteratur og sakprosa elever kan utvikle gjennom oppgavene i laereboker i norsk. Dette blir undersokt gjennom en kvalitativ innholdsanalyse av 1943 oppgaver fra tre laereboker for videregaende skole. Resultatene viser at det er klare forskjeller mellom oppgavene i de tre hoveddelene av laerebokene: Samlingene av skjonnlitteraere tekster er dominert av apne oppgaver der elevene inviteres til a uttrykke sine opplevelser og tolkninger av tekstene. I samlingene av sakprosatekster er det flere lukkede oppgaver der elevene skal avdekke tekstens mening gjennom a folge bestemte framgangsmater. I tilknytningen til selve laerebokteksten er det et stort antall lukkede oppgaver der elevene skal finne og gjengi informasjon som er eksplisitt uttrykt i teksten. Gjennom a arbeide med disse oppgavene kan elevene utvikle en forstaelse av at skjonnlitteraturen er rik pa potensielle meninger, mens sakprosaen er enkel og entydig. De kan ogsa fa inntrykk av at laerebokteksten er en spesiell sakprosasjanger man ikke trenger a analysere kritisk. Disse resultatene indikerer at oppgavekulturen i norskfaget er preget av eldre faglige forstaelser og ikke helt i trad med den gjeldende laereplanen. This article explores which understandings of fiction and non-fiction students may develop through the assignments in textbooks in Norwegian Language Arts. This is investigated through a qualitative content analysis of 1943 assignments from three textbooks for upper secondary school. The results show that there is a marked difference between the assignments in the three main parts of the textbooks: The fiction anthologies are dominated by open assignments where the students are invited to express their experiences with and interpretations of the texts. I the non-fiction anthologies, there are several closed assignments where the students are instructed to reveal the meaning of the text by following certain procedures. Connected to the main text in the textbook, there is a large number of assignments where the students are asked to find and repeat information which is explicitly stated in the text. Through these assignments the students may develop the understanding that fiction is rich with potential meanings, while non-fiction is simple and unambiguous. They may also get the impression that the textbook itself is a special non-fiction genre, which is unnecessary to analyse critically. These results indicate that the assignment culture in Norwegian Language Arts is characterised by older academic understandings and not fully in line with the current national curriculum.

The aim of this chapter is to explore how teachers use questions as an instructional tool in science and language arts classrooms, and how these questions enable dialogue between teachers and students and support student learning. The focus will be on investigating teachers’ use of questions and responses as an instructional tool during whole class sessions. There are many research reports on teachers’ use of questions as a key instructional tool (Barnes 2008; Chin 2007; Croom 2004; Mehan 1979) and also on how classroom dialogue is crucial to facilitate learning (Alexander 2008; Juzwik et al. 2008; Lemke 1993; Mercer and Littleton 2007; Nystrand et al. 1997). In this respect, scholars agree that teachers’ questioning can significantly improve the students’ performance. The analyses presented in this chapter draw on video recordings from teacher-led whole class instruction in lower secondary language arts and science classrooms. We are interested in exploring the role and type of teacher questions in these two distinct, and rather different, subject areas.

In solid tumors, human leucocyte antigen (HLA)-A2 has been suggested to be a risk factor and a negative prognostic factor. The HLA-A2 allele in Scandinavia has a high prevalence; it decreases with latitude and also with ovarian cancer mortality in Europe. Furthermore, an association of the HLA-A2 allele with severe prognosis in serous adenocarcinoma of the ovary in stages III-IV was found. Thirty-two unrelated Swedish women with relapsing or progressive ovarian cancer were analysed for the genotypes at the HLA-A, HLA-B, HLA-Cw, and HLA-DRB1 loci by the polymerase chain reaction/sequence-specific primer method. The frequencies of HLA alleles of healthy Swedish bone marrow donors provided by the coordinating centre of the Bone Marrow Donors Worldwide Registries, Leiden, the Netherlands were used as controls. When this cohort of epithelial ovarian cancer patients was compared with healthy Swedish donors, the frequency of HLA-A1 and HLA-A2 gene/phenotype appears, although not statistically significant, to be increased in patients with ovarian carcinoma, while HLA-A3 was decreased. HLA-A2 homozygotes were twofold higher in patients. The A2-B8 haplotype was significantly increased (corrected P value). A2-B5, A2-B15, A2-DRB1*03, A2-DRB1*04, A2-B15-Cw3, and A2-B8-DRB1*03 had odds ratio as well as the level of the lower confidence interval above 1 and significant P value only when considered as single, non-corrected analysis. HLA-B15 and HLA-Cw3 were only present in HLA-A2-positive patients showing that the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes were segregated. In this selected cohort with advanced disease, there are indications of an unusual overrepresentation of HLA class I and II genes/haplotypes as well as segregation for the HLA-A2-HLA-Cw3 and HLA-B15 haplotypes. These findings are presented as a descriptive analysis and need further investigations on a larger series of ovarian cancer patients to establish prognostic associations.

Tumor budding (single tumor cells or small tumor cell clusters) at the invasion front of colorectal cancer (CRC) is an adverse prognostic indicator linked to epithelial-mesenchymal transition. This study characterized the immunogenicity of tumor buds by analyzing the expression of the major histocompatibility complex (MHC) class I in the invasive tumor cell compartment. We hypothesized that maintenance of a functional MHC-I antigen presentation pathway, activation of CD8+ T-cells, and release of antitumoral effector molecules such as cytotoxic granule-associated RNA binding protein (TIA1) in the tumor microenvironment can counter tumor budding and favor prolonged patient outcome. Therefore, a well-characterized multipunch tissue microarray of 220 CRCs was profiled for MHC-I, CD8, and TIA1 by immunohistochemistry. Topographic expression analysis of MHC-I was performed using whole tissue sections (n = 100). Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations, mismatch repair (MMR) protein expression, and CpG-island methylator phenotype (CIMP) were investigated. Our results demonstrated that membranous MHC-I expression is frequently down-regulated in the process of invasion. Maintained MHC-I at the invasion front strongly predicted low-grade tumor budding (P = 0.0004). Triple-positive MHC-I/CD8/TIA1 in the tumor microenvironment predicted early T-stage (P = 0.0031), absence of lymph node metastasis (P = 0.0348), lymphatic (P = 0.0119) and venous invasion (P = 0.006), and highly favorable 5-year survival (90.9% vs 39.3% in triple-negative patients; P = 0.0032). MHC-I loss was frequent in KRAS-mutated, CD8+ CRC (P = 0.0228). No relationship was observed with CIMP, MMR, or BRAF mutation. In conclusion, tumor buds may evade immune recognition through downregulation of membranous MHC-I. A combined profile of MHC-I/CD8/TIA1 improves the prognostic value of antitumoral effector cells and should be preferred to a single marker approach.

Adoptive transfer of in vitro-expanded tumor-infiltrating lymphocytes (TIL) has shown great clinical benefit in patients with malignant melanoma. TIL therapy itself has little side effects, but conditioning chemo- or radiotherapy and postinfusion interleukin 2 (IL-2) injections are associated with severe adverse advents. We reasoned that combining TIL infusion with dendritic cell (DC) vaccination could circumvent the need for conditioning and IL-2 support and thus represent a milder treatment approach. Eight patients with stage IV melanoma were enrolled in the MAT01 study, consisting of vaccination with autologous tumor-lysate-loaded DC, followed by TIL infusion. Six of eight patients were treated according to protocol, while one patient received only TIL and one only DC. Treatments were well tolerated with a single grade 3 adverse event. The small study size precludes analysis of clinical responses, though interestingly one patient showed a complete remission and two had stable disease. Analysis of the infusion products revealed that mature DC were generated in all cases. TIL after expansion were CD3+ T cells, dominated by effector memory CD8+ cytotoxic T cells. Analysis of the T cell receptor repertoire revealed presence of highly dominant clones in most infusion products, and many of these could be detected in the circulation for weeks after T cell transfer. Here, we report the first combination of DC vaccination and TIL infusion in malignant melanoma. This combined treatment was safe and feasible, though after evaluating both clinical and immunological parameters, we expect that administration of lymphodepleting chemotherapy and IL-2 will likely increase treatment efficacy.

The major histocompatibility complex (MHC) comprises a set of genes that are essential to immunity and surveillance against neoplastic transformation. MHC antigens not only regulate antitumor immune responses in experimental animal models but also directly correlate with survival and prognosis of patients with various types of cancers. Effective recognition of tumor cells by effector T cells may be affected by the genotype and the extent of expression of human leukocyte antigen (HLA)-peptide complexes. Therefore, MHC antigens may serve as potential biomarkers for prognosis and allow selection of cancer patients for specific therapy. We describe PCR-based method to determine the HLA genotype in healthy individuals and patients using blood and tumor tissue as DNA source.

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.

Human papillomavirus (HPV) is an important factor for the development of tonsillar squamous cell carcinoma (TSCC). In addition, patients with HPV-positive TSCC have a better clinical outcome than patients with HPV-negative TSCC. Although, HPV is an important prognostic marker, additional biomarkers are needed to better predict clinical outcome to individualize treatment. Hence, we examined if classical HLA HLA-A,B,C and nonclassical HLA-E,G could serve as such marker. Formalin-fixed paraffin-embedded TSCC from 150 patients diagnosed 2000-2006, earlier analyzed for HPV DNA and p16(INK4a), and treated with intention to cure were evaluated for the expression of HLA-A,B,C and HLA-E,G by immunohistochemistry. For HPV-positive TSCC a low expression of HLA-A,B,C, whereas for HPV-negative TSCC, a normal expression of HLA-A,B,C was significantly correlated to a favorable clinical outcome. These correlations were more pronounced for membrane staining of HLA-A,B,C when compared with cytoplasmatic staining. No significant correlation was found between HLA-E,G and HPV status or clinical outcome. The unexpected contrasting correlation between HLA-A,B,C expression, and clinical outcome depending on HPV, indicates essential differences between HPV-positive and HPV-negative TSCC. Furthermore, our data demonstrate that for both HPV-positive and HPV-negative TSCC, the expression of HLA-A,B,C together with HPV may serve as a useful biomarker for predicting clinical outcome.

BACKGROUND: In recent years, evidence is accumulating that cancer cells develop strategies to escape immune recognition. HLA class I HC down-regulation is one of the most investigated. In addition, different HLA haplotypes are known to correlate to both risk of acquiring diseases and also prognosis in survival of disease or cancer. We have previously shown that patients with serous adenocarcinoma of the ovary in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A02* genotype. We aimed to study the relationship between HLA-A02* genotype in these patients and the subsequent HLA class I HC protein product defects in the tumour tissue. MATERIALS AND METHODS: One hundred and sixty-two paraffin-embedded tumour lesions obtained from Swedish women with epithelial ovarian cancer were stained with HLA class I heavy chain (HC) and β(2)-microglobulin (β(2)-m)-specific monoclonal antibodies (mAb). Healthy ovary and tonsil tissue served as a control. The HLA genotype of these patients was determined by PCR/sequence-specific primer method. The probability of survival was calculated using the Kaplan–Meier method, and the hazard ratio (HR) was estimated using proportional hazard regression. RESULTS: Immunohistochemical staining of ovarian cancer lesions with mAb showed a significantly higher frequency of HLA class I HC and β(2)-m down-regulation in patients with worse prognosis (WP) than in those with better prognosis. In univariate analysis, both HLA class I HC down-regulation in ovarian cancer lesions and WP were associated with poor survival. In multivariate Cox-analysis, the WP group (all with an HLA-A02* genotype) had a significant higher HR to HLA class I HC down-regulation. CONCLUSIONS: HLA-A02* is a valuable prognostic bio-marker in epithelial ovarian cancer. HLA class I HC loss and/or down-regulation was significantly more frequent in tumour tissues from HLA-A02* positive patients with serous adenocarcinoma surgical stage III–IV. In multivariate analysis, we show that the prognostic impact is reasonably correlated to the HLA genetic rather than to the expression of its protein products.

We have previously demonstrated an association of the human leukocyte antigen (HLA), HLA-A2 allele with ovarian and prostate cancer mortality as well as a segregation of the ancestral HLA haplotype (AHH) 62.1 [(A2) B15 Cw3 DRB1*04] in patients with stage III–IV serous ovarian cancer. The objective of the present study was to determine the role of the HLA phenotype on the prognosis in stage III–IV malignant melanoma patients. A cohort of metastatic malignant melanoma patients (n = 91), in stage III (n = 26) or IV (n = 65) were analysed for HLA-A, -B, -Cw and -DRB1 types by PCR/sequence-specific primer method. The frequencies of HLA alleles in the patients were compared to that of healthy Swedish bone marrow donors. The effect of HLA types on prognosis was defined by Kaplan–Meier and Cox analysis. The presence of the AHH 62.1 in clinical stage IV patients was significantly and independently associated with the worst survival rate recorded from the appearance of metastasis (HR = 2.14; CI = 1.02–4.4; P = 0.04). In contrast, the period from the primary diagnosis to metastasis was the longest in patients with this haplotype (HR = 0.40; CI = 0.17–0.90; P = 0.02). Melanoma patients in our cohort with 62.1 AHH which is associated with autoimmune diseases have an initial strong anti-tumour control with longer metastasis-free period. These patients have rapid progression after the appearance of metastasis, responding poorly to chemo- or/and immunotherapy. This apparently paradoxical clinical process could be due to the interplay between tumour clones escape and immune surveillance ending up with a rapid disease progression.

HLA abnormalities on tumour cells for immune escape have been widely described. In addition, cellular components of the tumour microenvironment, in particular myeloid derived suppressor cells (MDSC) and alternatively activated M2 tumour-associated macrophages (TAMs), are involved in tumour promotion, progression, angiogenesis and suppression of anti-tumour immunity. However, the role of HLA in these activities is poorly understood. This review details MHC class I characteristics and describes MHC class I receptors functions. This analysis established the basis for a reflection about the crosstalk among the tumour cells, the TAMs and the cells mediating an immune response. The tumour cells and TAMs exploit MHC class I molecules to modulate the surrounding immune cells. HLA A, B, C and G molecules down-regulate the macrophage myeloid activation through the interaction with the inhibitory LILRB receptors. HLA A, B, C are able to engage inhibitory KIR receptors negatively regulating the Natural Killer and cytotoxic T lymphocytes function while HLA-G induces the secretion of pro-angiogenic cytokines and chemokine thanks to an activator KIR receptor expressed by a minority of peripheral NK cells. The open conformer of classical MHC-I is able to interact with LILRA receptors described as being associated to the Th2-type cytokine response, triggering a condition for the M2 like TAM polarization. In addition, HLA-E antigens on the surface of the TAMs bind the inhibitory receptor CD94/NKG2A expressed by a subset of NK cells and activated cytotoxic T lymphocytes protecting from the cytolysis. Furthermore MHC class II expression by antigen presenting cells is finely regulated by factors provided with immunological capacities. Tumour-associated macrophages show an epigenetically controlled down-regulation of the MHC class II expression induced by the decoy receptor DcR3, a member of the TNFR, which further enhances the M2-like polarization. BAT3, a positive regulator of MHC class II expression in normal macrophages, seems to be secreted by TAMs, consequently lacking its intracellular function, it looks like acting as an immunosuppressive factor. In conclusion HLA could cover a considerable role in tumour-development orchestrated by tumour-associated macrophages.

BACKGROUND: The prevalence and role of human papillomavirus (HPV) in the aetiology of oesophageal squamous cell carcinoma is uncertain. Based on the presence of HPV in the oral cavity and its causal association with squamous cell carcinoma of the oropharynx, we hypothesised that HPV is more strongly associated with proximal than distal oesophageal squamous cell carcinoma. METHODS: A population-based study comparing HPV infection in relation to tumour site in patients diagnosed with oesophageal squamous cell carcinomas in the Stockholm County in 1999-2006. Multiplex polymerase chain reaction genotyping (PCR) with Luminex was conducted on pre-treatment endoscopic biopsies to identify type specify HPV. Carcinogenic activity of HPV was assessed by p16(INK4a) expression. Multivariable logistic regression was used to calculate odds ratios and 95% confidence intervals. RESULTS: Among 204 patients, 20 (10%) had tumours harbouring HPV DNA, almost all (90%) of HPV high-risk type, mainly HPV16. Tumours containing HPV were not overrepresented in the upper compared to the middle or lower third of the oesophagus (odds ratio 0.6, 95% confidence interval 0.2-1.9). P16(INK4a) expression was similarly common (24% and 16%) in the HPV-positive and HPV-negative groups. CONCLUSION: This study found a limited presence of HPV in oesophageal squamous cell carcinoma of uncertain oncogenic relevance and did not demonstrate that HPV was more strongly associated with proximal than distal tumours.

16544 Background: We have shown that the high prevalence of HLA-A2 in Scandinavia is associated to poor prognosis in advanced (stage III-IV) serous adenocarcinoma of the ovary. Our objective was to...

9050 Background: Current methods to predict survival in patients with metastatic malignant melanoma are limited. Our objective was to determine the impact of Human Leukocyte Antigen-type (HLA-type)...

21059 Background: HLA-A2 allele is highly prevalent in Scandinavia and decreases with latitude in Europe. Similarly, ovarian cancer mortality decreases with latitude in European countries. We have previously shown that HLA-A2 is associated to a severe prognosis in serous adenocarcinoma of the ovary in stage III-IV. Others have shown that lack of p21/Waf-1 and lymphocyte infiltration in this tumour is correlated to poorer prognosis as well. Here we analyse a possible linkage between these variables. Methods: Thirty eight Swedish women with epithelial ovarian cancer were analyzed for the HLA-A genotype locus by PCR/sequence-specific oligonucleotide hybridization procedure (PCR/SSOP). Tissue slides from paraffin embedded tumour material were stained with haematoxylin and antibodies anti-p21 and -p53. Results: Twenty two patients were HLA-A2 positive. Fourteen of them (37%) lacked lymphocyte infiltration wich differed significantly from HLA-A2 negative patients (n=4, 11 %) (p=0.002). All of the HLA-A2 positive patients, with serous adenocarcinoma that showed absence of tumour lymphocyte infiltration, did not express p21/waf-1 (n=7 18 %). No cases in the HLA-A2 negative group with the same parameters were identified (p=0.009). Conclusions: These observations identify a high risk group of patients by linking together the somatic expression of HLA-A2 to the lack of lymphocyte infiltration and absence of p21/Waf-1 expression at the tumour level as prognostic negative factors. The information might have a relevant impact on the patient management.. In order to obtain a stronger power the analysis will be extended to a larger group of patients. No significant financial relationships to disclose.