Characterization of the T cell receptor repertoire and melanoma tumor microenvironment upon combined treatment with ipilimumab and hTERT vaccination.

BACKGROUND: This clinical trial evaluated a novel telomerase-targeting therapeutic cancer vaccine, UV1, in combination with ipilimumab, in patients with metastatic melanoma. Translational research was conducted on patient-derived blood and tissue samples with the goal of elucidating the effects of treatment on the T cell receptor repertoire and tumor microenvironment. METHODS: The trial was an open-label, single-center phase I/IIa study. Eligible patients had unresectable metastatic melanoma. Patients received up to 9 UV1 vaccinations and four ipilimumab infusions. Clinical responses were assessed according to RECIST 1.1. Patients were followed up for progression-free survival (PFS) and overall survival (OS). Whole-exome and RNA sequencing, and multiplex immunofluorescence were performed on the biopsies. T cell receptor (TCR) sequencing was performed on the peripheral blood and tumor tissues. RESULTS: Twelve patients were enrolled in the study. Vaccine-specific immune responses were detected in 91% of evaluable patients. Clinical responses were observed in four patients. The mPFS was 6.7 months, and the mOS was 66.3 months. There was no association between baseline tumor mutational burden, neoantigen load, IFN-γ gene signature, tumor-infiltrating lymphocytes, and response to therapy. Tumor telomerase expression was confirmed in all available biopsies. Vaccine-enriched TCR clones were detected in blood and biopsy, and an increase in the tumor IFN-γ gene signature was detected in clinically responding patients. CONCLUSION: Clinical responses were observed irrespective of established predictive biomarkers for checkpoint inhibitor efficacy, indicating an added benefit of the vaccine-induced T cells. The clinical and immunological read-out warrants further investigation of UV1 in combination with checkpoint inhibitors. Trial registration Clinicaltrials.gov identifier: NCT02275416. Registered October 27, 2014. https://clinicaltrials.gov/ct2/show/NCT02275416?term=uv1&draw=2&rank=6.

Proteogenomic analysis of the autoreactive B cell repertoire in blood and tissues of patients with Sjögren's syndrome.

OBJECTIVE: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow. METHODS: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS. RESULTS: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification. CONCLUSION: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

Neoadjuvant immunotherapy leads to pathological responses in MMR-proficient and MMR-deficient early-stage colon cancers.

PD-1 plus CTLA-4 blockade is highly effective in advanced-stage, mismatch repair (MMR)-deficient (dMMR) colorectal cancers, yet not in MMR-proficient (pMMR) tumors. We postulated a higher efficacy of neoadjuvant immunotherapy in early-stage colon cancers. In the exploratory NICHE study (ClinicalTrials.gov: NCT03026140), patients with dMMR or pMMR tumors received a single dose of ipilimumab and two doses of nivolumab before surgery, the pMMR group with or without celecoxib. The primary objective was safety and feasibility; 40 patients with 21 dMMR and 20 pMMR tumors were treated, and 3 patients received nivolumab monotherapy in the safety run-in. Treatment was well tolerated and all patients underwent radical resections without delays, meeting the primary endpoint. Of the patients who received ipilimumab + nivolumab (20 dMMR and 15 pMMR tumors), 35 were evaluable for efficacy and translational endpoints. Pathological response was observed in 20/20 (100%; 95% exact confidence interval (CI): 86-100%) dMMR tumors, with 19 major pathological responses (MPRs, ≤10% residual viable tumor) and 12 pathological complete responses. In pMMR tumors, 4/15 (27%; 95% exact CI: 8-55%) showed pathological responses, with 3 MPRs and 1 partial response. CD8+PD-1+ T cell infiltration was predictive of response in pMMR tumors. These data indicate that neoadjuvant immunotherapy may have the potential to become the standard of care for a defined group of colon cancer patients when validated in larger studies with at least 3 years of disease-free survival data.

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

Consensus molecular subtype classification of colorectal adenomas.

Consensus molecular subtyping is an RNA expression-based classification system for colorectal cancer (CRC). Genomic alterations accumulate during CRC pathogenesis, including the premalignant adenoma stage, leading to changes in RNA expression. Only a minority of adenomas progress to malignancies, a transition that is associated with specific DNA copy number aberrations or microsatellite instability (MSI). We aimed to investigate whether colorectal adenomas can already be stratified into consensus molecular subtype (CMS) classes, and whether specific CMS classes are related to the presence of specific DNA copy number aberrations associated with progression to malignancy. RNA sequencing was performed on 62 adenomas and 59 CRCs. MSI status was determined with polymerase chain reaction-based methodology. DNA copy number was assessed by low-coverage DNA sequencing (n = 30) or array-comparative genomic hybridisation (n = 32). Adenomas were classified into CMS classes together with CRCs from the study cohort and from The Cancer Genome Atlas (n = 556), by use of the established CMS classifier. As a result, 54 of 62 (87%) adenomas were classified according to the CMS. The CMS3 'metabolic subtype', which was least common among CRCs, was most prevalent among adenomas (n = 45; 73%). One of the two adenomas showing MSI was classified as CMS1 (2%), the 'MSI immune' subtype. Eight adenomas (13%) were classified as the 'canonical' CMS2. No adenomas were classified as the 'mesenchymal' CMS4, consistent with the fact that adenomas lack invasion-associated stroma. The distribution of the CMS classes among adenomas was confirmed in an independent series. CMS3 was enriched with adenomas at low risk of progressing to CRC, whereas relatively more high-risk adenomas were observed in CMS2. We conclude that adenomas can be stratified into the CMS classes. Considering that CMS1 and CMS2 expression signatures may mark adenomas at increased risk of progression, the distribution of the CMS classes among adenomas is consistent with the proportion of adenomas expected to progress to CRC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.

Large variety in a panel of human colon cancer organoids in response to EZH2 inhibition.

EZH2 inhibitors have gained great interest for their use as anti-cancer therapeutics. However, most research has focused on EZH2 mutant cancers and recently adverse effects of EZH2 inactivation have come to light. To determine whether colorectal cancer cells respond to EZH2 inhibition and to explore which factors influence the degree of response, we treated a panel of 20 organoid lines derived from human colon tumors with different concentrations of the EZH2 inhibitor GSK126. The resulting responses were associated with mutation status, gene expression and responses to other drugs. We found that the response to GSK126 treatment greatly varied between organoid lines. Response associated with the mutation status of ATRX and PAX2, and correlated with BIK expression. It also correlated well with response to Nutlin-3a which inhibits MDM2-p53 interaction thereby activating p53 signaling. Sensitivity to EZH2 ablation depended on the presence of wild type p53, as tumor organoids became resistant when p53 was mutated or knocked down. Our exploratory study provides insight into which genetic factors predict sensitivity to EZH2 inhibition. In addition, we show that the response to EZH2 inhibition requires wild type p53. We conclude that a subset of colorectal cancer patients may benefit from EZH2-targeting therapies.

Prospective derivation of a living organoid biobank of colorectal cancer patients.

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.