Prognostic and immunological significance of tryptophan metabolic enzymes across endometrial cancer molecular subtypes.

Since the establishment of 4 molecular subgroups of endometrial carcinoma (EC), there has been significant interest in understanding molecular classification in the context of histologic features and diagnoses. ECs with undifferentiated, spindle, and/or sarcomatous components represent a diagnostically challenging subset of tumors with overlapping clinical and histologic features. We examined the clinicopathologic, morphologic, immunohistochemical, and molecular features of these tumors identified in our institutions' pathology databases using immunohistochemistry and targeted sequencing. Disease-specific survival (DSS) and progression-free survival (PFS) were analyzed using Kaplan-Meier curves and log-rank tests. One hundred sixty-two ECs were included: carcinosarcomas (UCS; n=96), dedifferentiated/undifferentiated EC (DDEC/UDEC; n=49), and grade 3 endometrioid EC with spindled growth (GR3spEEC) (n=17). All molecular subgroups were represented in all histologic subtypes and included 12 (7%) POLE -mutated ( POLE mut), 43 (27%) mismatch repair-deficient (MMRd), 77 (48%) p53-abnormal (p53abn), and 30 (19%) no specific molecular profile (NSMP) tumors. However, the molecular classification (irrespective of histologic diagnosis) was a significant predictor for both DSS ( P =0.008) and P≤0.0001). POLE mut EC showed an excellent prognosis with no recurrences or deaths from the disease. MMRd tumors also showed better outcomes relative to NSMP and p53abn tumors. In conclusion, molecular classification provides better prognostic information than histologic diagnosis for high-grade EC with undifferentiated and sarcomatous components. Our study strongly supports routine molecular classification of these tumors, with emphasis on molecular group, rather than histologic subtyping, in providing prognostication.

Novel developments in the diagnosis and treatment of endometrial cancer will likely improve the prognosis of early, advanced and recurrent tumors. Molecular pathology currently classifies endometrial carcinoma into 4 molecular subtypes with prognostic significance. POLE mutated tumors, amounting to about 7% of all endometrial cancer cases, dubbed "ultra-mutated", have an excellent prognosis in early stages - even without adjuvant therapy. Mismatch repair deficient (MMRd) tumors are called "hypermutated" and have an intermediate prognosis in early stages. In advanced stages, they are highly sensitive to immune checkpoint inhibitors which are an integral part of their treatment. The tumors with "no specific molecular profile" have a prognosis that is similar to MMRd endometrial cancers. Finally, TP53 mutated cancers have a dismal prognosis, and aggressive adjuvant therapy is indicated. The 2023 FIGO classification recognizes for the first time the prognostically favorable synchronous endometrial and ovarian carcinomas, the importance of lymph node metastases depending on size and pattern, and the relevance of peritoneal involvement inside versus outside the pelvis. In metastatic disease, in mismatch repair proficient cases, the combination of carboplatin and paclitaxel chemotherapy with durvalumab has been recently approved as first line therapy in the European Union, followed by maintenance therapy with the PARP inhibitor olaparib, in combination with durvalumab. For MMRd tumors, several immune checkpoint inhibitors in combination with chemotherapy or as monotherapy have been approved in recent years. Tumors that are overexpressing Her2/neu have an additional treatment option with trastuzumab.

Introduction/Purpose: Endometrial cancer (EC) is the most common gynecologic cancer in the US with increasing incidence and mortality. To proliferate and metastasize, tumor cells must evade immune detection and attack. Evasion of immune detection and establishment of immune tolerance is regulated by a specific type of T cells, known as regulatory T cells (Tregs), through activation of the transcription factor, Foxp3. Our previous data indicated that patients with EC exhibited peripheral inflammation, evident by decreased inducible Tregs (iTreg) and total Treg populations and increased inflammatory cytokines (IL-17A, IL-21, IL-22, TNF-α, IL-10 and IL-1β). However, analysis of the immune profiles within EC tumors showed elevated Foxp3 expression, which indicated an immune tolerant tumor microenvironment (TME). Catabolism of the amino acid, tryptophan (Trp), by tumor cells and specific immune cell populations has been reported to alter effector immune cell function by promoting Treg differentiation. Transport of Trp into cells is regulated by SLC3A2, which is part of the heterodimeric LAT1 transporter. Once Trp enters the cytoplasm, intracellular indoleamine 2,3-dioxygenase (IDO1) catabolizes Trp, to produce kynurenine (Kyn). Catabolism of Trp to Kyn can be blocked by BIN1 (a tumor suppressor, Myc interacting protein) which inhibits IDO1 enzymatic activity. Kyn is a known ligand of the aryl hydrocarbon receptor (AhR) which, when activated, can induce the expression of IDO1, in both an autocrine and paracrine fashion. Activation of the AhR by Kyn has been shown to regulate the polarization of CD4+ cells to Tregs through the production of stimulatory cytokines such as IL-4, IL-10 and TGF-b1. Based on our preliminary data and existing literature, we hypothesized that EC tumor cells promote Treg differentiation of infiltrating CD4+ cells, within the tumor microenvironment through cytokine regulation of tryptophan catabolism by IDO1. Methods: The study population (IRB# 16-493) includes postmenopausal patients who underwent a hysterectomy at Southern Illinois University School of Medicine, Department of Ob/Gyn, Division of Gynecologic Oncology. Endometrial tissue biopsies were pathologically analyzed to confirm the presence of EC. A total of 35 patients were analyzed for this study; 8 Controls and 27 EC. Tissue biopsies and plasma samples were collected during surgery. To investigate alterations in the Trp catabolism pathway, tumor expression of AhR, BIN1, IDO1 and SLC3A2 was determined using quantitative real-time PCR (qPCR). Additionally, we used ELISA and qPCR to define the plasma and tissue expression of inflammatory cytokines (IL-1β, IL-4, IL-6 and IFN-γ) which are known regulators of IDO1. To assess Treg infiltration into the TME, we measured FOXp3 expression and performed immunohistochemical staining for FOXp3 in tissue samples. EC patients were categorized by disease stage and tumor histotype was determined by pathological analysis. The Mann-Whitney U test was used to analyze differences among EC and control patients, and within EC tumors we analyzed differences by disease staging. Results: Of the 27 EC patients, 13 were stage I-II and 14 stage were III-IV. Tumor histotypes represented 16 endometrioid EC, 7 serous, 3 carcinosarcoma and 1 mixed EC. Compared to endometrial biopsies from control patients, IDO1 expression was significantly increased (p=0.0159) in stage I-II EC tumors, but no changes in SLC3A2, BIN1 or AhR. Stage III-IV EC tumors had significantly decreased BIN1 and SLC3A2 (p=0.0091 and p=0.0121, respectively), but no alteration in IDO1 or AhR expression. We found no differences in inflammatory cytokine expression in EC tumors compared to control patients. All EC tumors, regardless of stage displayed elevated Foxp3 expression (p=0.0204) compared to RORγt. Consistent with this, EC tumors had increased Treg localization compared to tissue biopsies from control patients. Conclusions: Early stage (I-II) EC tumors had an immune tolerant TME via increased Treg localization, which may be mediated through IDO1 stimulation of Treg differentiation. Late stage (III-IV) EC tumors also expressed increased Treg localization; however, this may not be mediated by IDO1 catabolism of Trp. Together, these data support that EC tumors establish an immune tolerant TME to evade immune recognition and promote tumor survival.

Simple SummaryIdentification of mismatch repair-deficient tumors (MMRd), which occur in up to 30% of all endometrial cancers (EC), has become unavoidable for therapeutic management, clinical decision making, and prognosis. The objective of this systematic review was to summarize our current knowledge of the role of immunohistochemistry (IHC) markers for identifying MMRd tumors in EC. IHC with the expression of four proteins and MLH1 promoter methylation remains the reference of choice for diagnosis because it is reproducible and applicable in routine clinical practice. Further studies are needed to evaluate IHC in comparison with molecular tests including artificial intelligence, in terms of both efficacy and medical/economic aspects.The objective of this systematic review was to summarize our current knowledge of the role of immunohistochemistry (IHC) markers for identifying mismatch repair-deficient (MMRd) tumors in endometrial cancer (EC). Identification of MMRd tumors, which occur in 13% to 30% of all ECs, has become critical for patients with colorectal and endometrial cancer for therapeutic management, clinical decision making, and prognosis. This review was conducted by two authors applying the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines using the following terms: “immunohistochemistry and microsatellite instability endometrial cancer” or “immunohistochemistry and mismatch repair endometrial cancer” or “immunohistochemistry and mismatch repair deficient endometrial cancer”. Among 596 retrieved studies, 161 fulfilled the inclusion criteria. Articles were classified and presented according to their interest for the diagnosis, prognosis, and theragnostics for patients with MMRd EC. We identified 10, 18, and 96 articles using IHC expression of two, three, or four proteins of the MMR system (MLH1, MSH2, MHS6, and PMS2), respectively. MLH1 promoter methylation was analyzed in 57 articles. Thirty-four articles classified MMRd tumors with IHC markers according to their prognosis in terms of recurrence-free survival (RFS), overall survival (OS), stage, grade, and lymph node invasion. Theragnostics were studied in eight articles underlying the important concentration of PD-L1 in MMRd EC. Even though the role of IHC has been challenged, it represents the most common, robust, and cheapest method for diagnosing MMRd tumors in EC and is a valuable tool for exploring novel biotherapies and treatment modalities.

Immunotherapy Sensitivity of Mismatch Repair-Deficient Cancer: Mutation Load Is Not Enough

5594 Background: Molecular subtyping of endometrial cancer (EC), unlike histopathological evaluation, offers an objective and reproducible classification system that has strong prognostic value and therapeutic implications. The Proactive Molecular risk classifier for Endometrial cancer (ProMisE) was developed by our team as a pragmatic, cost-effective, and clinically applicable molecular classifier for EC patients. ProMisE has four subtypes: (i) POLE mutant ( POLEmut), (ii) mismatch repair deficient (MMRd), (iii) p53 abnormal (p53abn) by immunohistochemistry, and (iv) NSMP (No Specific Molecular Profile), lacking any of the defining features of the other three subtypes. While ProMisE subtypes are associated with clinical outcomes, within each subtype, there are clinical/prognostic outliers. This is particularly true within the largest ProMisE subtype; NSMP (representing ̃50% of ECs), where a subset of patients experience a very aggressive disease course, comparable to what is observed in patients with p53abn ECs. Methods: We hypothesized that objective assessment of the digitized hematoxylin and eosin (H&E)-stained histopathology slides of the largest and most diverse EC subset, NSMP, could potentially identify clinical outcome outliers. As such, we developed an artificial intelligence (AI)-based image analysis model to identify the NSMP cases that had similar histopathological features to the p53abn subtype, as assessed by H&E stain. We used a discovery cohort of 182 and an external validation cohort of 195 NSMP ECs. Results: Our AI-based image analysis model, based on deep convolutional neural networks, identified 21 (11.5%) out of the 182 NSMP cases with similar histopathological features as p53abn cases. We refer to these cases as ‘p53abn-like’ NSMPs. Compared to the rest of the NSMP cases, these cases had markedly inferior disease-specific survival (DSS) (10-year DSS 58.9% vs. 93.1% ( p<3.44e-8)) and progression-free survival (PFS) (10-year PFS 55.1% vs. 91.4% ( p<3.76e-6)). These findings were confirmed in our validation cohort, with 10.7% of the 195 patients categorized as ‘p53abn-like’ tumors with 10-year DSS of 82% vs. 51.3% ( p<5.28e-5) and PFS of 89.3% vs. 56.6% (p<2.15e-4). Conclusions: Utilizing an AI-based approach for histopathology image analysis, we have discovered ‘p53abn-like’ NSMPs, a novel subtype of NSMP ECs with morphological features similar to p53abn cases. ‘p53abn-like’ NSMPs exhibit similar clinical behavior as p53abn, having noticeably inferior outcome compared to the rest of the NSMP cases in two independent cohorts. These findings warrant further molecular investigation of this novel subtype of EC to identify the biological underpinning and future therapeutic strategies.

Simple SummaryWe studied mismatch repair (MMR) deficiency as a predictive and prognostic biomarker in endometrial carcinoma. MMR deficiency was associated with poor outcome only when p53 aberrant and polymerase-ϵ mutant tumors were excluded from the MMR proficient subgroup, in accordance with molecular classification based on The Cancer Genome Atlas. MMR deficiency was associated with an increased risk of death in the absence of various clinicopathologic risk factors, but the outcome was not worsened when such risk factors were present. The proportion of pelvic relapses and lymphatic dissemination, defined as primary lymph node involvement or relapses in regional lymph nodes, were higher in the MMR deficient subgroup. In conclusion, the effect of MMR deficiency on the outcome of endometrial carcinoma depends on how MMR proficiency is defined. MMR deficiency is associated with an increased risk of death in the absence of established risk factors and a unique pattern of disease spread.The aggressiveness of mismatch repair (MMR) deficient endometrial carcinomas was examined in a single institution retrospective study. Outcomes were similar for MMR proficient (n = 508) and deficient (n = 287) carcinomas, identified by immunohistochemistry. In accordance with molecular classification based on The Cancer Genome Atlas (TCGA), tumors with abnormal p53 staining or polymerase-ϵ exonuclease domain mutation were excluded from the MMR proficient subgroup, termed as “no specific molecular profile” (NSMP). Compared with NSMP (n = 218), MMR deficiency (n = 191) was associated with poor disease-specific survival (p = 0.001). MMR deficiency was associated with an increased risk of cancer-related death when controlling for confounders (hazard ratio 2.0). In the absence of established clinicopathologic risk factors, MMR deficiency was invariably associated with an increased risk of cancer-related death in univariable analyses (hazard ratios ≥ 2.0). In contrast, outcomes for MMR deficient and NSMP subgroups did not differ when risk factors were present. Lymphatic dissemination was more common (p = 0.008) and the proportion of pelvic relapses was higher (p = 0.029) in the MMR deficient subgroup. Our findings emphasize the need for improved triage to adjuvant therapy and new therapeutic approaches in MMR deficient endometrial carcinomas.

Endometrial carcinomas with mismatch repair deficiency (MMRd) and no specific molecular profile (NSMP) are considered to have intermediate prognoses. However, potential prognostic differences between these molecular subgroups remain unclear due to the lack of standardized control for clinicopathologic factors. This study aims to evaluate outcomes of MMRd and NSMP endometrial carcinomas across guideline-based clinicopathologic risk groups. This study analyzed patients treated at a single tertiary center. Immunohistochemistry and polymerase-ϵ sequencing were performed for molecular classification. MLH1-deficient tumors underwent methylation-specific multiplex ligation-dependent probe amplification. Carcinomas were classified into clinicopathologic risk groups according to European guidelines. The analysis included 420 MMRd and 399 NSMP carcinomas. Among MMRd cases, 224 were subcategorized as MLH1-methylated or MLH1-non-methylated. Median follow-up was 71 months (range; 1-136). Survival differences were most notable in clinicopathologic medium-risk carcinomas, with the MMRd subgroup exhibiting poorer progression-free, disease-specific, and overall survival compared to NSMP. Adjusting for age and adjuvant therapy, MMRd still showed an association with progression-free survival. Both MLH1-methylated (n = 154) and MLH1-non-methylated tumors (n = 70) were associated with more aggressive clinicopathologic risk groups compared to NSMP, but only methylated tumors showed poorer outcomes. The distinct outcomes for MMRd and NSMP in the clinicopathologic medium-risk group suggest that uterine risk factors may worsen the prognosis for MMRd endometrial carcinomas. Advanced stage may be the primary factor contributing to poor outcomes in high-risk-advanced metastatic carcinomas. Clinicopathologic factors may particularly worsen the prognosis of MLH1-methylated carcinomas.

Introduction/BackgroundNearly 30% of unselected endometrial cancer (EC) are mismatch repair deficient (MMRd).The majority resulting from epigenetic changes due to MLH1 promoter hypermethylation (MLH1-PM) and only a fraction from mutations in...

While endometrial cancer (EC) has an overall favorable prognosis, some patients do poorly and may benefit from refinements of current classification systems. The TCGA-inspired pragmatic molecular classification tool Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) has been integrated into international guidelines for EC risk stratification and management. ProMisE stratifies ECs into four prognostic groups: POLEmut, NSMP (no specific molecular profile), MMRd (mismatch repair deficient), and p53abn, where POLEmut has the best prognosis and p53abn has the worst prognosis. Our objective was to determine if proteomic profiling could provide additional prognostic or predictive information for EC patients, across or within ProMisE molecular subtypes. Global proteome profiling of FFPE samples, that had clinicopathologic and outcome data, was performed on 184 ECs encompassing all four ProMisE subtypes, including replicate samples of the same tumor, and both biopsy and final hysterectomy specimens. To ensure representation of each subtype, we aimed for an approximately equal distribution; 40 (27 %) MMRd, 33 (22 %) POLEmut, 42 (28 %) NSMP and 33 (22 %) p53abn, rather than the population-based distributions. There was high reproducibility in the proteomic profiles of intra-tumor replicate samples, and between matched biopsy and hysterectomy tumor samples (Pearson’s correlation >0.9). Consensus clustering generated four clusters, named ‘Adhesion’, ‘Immune’, ‘Proliferation’, and ‘Metabolic’ based on proteins enriched in each cluster. The Proliferation Cluster had the worst outcomes and the highest proportion of stage III/IV, serous, and p53abn tumors than the other clusters. The Immune Cluster had the most favorable outcomes, despite having a relatively substantial proportion of stage III/IV, serous, and p53abn tumors. We correlated protein expression with common mutations, including ARID1A mutations that cause loss of ARID1A protein expression, found in up to 60% ECs. ARID1A positive tumors also express proteins in the retinoic acid signaling pathway, while ARID1A-deficient tumors express proteins indicative of neutrophil infiltration. Comparing molecular subtypes, we found p53abn ECs were enriched in proteins associated with poor outcomes in many tumor types, such as GRB7. We validated elevated GRB7 expression in p53abn tumors using immunohistochemistry and found an association with worse disease specific survival (DSS) across the whole cohort (HR=2.39). Nucleolin expression was associated with worse prognosis in the NSMP subtype (DSS HR=9.88), while BABAM1 expression was associated with better prognosis within p53abn tumors (DSS HR=2.43). Knockout of BABAM1 (part of the BRCA complex) in cell lines resulted in increased sensitivity to PARP inhibition. Proteomic analysis of EC identifies candidate prognostic markers that may further refine current molecular classification and help guide treatment decisions. New therapeutic interventions could be developed to target proteins and pathways identified by EC proteomic profiling. Citation Format: Dawn R. Cochrane, Gian Luca Negri, Jutta Huvila, Juliana Sobral de Barros, Forouh Kalantari, Nissreen Mohammad, David Farnell, Emily Thompson, Amy Lum, Sandra E. Spencer, Amy Jamieson, Samuel Leung, Derek Chiu, Martin Koebel, Stefan Kommoss, Friedrich Kommoss, Blake Gilks, Lien Hoang, David Huntsman, Gregg B. Morin, Jessica N. McAlpine. Proteomic profiling of endometrial carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR002.

ObjectiveTo assess the risk stratification of clinicopathologically and molecularly classified endometrial cancer based on estrogen receptor (ER) and L1 cell adhesion molecule (L1CAM) expression. MethodsThis was a retrospective study of patients who underwent primary treatment at a single tertiary center. Carcinomas were classified into 5 clinicopathological risk groups, as per European guidelines. Immunohistochemistry and polymerase-ϵ sequencing were conducted for molecular classification and determination of ER and L1CAM expression. ResultsData from 1044 patients were analyzed. The median follow-up was 67.5 months. In univariable analyses, ER expression correlated with improved disease-specific survival (DSS) in the “no specific molecular profile” (NSMP) (P < 0.001) and mismatch repair deficient (MMRd) (P = 0.002) subgroups. Negative L1CAM expression was associated with enhanced DSS in the NSMP subgroup alone (P < 0.001). ER (hazard ratio [HR] 0.18), but not L1CAM, exhibited prognostic significance within NSMP when controlling for parameters available at the time of diagnosis (tumor histotype, grade, age). ER and L1CAM were not independently associated with DSS within NSMP when controlling for parameters available after surgery (clinicopathological risk groups, age, adjuvant therapy). However, in high-risk–advanced-metastatic cases, both ER (HR 0.26) and L1CAM (HR 3.9) independently correlated with DSS. Similarly, within MMRd, ER was associated with improved DSS in high-risk–advanced-metastatic carcinomas (HR 0.42). ConclusionThe prognostic significance of ER and L1CAM varies across clinicopathological risk groups and molecular subgroups of endometrial cancer. Notably, risk assessment for high-risk–advanced-metastatic NSMP and MMRd subtype carcinomas can be refined by ER status.

Molecular typing guiding treatment and prognosis of endometrial cancer

e17615 Background: Management of endometrial carcinoma (EC) relies on prognostic risk group classification to help to determine the individual risk of recurrence and the need for adjuvant treatment after surgery. A molecular classification with four distinct prognostic EC subtypes based on genomic abnormalities - DNA polymerase epsilon ( POLE) mutated ( POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53abn) and no specific molecular profile (NSMP) - has emerged, that raises the possibility of a more precise tailoring of adjuvant therapy. This study aimed to describe the clinicopathological and molecular characteristics of an EC Portuguese cohort, assess its potential impact in patient management and evaluate its prognostic value. Methods: Multicentre, retrospective cohort study of 230 patients with EC diagnosed between 2019 and 2022. Sample processing, clinicopathological, treatment and follow-up data was collected. Molecular classification was obtained by p53 and mismatch repair proteins immunohistochemistry, and POLE next-generation sequencing. Results: Overall, 230 patients from two institutions were included. Median age at diagnosis was 68 years. The most frequent histology was endometrioid (n=163; 70.9%) and most were low-grade (n=129, 56.1%).At diagnosis, disease was confined to the uterus in the majority of patients (FIGO stage I/II, n=153, 66.5%). Most had surgery upfront (n=196, 85.2%) and, of those, 120 (52.2%) received adjuvant treatment. 26 (11.3%) had metastatic disease. Median follow-up time was 15.4 months. Regarding molecular subgroups, the majority were classified as NSMP (n=87, 37.8%), followed by p53abn (n=73, 31.7%), MMRd (n=56, 24.3%), and POLEmut (n=14, 6.1%); 11 (4.8%) were multiple-classifier. Integration of these results led to a change in adjuvant treatment in 3 patients. Median disease-specific survival (DSS) was 108 months. Factors that significantly influenced survival included age (p=0.030), histological type (p&lt;0.001), grade (p&lt;0.001) and FIGO stage (p=0.006). Overall, 35 (15.2%) patients developed recurrences. Median recurrence-free survival (RFS) was 46.7 months. Kaplan-Meier survival analysis showed that molecular alterations were significantly associated both with DSS and RFS (p=0.0092 and p=0.0139, respectively). Tumours with p53abn had the worst prognosis, and patients with POLEmut tumours experienced an excellent prognosis. Comparative measures (BIC, C-index, log-likelihood) showed that considering the molecular classification in establishing risk groups for adjuvant treatment had prognostic significance. Conclusions: The molecular classification has prognostic value and should be considered in adjuvant treatment decisions. It allows for a more personalised approach, helps to reduce under and overtreatment, and therefore reduce patient morbidity associated with treatment toxicities, and healthcare related costs.

Upregulation of the Kynurenine Pathway (KP) in Glioblastoma (GBM) plays an important role in driving its treatment-resistant immunosuppressive microenvironment. Factors driving this exaggerated pathway remain poorly understood. Our aim was to explore the correlation between key KP markers; IDO1, IDO2, TDO2, its primary effector target aryl hydrocarbon receptor (AhR) and a comprehensive set of clinical- and tumour characteristics. Tissue samples from 108 newly diagnosed GBM patients were analyzed for the expression of TDO2, IDO1, IDO2, and AhR using immunohistochemistry and QuPath software. Exploratory analyses were conducted to evaluate correlations between KP marker expression and clinical, radiological, and molecular data. IDO1 expression was primarily correlated with inflammatory blood markers, while TDO2 was correlated with patient age, gender, smoking habit and medication use. In contrast, AhR and IDO2 demonstrated hardly any correlations with clinical or tumour characteristics. Notably, IDO2 exhibited a strong association with AhR expression and tumour cell density, with no observed correlation between AhR and either IDO1 or TDO2. We validated the inflammatory influences on IDO1 expression and found that TDO2 was mostly correlated with medication and patient characteristics. We could not confirm IDO1 and TDO2 as most prominent drivers of AhR activity in the KP. However, we found a strong correlation between IDO2-AhR which may be responsible for the sustained and enhanced immunosuppression within the tumour microenvironment. This could explain recent failures of IDO1 and TDO2 antagonists and might redirect future studies to intervene in the kynurenine-AhR-IDO2 axis.

Immunohistochemistry (IHC) and/or MSI-PCR (microsatellite instability-polymerase chain reaction) tests are performed routinely to detect mismatch repair deficiency (MMR-D). Classical MMR-D tumors present a loss of MLH1/PMS2 or MSH2/MSH6 with MSI-High. Other profiles of MMR-D tumors have been described but have been rarely studied. In this study, we established a classification of unusual MMR-D tumors and determined their frequency and clinical impact. All MMR-D tumors identified between 2007 and 2017 were selected. Any profile besides the classical MMR-D phenotype was defined as unusual. For patients with unusual MMR-D tumors, IHC, and PCR data were reviewed, the tumor mutation burden (TMB) was evaluated and clinical and genetic features were collected. Of the 4948 cases of MMR testing, 3800 had both the available IHC and MSI-PCR results and 585 of these had MMR-D. After reviewing the IHC and PCR, 21% of the cases initially identified as unusual MMR-D were reclassified, which resulted in a final identification of 89 unusual MMR-D tumors (15%). Unusual MMR-D tumors were more often associated with non-CRC than classical MMR-D tumors. Unusual MMR-D tumors were classified into four sub-groups: i) isolated loss of PMS2 or MSH6, ii) classical loss of MLH1/PMS2 or MSH2/MSH6 without MSI, iii) four MMR proteins retained with MSI and, iv) complex loss of MMR proteins, with clinical characteristics for each sub-group. TMB-high or -intermediate was shown in 96% of the cancers studied (24/25), which confirmed MMR deficiency. Genetic syndromes were identified in 44.9% (40/89) and 21.4% (106/496) of patients with unusual and classical MMR-D tumors, respectively (P < 0.001). Five patients treated with an immune checkpoint inhibitor (ICI) had a prolonged clinical benefit. Our classification of unusual MMR-D phenotype helps to identify MMR deficiency. Unusual MMR-D phenotype occurs in 15% of MMR-D tumors. A high frequency of genetic syndromes was noted in these patients who could benefit from ICI.