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Introduction: The Neoadjuvant rectal (NAR) score is a new surrogate endpoint to be used in clinical trials for early determination of treatment response to different preoperative therapies. The aim is to further validate the NAR-score, primarily developed using chemoradiotherapy (CRT) with a delay to surgery 6-8 weeks, and explore its value using other schedules. Materials and Methods: The study included all 9978 patients diagnosed with non-metastasized RC in 2007-2015 that had undergone surgery and was registered in the Swedish Colorectal Cancer Registry. The patients of interest had either short-course radiotherapy (scRT)/CRT + delayed surgery, longcourse radiotherapy (RT) + delayed surgery, (C)RT + additional chemotherapy, primary surgery, or scRT + immediate surgery. The scRT/CRT + delayed surgery groups were further divided based on time to surgery. Results: Mean NAR-score differed significantly (p < 0.0001) between different treatments. (C) RT + additional chemotherapy had the lowest mean score of 16.3 and CRT + delayed surgery had 17.7. There was a significant difference (p < 0.05) in overall survival (OS) and time to recurrence (TTR) of patients with a Low NAR-score (16) for both CRT- and scRT, with a stronger correlation for CRT-patients. C-index for the NAR-score model (0.623) was not superior to when only pathological T- and N-stage was used (0.646). Conclusions: The NAR-score is prognostic, but it is not better than pT- and pN-stage. However, the NARscore can still discriminate between two treatments that have different cell killing effect and may still be of value in clinical trials as an easier method than pT- and N-stage.

Background Identification of biomarkers associated with benefit of adjuvant chemotherapy in stage II/III colon cancer is an important task. Methods Vessel density (VD) and tumour stroma were analysed in a randomised-trial-derived discovery cohort (n = 312) and in a stage II/III group of a population-based validation cohort (n = 85). VD was scored separately in the tumour centre, invasive margin and peritumoral stroma compartments and quantitated as VD/total analysed tissue area or VD/stroma area. Results High stroma-normalised VD in the invasive margin was associated with significantly longer time to recurrence and overall survival (OS) (p = 0.002 and p = 0.006, respectively) in adjuvant-treated patients of the discovery cohort, but not in surgery-only patients. Stroma-normalised VD in the invasive margin and treatment effect were significantly associated according to a formal interaction test (p = 0.009). Similarly, in the validation cohort, high stroma-normalised VD was associated with OS in adjuvant-treated patients, although statistical significance was not reached (p = 0.051). Conclusion Through the use of novel digitally scored vessel-density-related metrics, this exploratory study identifies stroma-normalised VD in the invasive margin as a candidate marker for benefit of adjuvant 5-FU-based chemotherapy in stage II/III colon cancer. The findings, indicating particular importance of vessels in the invasive margin, also suggest biological mechanisms for further exploration.

Immune cells in the microenvironment shape tumor development and progression. The prognostic value of T-cell-based immune scores exceeds those of clinical parameters in colon cancer, but reflects only a part of the anti-tumor immune response. Here, we assessed 15 distinct immune cell classes and identified a simple prognostic signature based on pro- and anti-tumoral immune cells in the tumor microenvironment. The ratio of cytotoxic lymphocytes to tumor supportive macrophages predicted survival better than the state-of-art immune score in colon cancer and had the highest relative contribution to survival prediction when compared to established clinical parameters. This signature was prognostic also in other cancers with high mutation burden, such as those of the lung, bladder, esophagus, and melanomas, supporting broad clinical applicability.One Sentence SummaryThe CD8/M2 ratio in tumor tissue defines prognosis in immunogenic cancers

Breast cancer (BC) is a genetically heterogeneous disease with high prevalence in Northern Europe. However, there has been no detailed investigation into the Scandinavian somatic landscape. Here, in a homogeneous Swedish cohort, we describe the somatic events underlying BC, leveraging a targeted next-generation sequencing approach. We designed a 20.5 Mb array targeting coding and regulatory regions of genes with a known role in BC (n = 765). The selected genes were either from human BC studies (n = 294) or from within canine mammary tumor associated regions (n = 471). A set of predominantly estrogen receptor positive tumors (ER + 85%) and their normal tissue counterparts (n = 61) were sequenced to ~ 140 × and 85 × mean target coverage, respectively. MuTect2 and VarScan2 were employed to detect single nucleotide variants (SNVs) and copy number aberrations (CNAs), while MutSigCV (SNVs) and GISTIC (CNAs) algorithms estimated the significance of recurrent somatic events. The significantly mutated genes (q ≤ 0.01) were PIK3CA (28% of patients), TP53 (21%) and CDH1 (11%). However, histone modifying genes contained the largest number of variants (KMT2C and ARID1A, together 28%). Mutations in KMT2C were mutually exclusive with PI3KCA mutations (p ≤ 0. 001) and half of these affect the formation of a functional PHD domain. The tumor suppressor CDK10 was deleted in 80% of the cohort while the oncogene MDM4 was amplified. Mutational signature analyses pointed towards APOBEC deaminase activity (COSMIC signature 2) and DNA mismatch repair (COSMIC signature 6). We noticed two significantly distinct patterns related to patient age; TP53 being more mutated in the younger group (29% vs 9% of patients) and CDH23 mutations were absent from the older group. The increased somatic mutation prevalence in the histone modifying genes KMT2C and ARID1A distinguishes the Swedish cohort from previous studies. KMT2C regulates enhancer activation and assists tumor proliferation in a hormone-rich environment, possibly pointing to a role in ER + BC, especially in older cases. Finally, age of onset appears to affect the mutational landscape suggesting that a larger age-diverse population incorporating more molecular subtypes should be studied to elucidate the underlying mechanisms.

BACKGROUND The completeness and accuracy of the registration of synchronous metastases and recurrences in the Swedish Colorectal Cancer Registry has not been investigated. Knowing how accurate these parameters are in the registry is a prerequisite to adequately measure the current recurrence risk. METHODS All charts for patients diagnosed with stage I-III colorectal cancer (CRC) in two regions were reviewed. In one of the regions, all registrations of synchronous metastases were similarly investigated. After the database had been corrected, recurrence risk in colon cancer was calculated stratified by risk group as suggested by ESMO in 2020. RESULTS In patients operated upon more than five years ago (N = 1235), there were 20 (1.6%) recurrences not reported. In more recent patients, more recurrences were unreported (4.0%). Few synchronous metastases were wrongly registered (3.6%) and, likewise, few synchronous metastases were not registered (about 1%). The five-year recurrence risk in stage II was 6% for low-risk, 11% for intermediate risk, and 23% for high-risk colon cancer patients. In stage III, it was 25% in low- and 45% in high-risk patients. Incorporation of risk factors in stage III modified the risks substantially even if this is not considered by ESMO. Adjuvant chemotherapy lowered the risk in stage III but not to any relevant extent in stage II. CONCLUSION The registration of recurrences in the registry after 5 years is accurate to between 1 and 2% but less accurate earlier. A small number of unreported recurrences and falsely reported recurrences were discovered in the chart review. The recurrence risk in this validated and updated patient series matches what has been recently reported, except for the risk of recurrence in stage II low risk colon cancers which seem to be even a few percentage points lower (6 vs. 9%).

Background Prediction models are useful tools in the clinical management of colon cancer patients, particularly when estimating the recurrence rate and, thus, the need for adjuvant treatment. However, the most used models (MSKCC, ACCENT) are based on several decades-old patient series from clinical trials, likely overestimating the current risk of recurrence, especially in low-risk groups, as outcomes have improved over time. The aim was to develop and validate an updated model for the prediction of recurrence within 5 years after surgery using routinely collected clinicopathologic variables. Material and methods A population-based cohort from the Swedish Colorectal Cancer Registry of 16,134 stage I–III colon cancer cases was used. A multivariable model was constructed using Cox proportional hazards regression. Three-quarters of the cases were used for model development and one quarter for internal validation. External validation was performed using 12,769 stage II–III patients from the Norwegian Colorectal Cancer Registry. The model was compared to previous nomograms. Results The nomogram consisted of eight variables: sex, sidedness, pT-substages, number of positive and found lymph nodes, emergency surgery, lymphovascular and perineural invasion. The area under the curve (AUC) was 0.78 in the model, 0.76 in internal validation, and 0.70 in external validation. The model calibrated well, especially in low-risk patients, and performed better than existing nomograms in the Swedish registry data. The new nomogram’s AUC was equal to that of the MSKCC but the calibration was better. Conclusion The nomogram based on recently operated patients from a population registry predicts recurrence risk more accurately than previous nomograms. It performs best in the low-risk groups where the risk-benefit ratio of adjuvant treatment is debatable and the need for an accurate prediction model is the largest. publishedVersion

Therapies targeting somatic bystander genetic events represent a new avenue for cancer treatment. We recently identified a subset of colorectal cancer (CRC) patients who are heterozygous for a wild-type and a low activity allele (NAT2*6) but lack the wild-type allele in their tumors due to loss of heterozygosity (LOH) at 8p22. These tumors were sensitive to treatment with a cytotoxic substrate of NAT2 (6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine, APA), and pointed to NAT2 loss being a therapeutically exploitable vulnerability of CRC tumors. To better estimate the total number of treatable CRC patients, we here determined whether tumor cells retaining also other NAT2 low activity variants after LOH respond to APA treatment. The prevalent low activity alleles NAT2*5 and NAT2*14, but not NAT2*7, were found to be low metabolizers with high sensitivity to APA. By analysis of two different CRC patient cohorts, we detected heterozygosity for NAT2 alleles targetable by APA, along with allelic imbalances pointing to LOH, in ~ 24% of tumors. Finally, to haplotype the NAT2 locus in tumor and patient-matched normal samples in a clinical setting, we develop and demonstrate a long-read sequencing based assay. In total, > 79.000 CRC patients per year fulfil genetic criteria for high sensitivity to a NAT2 LOH therapy and their eligibility can be assessed by clinical sequencing.

Breast cancer (BC) is a genetically heterogeneous disease with high prevalence in Northern Europe. However, there has been no detailed investigation into the Scandinavian somatic landscape. Here, in a homogeneous Swedish cohort, we describe the somatic events underlying BC, leveraging a targeted next-generation sequencing approach. We designed a 20.5 Mb array targeting coding and regulatory regions of genes with a known role in BC (n = 765). The selected genes were either from human BC studies (n = 294) or from within canine mammary tumor associated regions (n = 471). A set of predominantly estrogen receptor positive tumors (ER + 85%) and their normal tissue counterparts (n= 61) were sequenced to ~ 140 × and 85 × mean target coverage, respectively. MuTect2 and VarScan2 were employed to detect single nucleotide variants (SNVs) and copy number aberrations (CNAs), while MutSigCV (SNVs) and GISTIC (CNAs) algorithms estimated the significance of recurrent somatic events. The significantly mutated genes (q ≤ 0.01) were PIK3CA (28% of patients), TP53 (21%) and CDH1 (11%). However, histone modifying genes contained the largest number of variants (KMT2C and ARID1A, together 28%). Mutations in KMT2C were mutually exclusive with PI3KCA mutations (p ≤ 0. 001) and half of these affect the formation of a functional PHD domain. The tumor suppressor CDK10 was deleted in 80% of the cohort while the oncogene MDM4 was amplified. Mutational signature analyses pointed towards APOBEC deaminase activity (COSMIC signature 2) and DNA mismatch repair (COSMIC signature 6). We noticed two significantly distinct patterns related to patient age; TP53 being more mutated in the younger group (29% vs 9% of patients) and CDH23 mutations were absent from the older group. The increased somatic mutation prevalence in the histone modifying genes KMT2C and ARID1A distinguishes the Swedish cohort from previous studies. KMT2C regulates enhancer activation and assists tumor proliferation in a hormone-rich environment, possibly pointing to a role in ER + BC, especially in older cases. Finally, age of onset appears to affect the mutational landscape suggesting that a larger age-diverse population incorporating more molecular subtypes should be studied to elucidate the underlying mechanisms.

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. Abstract 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.

Background: We have reported that BRAF V600E mutations and microsatellite instability-high (MSI-H) are more prevalent in a population-based cohort of metastatic colorectal cancer (mCRC) patients than has been reported from clinical trials or hospital-based patient groups. The aim was to explore if other mutations in mCRC differ in prevalence between these cohorts in relation to mismatch repair status and primary tumor location and if presence of bone or brain metastases is associated with any mutations. Material and methods: A population-based cohort of 798 mCRC patients from three regions in Scandinavia was used. Forty-four cancer related genes were investigated in a custom designed Ampliseq hotspot panel. Differences in survival were analyzed using the Kaplan–Meier estimator and the Cox regression analysis. Results: Determination of mutations was possible in 449/501 patients for 40/44 genes. Besides BRAF V600E, seen in 19% of the tumors, none of the other mutations appeared more prevalent than in trial cohorts. BRAF V600E and MSI-H, seen in 8%, were associated with poor prognosis as was right-sided primary tumor location (39%) when compared to left-sided and rectum together; however, in a multivariable regression, only the BRAF mutation retained its statistical significance. No other mutations were associated with poor prognosis. ERBB2 alterations were more common if bone metastases were present at diagnosis (17% vs. 4%, p = .011). No association was found for brain metastases. Fifty-two percent had an alteration that is treatable with an FDA-approved targeted therapy, chiefly by EGFR-inhibitor for RAS wild-type and a check-point inhibitor for MSI-H tumors. Conclusions: Right-sided tumor location, BRAF V600E mutations, but no other investigated mutation, and MSI-H are more commonly seen in an unselected cohort than is reported from clinical patient cohorts, likely because they indicate poor prognosis. Half of the patients have a tumor that is treatable with an already FDA-approved targeted drug for mCRC.

Background The purpose of this study was to investigate whether pT3–4 and pN-subclassifications, lymph-node ratio (LNR), tumour deposits, pre- and postoperative carcinoembryonic antigen (CEA), and C-reactive protein (CRP)—all parameters commonly collected in clinical management—add information about recurrence risk against a background of routine clinicopathological parameters as defined by the NCCN. Methods The prospective cohort consisted of all 416 patients diagnosed with colon cancer stage I–III in Uppsala County between 2010 and 2015. Cox proportional hazard models were used to calculate hazard ratios for time to recurrence and overall survival. The results were compared with the entire Swedish population concerning parameters recorded in the national quality registry, SCRCR, during the same time period. Results The Uppsala cohort was representative of the entire Swedish cohort. In unadjusted analyses, pT3-subclassification, pN-subclassification, LNR, tumour deposits, elevated postoperative CEA, and preoperative CRP correlated with recurrence. After adjusting for T-, N-stage, and NCCN risk factors, pN-subclassification, sidedness, and elevated postoperative CEA levels correlated with recurrence. Survival correlated with parameters associated with recurrence, LNR, and elevated postoperative CRP. Conclusions Additional information on recurrence risk is available from several routinely recorded parameters, but most of the risk is predicted by the commonly used clinicopathological parameters.

Unstable and dysfunctional tumor vasculature promotes cancer progression and spread. Signal transduction by the pro-angiogenic vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) is modul ...

Background Pre-operative radiotherapy (RT) or chemoradiotherapy (CRT) is frequently used prior to rectal cancer surgery to improve local control and survival. The treatment is administered according to guidelines, but these recommendations vary significantly between countries. Based on the stage distribution and risk factors of rectal cancers as determined by magnetic resonance imaging (MRI) in an unselected Swedish population, the use of RT/CRT according to 15 selected guidelines is described. Materials and methods Selected guidelines from different countries and regions were applied to a well-characterized unselected population-based material of 686 primary non-metastatic rectal cancers staged by MRI. The fraction of patients assigned to surgery alone or surgery following pre-treatment with (C)RT was determined according to the respective guideline. RT/CRT administered to rectal cancer patients for other reasons, for example, for organ preservation or palliation, was not considered. Results The fraction of patients with a clear recommendation for pre-treatment with (C)RT varied between 38% and 77% according to the different guidelines. In most guidelines, CRT was recommended to all patients who were not operated directly, and, in others, short-course RT was also recommended to patients with intermediate risk tumours. If only non-resectable or difficult to resect tumours were recommended pre-treatment, as stated in many Japanese publications, 9% would receive CRT followed by a delay to surgery. Conclusions According to most guidelines, well over 50% of primary non-metastatic rectal cancer patients from a general population, in which screening for colorectal cancer is not practised, are recommended treatment with pre-operative/neo-adjuvant therapy.

Purpose: Oestrogen receptor-positive (ER+) and human epidermal receptor 2-negative (HER2-) breast cancers are classified as Luminal A or B based on gene expression, but immunohistochemical markers are used for surrogate subtyping. The aims of this study were to examine the agreement between molecular subtyping (MS) and surrogate subtyping and to identify subgroups consisting mainly of Luminal A or B tumours. Methods: The cohort consisted of 2063 patients diagnosed between 2013-2017, with primary ER+/HER2- breast cancer, analysed by RNA sequencing. Surrogate subtyping was performed according to three algorithms (St. Gallen 2013, Maisonneuve and our proposed Grade-based classification). Agreement (%) and kappa statistics (kappa) were used as concordance measures and ROC analysis for luminal distinction. Ki67, progesterone receptor (PR) and histological grade (HG) were further investigated as surrogate markers. Results: The agreement rates between the MS and St. Gallen 2013, Maisonneuve and Grade-based classifications were 62% (kappa = 0.30), 66% (kappa = 0.35) and 70% (kappa = 0.41), respectively. PR did not contribute to distinguishing Luminal A from B tumours (auROC = 0.56). By classifying HG1-2 tumours as Luminal A-like and HG3 as Luminal B-like, agreement with MS was 80% (kappa = 0.46). Moreover, by combining HG and Ki67 status, a large subgroup of patients (51% of the cohort) having > 90% Luminal A tumours could be identified. Conclusions: Agreement between MS and surrogate classifications was generally poor. However, a post hoc analysis showed that a combination of HG and Ki67 could identify patients very likely to have Luminal A tumours according to MS.

Multigene expression signatures provide a molecular subdivision of early breast cancer associated with patient outcome. A gap remains in the validation of such signatures in clinical treatment groups of patients within population-based cohorts of unselected primary breast cancer representing contemporary disease stages and current treatments. A cohort of 3520 resectable breast cancers with RNA sequencing data included in the population-based SCAN-B initiative (ClinicalTrials.gov ID NCT02306096) were selected from a healthcare background population of 8587 patients diagnosed within the years 2010–2015. RNA profiles were classified according to 19 reported gene signatures including both gene expression subtypes (e.g. PAM50, IC10, CIT) and risk predictors (e.g. Oncotype DX, 70-gene, ROR). Classifications were analyzed in nine adjuvant clinical assessment groups: TNBC-ACT (adjuvant chemotherapy, n = 239), TNBC-untreated (n = 82), HER2+/ER− with anti-HER2+ ACT treatment (n = 110), HER2+/ER+ with anti-HER2 + ACT + endocrine treatment (n = 239), ER+/HER2−/LN− with endocrine treatment (n = 1113), ER+/HER2−/LN− with endocrine + ACT treatment (n = 243), ER+/HER2−/LN+ with endocrine treatment (n = 423), ER+/HER2−/LN+ with endocrine + ACT treatment (n = 433), and ER+/HER2−/LN− untreated (n = 200). Gene signature classification (e.g., proportion low-, high-risk) was generally well aligned with stratification based on current immunohistochemistry-based clinical practice. Most signatures did not provide any further risk stratification in TNBC and HER2+/ER– disease. Risk classifier agreement (low-, medium/intermediate-, high-risk groups) in ER+ assessment groups was on average 50–60% with occasional pair-wise comparisons having

Pre-operative radiotherapy (RT) or chemo-radiotherapy (CRT) are sometimes recommended prior to rectal cancer surgery, but guideline recommendations vary. The aim was to describe stage distribution and other important characteristics required for the treatment decision of patients with primary rectal cancers utilizing magnetic resonance imaging (MRI) in an unselected population.All 796 histopathologically verified rectal adenocarcinomas diagnosed 2010-2015 in two counties in Sweden (population 630,000 in 2015) were identified. Staging with pelvic MRI unless contraindications were present, treatment and pathology followed Swedish guidelines.Twenty-three % of cases (n = 186) had distant metastases at diagnosis, demonstrating more advanced tumor and nodal stages when compared with non-metastatic patients (p 0.001), and they more often displayed MRI-identified mucinous features and extramural vascular invasion (EMVI) than non-metastatic tumors (p 0.001 for both). In non-metastatic patients, 8% displayed clinical stage T1 (cT1), 21% cT2, and 53% cT3; one-third of the latter threatened or involved the mesorectal fascia (MRF+). Almost 20% had stage cT4 (4% cT4a, 14% cT4b) of which 50% were considered "non-resectable". EMVI was seen in 33% of cT3M0 tumors and in 48% of cT4M0 tumors.In an unselected population, approximately 80% of primary rectal cancers are referred to as "locally advanced" (stage II-III, or cT3-4 or N+), meaning that they, according to many international guidelines, are recommended neo-adjuvant treatment. This study provides a detailed description of the clinical stages and presence of characteristics identifiable on MRI which are of importance when assessing the needs for RT/CRT, when using different guidelines.

Modeling the cancer transcriptome Recent initiatives such as The Cancer Genome Atlas have mapped the genome-wide effect of individual genes on tumor growth. By unraveling genomic alterations in tumors, molecular subtypes of cancers have been identified, which is improving patient diagnostics and treatment. Uhlen et al. developed a computer-based modeling approach to examine different cancer types in nearly 8000 patients. They provide an open-access resource for exploring how the expression of specific genes influences patient survival in 17 different types of cancer. More than 900,000 patient survival profiles are available, including for tumors of colon, prostate, lung, and breast origin. This interactive data set can also be used to generate personalized patient models to predict how metabolic changes can influence tumor growth. Science , this issue p. eaan2507

Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant of cancer-associated fibroblasts (CAF). Elevated expression of PDGF receptors on stromal CAFs is associated with metastasis and poor prognosis, but mechanism(s) that underlie these connections are not understood. Here, we report the identification of the secreted glycoprotein stanniocalcin-1 (STC1) as a mediator of metastasis by PDGF receptor function in the setting of colorectal cancer. PDGF-stimulated fibroblasts increased migration and invasion of cocultured colorectal cancer cells in an STC1-dependent manner. Analyses of human colorectal cancers revealed significant associations between stromal PDGF receptor and STC1 expression. In an orthotopic mouse model of colorectal cancer, tumors formed in the presence of STC1-deficient fibroblasts displayed reduced intravasation of tumor cells along with fewer and smaller distant metastases formed. Our results reveal a mechanistic basis for understanding the contribution of PDGF-activated CAFs to cancer metastasis. Cancer Res; 73(4); 1287–97. ©2012 AACR.

Background: The recent successes of immunotherapy in non-small cell lung cancer (NSCLC) raise the interest to the deeper understanding of the diversity of the immune microenvironment. Meticulous characterization of the immune cell infiltrates is crucial for patients’ prognosis and the prediction therapeutic effects. The aim of this study was to analyze expression profiles of the immune cells and their linkage to clinical parameters in NSCLC patients. Methods: A tissue microarray of the NSCLC cohort comprised of 55 cases was used in the study. The staining was performed with the antibodies against CD8, CD20, CD4, FoxP3, CD45RO and pan-cytokeratin together with immuno-fluorescent markers (Opal, Perkin Elmer) and digital images acquired by a multispectral imaging system (Vectra 3, PerkinElmer). Marker expression pattern was evaluated on a cell level in total sample area, or in stroma and tumor area separately. Conventional immunohistochemical techniques and RNAseq data were used to validate the method. Results: Marker expression intensities were used to identify 5 non-epithelial cell classes, which express CD8, CD20, CD4, FoxP3, CD45RO. Relative abundance of marker-positive cells in individual cases were subjected to hierarchical clustering analysis, for the discovery of distinct immune cell infiltration patterns. The resulted clusters were exploratively analyzed with regard to survival associations. Patients with specific immune profile with high infiltration of FoxP3+ cells, moderate infiltration of CD4+ and CD8+ cells and low quantity of CD20+ and CD45RO+ cells in the stroma were associated with longer survival (median 87 vs. 54 month, log-rank p=0.050). Conclusion: Multi-marker analysis on single-cell levels with the fluorescence multiplexed IHC technique provides a valuable basic immune profile of cancer patients. Clustering analysis of the expression of immune markers identified a group of patients with improved survival in NSCLC independent of histology. Note: This abstract was not presented at the conference. Citation Format: Artur Mezheyeuski, Christian Bergsland, Max Backman, Tobias Sjöblom, Ragnhild A. Lothe, Jarle Bruun, Patrick Micke. Digital multiplex immunofluorescence analysis identifies immune profiles in the tumor stroma associated with clinical outcome [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B30.

Mutanome engineered RNA immuno-therapy (MERIT) for patients with triple negative breast cancer

This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF beta-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF alpha- and beta-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF beta-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF beta-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF beta-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF beta-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF alpha- and beta-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF beta-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors.

The benefits of combining PDGF antagonists and VEGF antagonists in a mouse model of pancreatic islet cancer are shown in a report by Bergers et al.: Bergers, G., Song, S., Meyer-Morse, N., Bergsland, E., and Hanahan, D. (2003). Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest. 111, 1287–1295.

The majority of metastatic cancers remain incurable since the current methods of treatment often fail to target the heterogeneous nature of each individual patient's tumor. Personalized approaches targeting each individual patient's tumor may therefore bring significant improvements. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically validate a pioneering RNA-based immunotherapy concept for the treatment of triple negative breast cancer (TNBC) by targeting shared tumor antigens and individual neo-antigens in TNBC patients. MERIT combines two personalized treatment concepts: (i) treatment with vaccines containing “off-the-shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode shared breast cancer tumor antigens expressed in the respective patient's tumor; (ii) treatment with mRNAs engineered on-demand that encode patient-specific mutated neo-antigens identified by next generation sequencing (NGS) and ranked according to the predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation, which specifically targets APCs and consequently induces antigen-specific T cell responses. MERIT is a multi-center phase I trial (NCT02316457) conducted in four European countries to assess the feasibility, safety and biological efficacy of this personalized immunotherapy. TNBC patients (pT1cN0M0 - anyTanyNM0) after surgery and adjuvant chemotherapy will be allocated to one of two study arms. Patients in ARM1 will receive eight vaccination cycles with a personalized set of shared tumor antigens selected from the WAREHOUSE that correspond to the patient tumor's antigen-expression profile. Patients in ARM2 will be first treated with the personalized WAREHOUSE vaccine approach followed by six vaccination cycles of on-demand manufactured MUTANOME vaccine targeting the unique mutation signature of the individual patient. During the clinical trial, patients will receive the individualized combination of the RNAs in parallel to standard radiotherapy. The clinical trial is approved and the study start is planned for Q1 2016. The consortium has built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept, which covers the whole individualized drug development cycle from target discovery, validation to GMP manufacturing and drug release for each individual patient. We will present the therapeutic concept and study protocol as well as the methodologies required for this highly innovative phase I trial. The personalized immunotherapy overcomes the current limitations of fixed, off-the-shelf therapeutics and thus might increase the clinical benefit for TNBC patients. This project is a collaborative effort of five partners from academia and industry funded by the European Commission's FP7 and led by BioNTech AG. Citation Format: Sandra Heesch, Valesca Bukur, Janina Buck, Jan Diekmann, Mustafa Diken, Kerstin Ewen, Heinrich Haas, Alexandra Kemmer-Brueck, Björn-Philipp Kloke, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Anna Paruzynski, Kathrin Schultheiß, Doreen Schwarck, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Özlem Türeci, Ugur Sahin. MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial. [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 CT020.

The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically and industrially validate a pioneering RNA-based immunotherapy concept that targets individual tumor antigens and tumor-specific mutations in triple negative breast cancer (TNBC) patients. This biomarker-guided, personalized therapy is a collaborative effort of five partners from academia and industry and is funded by the European Commission's FP7 and led by BioNTech AG. TNBC is an aggressive, molecularly heterogeneous cancer that accounts for 20% of all breast cancer patients. The 5-year survival rate is less than 80%. The molecular heterogeneity across TNBCs results in a lack of common targetable molecular alterations, and thus targeted therapies frequently fail to provide clinical benefit. The MERIT concept attempts to address this unmet medical need. The personalized treatment consists in (i) injecting vaccines containing “off the shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode tumor specific antigens expressed in the respective patient's tumor; and (ii) thereafter mRNAs engineered on-demand that encode patient-specific sequence stretches incorporating non-synonymous mutations identified by next generation sequencing (NGS) and ranked by predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation and are selectively delivered to splenic APCs. The encoded antigens are translated into proteins that are rapidly processed. Subsequent peptide presentation on the surface of APCs induces antigen-specific T cell responses. The central part of the MERIT project, a multi-center first in human trial, will assess the feasibility, safety and biological efficacy of this innovative personalized immunotherapy in TNBC patients. After discussing the regulatory challenges with the German national regulatory agency (PEI), a phase I study is now in preparation. The trial will start in Q2 2015 in five academic centers in Europe and will recruit thirty TNBC patients. Furthermore, the project includes a comprehensive T-cell immunomonitoring and biomarker program. Moreover, an extensive research program will address the optimization of algorithms for improved prediction of immunogenic mutations. Additionally, compounds to enhance vaccine efficacy will be developed and improved to support further clinical development. We have established a RNA delivery platform as well as a MERIT WAREHOUSE containing mRNAs coding for a selection of TNBC specific antigens. Additionally, we have built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept. We will describe the therapeutic concept and the critical skills, and methodologies required for this project, including cancer genomics, NGS, bioinformatics, tumor immunomics, industrial drug development, GMP manufacturing, clinical immunotherapy and immunological monitoring. Citation Format: Sandra Heesch, Cedrik M. Britten, Valesca Bukur, Janina Buck, John Castle, Jan Diekmann, Mustafa Diken, Katrin Frenzel, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Heinrich Haas, Alexandra Kemmer-Brueck, Bjoern-Philipp Kloke, Burkhard Otte, Anna Paruzynski, Sebastian Petri, Doreen Schwarck-Kokarakis, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Oezlem Tuereci, Ugur Sahin. The Mutanome Engineered RNA Immuno-Therapy (MERIT) project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT201. doi:10.1158/1538-7445.AM2015-CT201

Background— Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach. Methods and Results— To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg −1 per d −1 ), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15×10 10 pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI571 therapy, the circulating concentrations were able to suppress PDGF receptor phosphorylation. At 3 weeks, the treatment with STI571 led to a transient decrease in intralesion macrophages and to an increase in intimal smooth muscle cell apoptosis. VEGF-C application reduced neointima formation and accelerated reendothelialization. However, none of the therapies alone reduced intimal thickening at a 6-week time point, whereas the combined treatment led to a persistent reduction (55% versus control) in lesion size at this time point. Conclusions— Our study provides one of the first successful examples of gene therapy combined with a pharmacological treatment to modulate 2 distinct ligand-receptor signaling systems and suggests combination of local VEGF-C gene therapy with systemic inhibition of PDGF signaling as a novel principle to prevent intimal hyperplasia after vascular manipulations.