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.

Enhanced Immunogenicity of Mitochondrial-Localized Proteins in Cancer Cells.

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.

Responsiveness to Immune Checkpoint Inhibitors Is Associated With a Peripheral Blood T-Cell Signature in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Although most patients with microsatellite instable (MSI) metastatic castration-resistant prostate cancer (mCRPC) respond to immune checkpoint blockade (ICB), only a small subset of patients with microsatellite stable (MSS) tumors have similar benefit. Biomarkers defining ICB-susceptible subsets of patients with MSS mCRPC are urgently needed. METHODS: Using next-generation T-cell repertoire sequencing, we explored immune signatures in 54 patients with MSS and MSI mCRPC who were treated with or without ICB. We defined subset-specific immune metrics as well as T-cell clusters and correlated the signatures with treatment benefit. RESULTS: Consistent overlaps between tumor and peripheral T-cell repertoires suggested that blood was an informative material to identify relevant T-cell signatures. We found considerably higher blood T-cell richness and diversity and more shared T-cell clusters with low generation probability (pGen) in MSI versus MSS mCRPC, potentially reflecting more complex T-cell responses because of a greater neoepitope load in the MSI subset. Interestingly, patients with MSS mCRPC with shared low pGen T-cell clusters showed significantly better outcomes with ICB, but not with other treatments, compared with patients without such clusters. Blood clearance of T-cell clusters on ICB treatment initiation seemed to be compatible with T-cell migration to the primary tumor or metastatic sites during the process of clonal replacement as described for other tumors receiving ICB. CONCLUSION: The MSI mCRPC subset shows a distinct T-cell signature that can be detected in blood. This signature points to immune parameters that could help identify a subset of patients with MSS mCRPC who may have an increased likelihood of responding to ICB or to combination approaches including ICB.

Tumor organoid-T-cell coculture systems.

T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8+ T-cell populations can be obtained from ~33-50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient.

T cell receptor next-generation sequencing reveals cancer-associated repertoire metrics and reconstitution after chemotherapy in patients with hematological and solid tumors.

The dynamics of immunoaging and the onset of immunoparesis in healthy individuals and cancer patients has been controversially discussed. Moreover, the role of chemotherapy on T cell regeneration needs further elucidation in light of novel immunotherapies that have become standard of care for many elderly cancer patients. We used next-generation immunosequencing to study T cell receptor (TCR) repertoire metrics on 346 blood samples from healthy individuals and cancer patients producing a dataset with around 8.8 million TCR reads. This analysis showed that decline of T cell diversity and increase in T cell clonality is a continuous process beginning in healthy individuals over 40 years of age. Untreated patients with both hematological and solid tumors showed blood TCR repertoires with significantly lower diversity and higher clonality as compared to healthy individuals across all decades. Loss in T cell diversity was essentially driven by a loss in richness in aging healthy individuals, while in cancer patients a loss in repertoire evenness was an additional contributing factor. Interestingly, chemotherapy did not impair the regeneration of blood TCR repertoire diversity to pre-treatment age-specific levels. Surprisingly, even patients over the age of 70 years receiving highly T cell toxic therapies reestablished their pre-treatment T cell diversity suggesting rebound thymic activity rather than recovery of T cell counts by peripheral expansion only. Taken together, these data suggest that human TCR repertoire metrics gradually deteriorate in the aging individual, but age-specific TCR metrics are restored after T cell depleting therapy even in elderly cancer patients.

High-Throughput Immunogenetics Reveals a Lack of Physiological T Cell Clusters in Patients With Autoimmune Cytopenias.

Autoimmune cytopenias (AIC) such as immune thrombocytopenia or autoimmune hemolytic anemia are claimed to be essentially driven by a dysregulated immune system. Using next-generation immunosequencing we profiled 59 T and B cell repertoires (TRB and IGH) of 25 newly diagnosed patients with primary or secondary (lymphoma-associated) AIC to test the hypothesis if these patients present a disease-specific immunological signature that could reveal pathophysiological clues and eventually be exploited as blood-based biomarker. Global TRB and IGH repertoire metrics as well as VJ gene usage distribution showed uniform characteristics for all lymphoma patients (high clonality and preferential usage of specific TRBV- and TRBJ genes), but no AIC-specific signature. Since T cell immune reactions toward antigens are unique and polyclonal, we clustered TCRß clones in-silico based on target recognition using the GLIPH (grouping of lymphocyte interactions by paratope hotspots) algorithm. This analysis revealed a considerable lack of physiological T cell clusters in patients with primary AIC. Interestingly, this signature did not discriminate between the different subentities of AIC and was also found in an independent cohort of 23 patients with active autoimmune hepatitis. Taken together, our data suggests that the identified T cell cluster signature could represent a blood biomarker of autoimmune conditions in general and should be functionally validated in future studies.

Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers.

Infiltration of human cancers by T cells is generally interpreted as a sign of immune recognition, and there is a growing effort to reactivate dysfunctional T cells at such tumor sites1. However, these efforts only have value if the intratumoral T cell receptor (TCR) repertoire of such cells is intrinsically tumor reactive, and this has not been established in an unbiased manner for most human cancers. To address this issue, we analyzed the intrinsic tumor reactivity of the intratumoral TCR repertoire of CD8+ T cells in ovarian and colorectal cancer-two tumor types for which T cell infiltrates form a positive prognostic marker2,3. Data obtained demonstrate that a capacity to recognize autologous tumor is limited to approximately 10% of intratumoral CD8+ T cells. Furthermore, in two of four patient samples tested, no tumor-reactive TCRs were identified, despite infiltration of their tumors by T cells. These data indicate that the intrinsic capacity of intratumoral T cells to recognize adjacent tumor tissue can be rare and variable, and suggest that clinical efforts to reactivate intratumoral T cells will benefit from approaches that simultaneously increase the quality of the intratumoral TCR repertoire.

Neoadjuvant versus adjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma.

Adjuvant ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) both improve relapse-free survival of stage III melanoma patients1,2. In stage IV disease, the combination of ipilimumab + nivolumab is superior to ipilimumab alone and also appears to be more effective than nivolumab monotherapy3. Preclinical work suggests that neoadjuvant application of checkpoint inhibitors may be superior to adjuvant therapy4. To address this question and to test feasibility, 20 patients with palpable stage III melanoma were 1:1 randomized to receive ipilimumab 3 mg kg-1 and nivolumab 1 mg kg-1, as either four courses after surgery (adjuvant arm) or two courses before surgery and two courses postsurgery (neoadjuvant arm). Neoadjuvant therapy was feasible, with all patients undergoing surgery at the preplanned time point. However in both arms, 9/10 patients experienced one or more grade 3/4 adverse events. Pathological responses were achieved in 7/9 (78%) patients treated in the neoadjuvant arm. None of these patients have relapsed so far (median follow-up, 25.6 months). We found that neoadjuvant ipilimumab + nivolumab expand more tumor-resident T cell clones than adjuvant application. While neoadjuvant therapy appears promising, with the current regimen it induced high toxicity rates; therefore, it needs further investigation to preserve efficacy but reduce toxicity.

Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids.

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.