Publications by authors named "Marcus O Butler"

67 Publications

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4 T cells.

Nat Biotechnol 2021 Mar 1. Epub 2021 Mar 1.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4 T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a K of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4 T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4 T-cell responses.
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http://dx.doi.org/10.1038/s41587-021-00836-4DOI Listing
March 2021

Biologic subtypes of melanoma predict survival benefit of combination anti-PD1+anti-CTLA4 immune checkpoint inhibitors versus anti-PD1 monotherapy.

J Immunother Cancer 2021 Jan;9(1)

Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

Purpose: Anti-programmed cell death protein 1 (PD1)±anti-cytotoxic T-lymphocyte associated protein 4 (CTLA4) immune checkpoint inhibitors (ICIs) are standard therapeutic options for metastatic melanoma. We assessed whether biologic subtype according to primary tumor type or genomic subtype can function as predictive biomarkers for anti-PD1±anti-CTLA4 ICI in patients with advanced melanoma.

Methods: We performed a single-center retrospective cohort analysis of patients who received anti-PD1±anti-CTLA4 ICI for advanced melanoma between 2012 and 2019. Primary tumor type, and mutation status, and other covariates were abstracted from chart review. Log-rank tests and multivariable Cox regression models were used to assess differences in clinical progression-free (cPFS) and overall survival (OS).

Results: We identified 230 patients who received 249 lines of anti-PD1±anti-CTLA4 ICI for unresectable or metastatic disease. Of these patients, 74% were cutaneous, 11% mucosal, 8% unknown primary and 7% acral. and mutations were identified in 35% and 28% of patients, respectively. In multivariable analyses of the entire cohort, acral or mucosal primary tumor type, >3 metastatic sites, elevated LDH were predictive of shorter cPFS and OS. Combination ICI therapy was associated with longer cPFS (HR 0.57, 95% CI 0.38 to 0.86, p=0.007) and OS (HR 0.42, 95% CI 0.28 to 0.65, p<0.001). Combination ICI was significantly associated with longer OS in unknown primary and mucosal melanoma. There was a non-significant trend toward longer OS with anti-PD1+anti-CTLA4 in cutaneous melanoma, but not in acral melanoma. In multivariable analyses, combination ICI was associated with longer OS in (HR 0.24, 95% CI 0.10 to 0.62, p=0.003, n=69) and V600E/K (HR 0.47, 95% CI 0.24 to 0.90, p=0.024, n=86) mutant melanoma but not wild-type (n=94) melanoma.

Conclusions: In our cohort, primary melanoma tumor type and genomic subtype were independent predictive markers of cPFS and OS for patients with metastatic melanoma receiving anti-PD1 ICI. Primary tumor type and genomic subtype-including NRAS-should be further evaluated in prospective clinical trials to determine their value as predictive markers. Biologic subtypes may facilitate clinical decision-making when recommending combination ICI treatment (anti-PD1±anti-CTLA4) versus anti-PD1 alone for patients with metastatic melanoma.
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http://dx.doi.org/10.1136/jitc-2020-001642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831745PMC
January 2021

Clinical impact of COVID-19 on patients with cancer treated with immune checkpoint inhibition.

J Immunother Cancer 2021 01;9(1)

European Institute of Oncology, Milan, Italy.

Background: Patients with cancer who are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to develop severe illness and die compared with those without cancer. The impact of immune checkpoint inhibition (ICI) on the severity of COVID-19 illness is unknown. The aim of this study was to investigate whether ICI confers an additional risk for severe COVID-19 in patients with cancer.

Methods: We analyzed data from 110 patients with laboratory-confirmed SARS-CoV-2 while on treatment with ICI without chemotherapy in 19 hospitals in North America, Europe and Australia. The primary objective was to describe the clinical course and to identify factors associated with hospital and intensive care (ICU) admission and mortality.

Findings: Thirty-five (32%) patients were admitted to hospital and 18 (16%) died. All patients who died had advanced cancer, and only four were admitted to ICU. COVID-19 was the primary cause of death in 8 (7%) patients. Factors independently associated with an increased risk for hospital admission were ECOG ≥2 (OR 39.25, 95% CI 4.17 to 369.2, p=0.0013), treatment with combination ICI (OR 5.68, 95% CI 1.58 to 20.36, p=0.0273) and presence of COVID-19 symptoms (OR 5.30, 95% CI 1.57 to 17.89, p=0.0073). Seventy-six (73%) patients interrupted ICI due to SARS-CoV-2 infection, 43 (57%) of whom had resumed at data cut-off.

Interpretation: COVID-19-related mortality in the ICI-treated population does not appear to be higher than previously published mortality rates for patients with cancer. Inpatient mortality of patients with cancer treated with ICI was high in comparison with previously reported rates for hospitalized patients with cancer and was due to COVID-19 in almost half of the cases. We identified factors associated with adverse outcomes in ICI-treated patients with COVID-19.
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http://dx.doi.org/10.1136/jitc-2020-001931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817383PMC
January 2021

PD-L1 blockade in combination with inhibition of MAPK oncogenic signaling in patients with advanced melanoma.

Nat Commun 2020 12 7;11(1):6262. Epub 2020 Dec 7.

Gustave Roussy Cancer Campus and Paris-Saclay University, Villejuif, France.

Combining PD-L1 blockade with inhibition of oncogenic mitogen-activated protein kinase (MAPK) signaling may result in long-lasting responses in patients with advanced melanoma. This phase 1, open-label, dose-escalation and -expansion study (NCT02027961) investigated safety, tolerability and preliminary efficacy of durvalumab (anti-PD-L1) combined with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for patients with BRAF-mutated melanoma (cohort A, n = 26), or durvalumab and trametinib given concomitantly (cohort B, n = 20) or sequentially (cohort C, n = 22) for patients with BRAF-wild type melanoma. Adverse events and treatment discontinuation rates were more common than previously reported for these agents given as monotherapy. Objective responses were observed in 69.2% (cohort A), 20.0% (cohort B) and 31.8% (cohort C) of patients, with evidence of improved tumor immune infiltration and durable responses in a subset of patients with available biopsy samples. In conclusion, combined MAPK inhibition and anti-PD-L1 therapy may provide treatment options for patients with advanced melanoma.
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http://dx.doi.org/10.1038/s41467-020-19810-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721806PMC
December 2020

Adjuvant nivolumab versus ipilimumab in resected stage IIIB-C and stage IV melanoma (CheckMate 238): 4-year results from a multicentre, double-blind, randomised, controlled, phase 3 trial.

Lancet Oncol 2020 11 19;21(11):1465-1477. Epub 2020 Sep 19.

Laura and Isaac Perlmutter Cancer Center, NYU Langone Health, New York, NY, USA.

Background: Previously, findings from CheckMate 238, a double-blind, phase 3 adjuvant trial in patients with resected stage IIIB-C or stage IV melanoma, showed significant improvements in recurrence-free survival and distant metastasis-free survival with nivolumab versus ipilimumab. This report provides updated 4-year efficacy, initial overall survival, and late-emergent safety results.

Methods: This multicentre, double-blind, randomised, controlled, phase 3 trial was done in 130 academic centres, community hospitals, and cancer centres across 25 countries. Patients aged 15 years or older with resected stage IIIB-C or IV melanoma and an Eastern Cooperative Oncology Group performance status of 0 or 1 were randomly assigned (1:1) to receive nivolumab or ipilimumab via an interactive voice response system and stratified according to disease stage and baseline PD-L1 status of tumour cells. Patients received intravenous nivolumab 3 mg/kg every 2 weeks or intravenous ipilimumab 10 mg/kg every 3 weeks for four doses, and then every 12 weeks until 1 year of treatment, disease recurrence, unacceptable toxicity, or withdrawal of consent. The primary endpoint was recurrence-free survival by investigator assessment, and overall survival was a key secondary endpoint. Efficacy analyses were done in the intention-to-treat population (all randomly assigned patients). All patients who received at least one dose of study treatment were included in the safety analysis. The results presented in this report reflect the 4-year update of the ongoing study with a database lock date of Jan 30, 2020. This study is registered with ClinicalTrials.gov, NCT02388906.

Findings: Between March 30 and Nov 30, 2015, 906 patients were assigned to nivolumab (n=453) or ipilimumab (n=453). Median follow-up was 51·1 months (IQR 41·6-52·7) with nivolumab and 50·9 months (36·2-52·3) with ipilimumab; 4-year recurrence-free survival was 51·7% (95% CI 46·8-56·3) in the nivolumab group and 41·2% (36·4-45·9) in the ipilimumab group (hazard ratio [HR] 0·71 [95% CI 0·60-0·86]; p=0·0003). With 211 (100 [22%] of 453 patients in the nivolumab group and 111 [25%] of 453 patients in the ipilimumab group) of 302 anticipated deaths observed (about 73% of the originally planned 88% power needed for significance), 4-year overall survival was 77·9% (95% CI 73·7-81·5) with nivolumab and 76·6% (72·2-80·3) with ipilimumab (HR 0·87 [95% CI 0·66-1·14]; p=0·31). Late-emergent grade 3-4 treatment-related adverse events were reported in three (1%) of 452 and seven (2%) of 453 patients. The most common late-emergent treatment-related grade 3 or 4 adverse events reported were diarrhoea, diabetic ketoacidosis, and pneumonitis (one patient each) in the nivolumab group, and colitis (two patients) in the ipilimumab group. Two previously reported treatment-related deaths in the ipilimumab group were attributed to study drug toxicity (marrow aplasia in one patient and colitis in one patient); no further treatment-related deaths were reported.

Interpretation: At a minimum of 4 years' follow-up, nivolumab demonstrated sustained recurrence-free survival benefit versus ipilimumab in resected stage IIIB-C or IV melanoma indicating a long-term treatment benefit with nivolumab. With fewer deaths than anticipated, overall survival was similar in both groups. Nivolumab remains an efficacious adjuvant treatment for patients with resected high-risk melanoma, with a safety profile that is more tolerable than that of ipilimumab.

Funding: Bristol Myers Squibb and Ono Pharmaceutical.
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http://dx.doi.org/10.1016/S1470-2045(20)30494-0DOI Listing
November 2020

An open-label, phase II multicohort study of an oral hypomethylating agent CC-486 and durvalumab in advanced solid tumors.

J Immunother Cancer 2020 08;8(2)

Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

Purpose: To evaluate whether administration of the oral DNA hypomethylating agent CC-486 enhances the poor response rate of immunologically 'cold' solid tumors to immune checkpoint inhibitor durvalumab.

Experimental Design: PD-L1/PD-1 inhibitor naïve patients with advanced microsatellite stable colorectal cancer; platinum resistant ovarian cancer; and estrogen receptor positive, HER2 negative breast cancer were enrolled in this single-institution, investigator-initiated trial. Two 28 day regimens, regimen A (CC-486 300 mg QD Days 1-14 (cycles 1-3 only) in combination with durvalumab 1500 mg intravenous day 15) and regimen B (CC-486 100 mg QD days 1-21 (cycle 1 and beyond), vitamin C 500 mg once a day continuously and durvalumab 1500 mg intravenous day 15) were investigated. Patients underwent paired tumor biopsies and serial peripheral blood mononuclear cells (PBMCs) collection for immune-profiling, transcriptomic and epigenomic analyzes.

Results: A total of 28 patients were enrolled, 19 patients treated on regimen A and 9 on regimen B. The combination of CC-486 and durvalumab was tolerable. Regimen B, with a lower dose of CC-486 extended over a longer treatment course, showed less grade 3/4 adverse effects. Global LINE-1 methylation assessment of serial PBMCs and genome-wide DNA methylation profile in paired tumor biopsies demonstrated minimal changes in global methylation in both regimens. The lack of robust tumor DNA demethylation was accompanied by an absence of the expected 'viral mimicry' inflammatory response, and consequently, no clinical responses were observed. The disease control rate was 7.1%. The median progression-free survival was 1.9 months (95% CI 1.5 to 2.3) and median overall survival was 5 months (95% CI 4.5 to 10).

Conclusions: The evaluated treatment schedules of CC-486 in combination with durvalumab did not demonstrate robust pharmacodynamic or clinical activity in selected immunologically cold solid tumors. Lessons learned from this biomarker-rich study should inform continued drug development efforts using these agents.

Trial Registration Number: NCT02811497.
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http://dx.doi.org/10.1136/jitc-2020-000883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406114PMC
August 2020

PD-1 Blockade in Anaplastic Thyroid Carcinoma.

J Clin Oncol 2020 08 4;38(23):2620-2627. Epub 2020 May 4.

Oregon Health & Science University, Portland, OR.

Purpose: Anaplastic thyroid carcinoma is an aggressive malignancy that is almost always fatal and lacks effective systemic treatment options for patients with -wild type disease. As part of a phase I/II study in patients with advanced/metastatic solid tumors, patients with anaplastic thyroid carcinoma were treated with spartalizumab, a humanized monoclonal antibody against the programmed death-1 (PD-1) receptor.

Methods: We enrolled patients with locally advanced and/or metastatic anaplastic thyroid carcinoma in a phase II cohort of the study. Patients received 400 mg spartalizumab intravenously, once every 4 weeks. The overall response rate was determined according to RECIST v1.1.

Results: Forty-two patients were enrolled. Adverse events were consistent with those previously observed with PD-1 blockade. Most common treatment-related adverse events were diarrhea (12%), pruritus (12%), fatigue (7%), and pyrexia (7%). The overall response rate was 19%, including three patients with a complete response and five with a partial response. Most patients had baseline tumor biopsies positive for PD-L1 expression (n = 28/40 evaluable), and response rates were higher in PD-L1-positive (8/28; 29%) versus PD-L1-negative (0/12; 0%) patients. The highest rate of response was observed in the subset of patients with PD-L1 ≥ 50% (6/17; 35%). Responses were seen in both -nonmutant and -mutant patients and were durable, with a 1-year survival of 52.1% in the PD-L1-positive population.

Conclusion: To our knowledge, this is the first clinical trial to show responsiveness of anaplastic thyroid carcinoma to PD-1 blockade.
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http://dx.doi.org/10.1200/JCO.19.02727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476256PMC
August 2020

Survival in Early Phase Immuno-Oncology Trials: Development and Validation of a Prognostic Index.

JNCI Cancer Spectr 2019 Dec 19;3(4):pkz071. Epub 2019 Sep 19.

Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada.

Background: Immuno-oncology (IO) is rapidly evolving in early drug development. We aimed to develop and prospectively validate a prognostic index for patients treated in IO phase I trials to assist with patient selection.

Methods: The development cohort included 192 advanced solid tumor patients treated in 13 IO phase I trials, targeting immune checkpoint and/or co-stimulatory molecules. A prognostic scoring system was developed from multivariate survival analysis of 10 clinical factors, and subsequently validated in two independent validation cohorts (n = 152 and n = 80).

Results: In the development cohort, median age was 57.5 years (range = 20.4-84.8 years). Median progression-free survival and overall survival (OS) were 13.4 and 73.6 weeks, respectively, 90-day mortality was 16%, and overall response rate was 20%. In multivariate analysis, Eastern Cooperative Oncology Group performance status greater than or equal to 1 (hazard ratio [HR] = 3.2, 95% confidence interval [CI] = 1.8 to 5.7;  < .001), number of metastatic sites greater than 2 (HR = 2.0, 95% CI = 1.3 to 3.1;  = .003), and albumin less than the lower limit of normal (HR = 1.8, 95% CI = 1.2 to 2.7;  = .007) were independent prognostic factors; comprising the Princess Margaret Immuno-oncology Prognostic Index (PM-IPI). Patients with a score of 2-3 compared with patients with a score of 0-1 had shorter OS (HR = 3.4, 95% CI = 1.9 to 6.1;  < .001), progression-free survival (HR = 2.3, 95% CI = 1.7 to 3.2;  < .001), higher 90-day mortality (odds ratio = 8.1, 95% CI = 3.0 to 35.4;  < .001), and lower overall response rate (odds ratio = 0.4, 95% CI = 0.2 to 0.8; = .019). The PM-IPI retained prognostic ability in both validation cohorts and performed better than previously published phase I prognostic scores for predicting OS in all three cohorts.

Conclusions: The PM-IPI is a validated prognostic score for patients treated in phase I IO trials and may aid in improving patient selection.
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http://dx.doi.org/10.1093/jncics/pkz071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050022PMC
December 2019

Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.

Cancer Immunol Res 2020 07 22;8(7):926-936. Epub 2020 Apr 22.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the , and genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with and double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0508DOI Listing
July 2020

Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma.

Elife 2020 04 21;9. Epub 2020 Apr 21.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Canada.

HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.
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http://dx.doi.org/10.7554/eLife.53244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7234812PMC
April 2020

A first-in-human phase 1 dose escalation study of spartalizumab (PDR001), an anti-PD-1 antibody, in patients with advanced solid tumors.

J Immunother Cancer 2020 03;8(1)

Sarah Cannon Research Institute, Nashville, Tennessee, USA.

Background: Spartalizumab is a humanized IgG4κ monoclonal antibody that binds programmed death-1 (PD-1) and blocks its interaction with PD-L1 and PD-L2. This phase 1/2 study was designed to assess the safety, pharmacokinetics, and preliminary efficacy of spartalizumab in patients with advanced or metastatic solid tumors.

Methods: In the phase 1 part of the study, 58 patients received spartalizumab, intravenously, at doses of 1, 3, or 10 mg/kg, administered every 2 weeks (Q2W), or 3 or 5 mg/kg every 4 weeks (Q4W).

Results: Patients had a wide range of tumor types, most commonly sarcoma (28%) and metastatic renal cell carcinoma (10%); other tumor types were reported in ≤3 patients each. Most patients (93%) had received prior antineoplastic therapy (median three prior lines) and two-thirds of the population had tumor biopsies negative for PD-L1 expression at baseline. The maximum tolerated dose was not reached. The recommended phase 2 doses were selected as 400 mg Q4W or 300 mg Q3W. No dose-limiting toxicities were observed, and adverse events included those typical of other PD-1 antibodies. The most common treatment-related adverse events of any grade were fatigue (22%), diarrhea (17%), pruritus (14%), hypothyroidism (10%), and nausea (10%). Partial responses occurred in two patients (response rate 3.4%); one with atypical carcinoid tumor of the lung and one with anal cancer. Paired tumor biopsies from patients taken at baseline and on treatment suggested an on-treatment increase in CD8+ lymphocyte infiltration in patients with clinical benefit.

Conclusions: Spartalizumab was well tolerated at all doses tested in patients with previously treated advanced solid tumors. On-treatment immune activation was seen in tumor biopsies; however, limited clinical activity was reported in this heavily pretreated, heterogeneous population. The phase 2 part of this study is ongoing in select tumor types.

Trial Registration Number: NCT02404441.
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http://dx.doi.org/10.1136/jitc-2020-000530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073791PMC
March 2020

Cancer patients' experiences with immune checkpoint modulators: A qualitative study.

Cancer Med 2020 05 2;9(9):3015-3022. Epub 2020 Mar 2.

Princess Margaret Cancer Centre, University Health Network, Toronto, Canada.

Background: Minimal qualitative data exist on the experiences of cancer patients treated with immune checkpoint inhibitors or costimulatory antibodies. Understanding the day to day experiences of patients being treated with immune checkpoint modulators, and how these relate to their health-related quality of life, can inform future research and lead to better clinical decision-making and care. We report here the first in depth qualitative study to consider patients' diverse and complex experiences with immune checkpoint modulators, with a focus on side effects and how these impact daily life.

Methods: This single-center qualitative study was based on focus groups and semistructured interviews. Patients who were being treated or who had been treated with immune checkpoint modulators within the last year for a range of cancer diagnoses were recruited. Interpretive description informed our inductive, iterative approach to analysis.

Results: Eight themes were identified, characterizing the complexity of these patients' lived experiences: major categories of side effects experienced and how they impacted patient well-being; the heterogeneous nature of side effects experienced; living with uncertainty; reframing the meaning and severity of SEs; focus on survival, hope, and being positive; acceptance and adaptation; feeling supported; and faith in medical innovation. Throughout their accounts, participants highlighted the profound impact that immune checkpoint modulators had on their daily lives.

Conclusion: This is the first in-depth qualitative study into patient accounts of their experiences of treatment with immune checkpoint modulators, related side effects, and how it impacted their daily lives. This research is an integral initial step in developing an instrument that will assess treatment-related side effects in patients treated with this form of therapy.
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http://dx.doi.org/10.1002/cam4.2940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196048PMC
May 2020

Phase II Trial of Cabozantinib in Recurrent/Metastatic Endometrial Cancer: A Study of the Princess Margaret, Chicago, and California Consortia (NCI9322/PHL86).

Clin Cancer Res 2020 06 28;26(11):2477-2486. Epub 2020 Jan 28.

Princess Margaret Cancer Centre, Toronto, Ontario, Canada.

Purpose: The relevance of the MET/hepatocyte growth factor pathway in endometrial cancer tumor biology supports the clinical evaluation of cabozantinib in this disease.

Patients And Methods: PHL86/NCI#9322 (NCT01935934) is a single arm study that evaluated cabozantinib (60 mg once daily) in women with endometrial cancer with progression after chemotherapy. Coprimary endpoints were response rate and 12-week progression-free-survival (PFS). Patients with uncommon histology endometrial cancer (eg, carcinosarcoma and clear cell) were enrolled in a parallel exploratory cohort.

Results: A total of 102 patients were accrued. Among 36 endometrioid histology patients, response rate was 14%, 12-week PFS rate was 67%, and median PFS was 4.8 months. In serous cohort of 34 patients, response rate was 12%, 12-week PFS was 56%, and median PFS was 4.0 months. In a separate cohort of 32 patients with uncommon histology endometrial cancer (including carcinosarcoma), response rate was 6% and 12-week PFS was 47%. Six patients were on treatment for >12 months, including two for >30 months. Common cabozantinib-related toxicities (>30% patients) included hypertension, fatigue, diarrhea, nausea, and hand-foot syndrome. Gastrointestinal fistula/perforation occurred in four of 70 (6%) patients with serous/endometrioid cancer and five of 32 (16%) patients in exploratory cohort. We observed increased frequency of responses with somatic mutation [four partial responses (PRs) in 10 patients, median PFS 7.6 months] and concurrent and mutations (three PRs in 12 patients, median PFS 5.9 months).

Conclusions: Cabozantinib has activity in serous and endometrioid histology endometrial cancer. These results support further evaluation in genomically characterized patient cohorts.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269808PMC
June 2020

Development of the Functional Assessment of Cancer Therapy-Immune Checkpoint Modulator (FACT-ICM): A toxicity subscale to measure quality of life in patients with cancer who are treated with ICMs.

Cancer 2020 04 8;126(7):1550-1558. Epub 2020 Jan 8.

Applied Health Research Centre, Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada.

Background: Patients with cancer who are treated with immune checkpoint modulators (ICMs) have their health-related quality of life (HRQOL) measured using general patient-reported outcome (PRO) tools. To the authors' knowledge, no instrument has been developed to date specifically for patients treated with ICMs. The objective of the current study was to develop a toxicity subscale PRO instrument for patients treated with ICMs to assess HRQOL.

Methods: Input was collected from a systematic review as well as patients and physicians experienced with ICM treatment. Descriptive thematic analysis was used to evaluate the qualitative data obtained from patient focus groups and interviews, which informed an initial list of items that described ICM side effects and their impact on HRQOL. These inputs informed item generation and/or reduction to develop a toxicity subscale.

Results: Focus groups and individual interviews with 37 ICM-treated patients generated an initial list of 176 items. After a first round of item reduction that produced a shortened list of 76 items, 16 physicians who care for patients who are treated with ICMs were surveyed with a list of 49 patient-reported side effects and 11 physicians participated in follow-up interviews. A second round of item reduction was informed by the physician responses to produce a list of 25 items.

Conclusions: To the authors' knowledge, this 25-item list is the first HRQOL-focused toxicity subscale for patients treated with ICMs and was developed in accordance with US Food and Drug Administration guidelines, which prioritize patient input in developing PRO tools. The subscale will be combined with the Functional Assessment of Cancer Therapy-General (FACT-G) to form the FACT-ICM. Prior to recommending the formal use of this PRO instrument, the authors will evaluate its validity and reliability in longitudinal studies involving substantially more patients.
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http://dx.doi.org/10.1002/cncr.32692DOI Listing
April 2020

GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment.

Sci Immunol 2019 12;4(42)

Tumor Immunotherapy Program, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8 T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.
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http://dx.doi.org/10.1126/sciimmunol.aax8189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201901PMC
December 2019

Randomized Phase II Trial and Tumor Mutational Spectrum Analysis from Cabozantinib versus Chemotherapy in Metastatic Uveal Melanoma (Alliance A091201).

Clin Cancer Res 2020 02 26;26(4):804-811. Epub 2019 Sep 26.

Columbia University Herbert Irving Comprehensive Cancer Center, New York, New York.

Purpose: The surface receptor MET is highly expressed on primary uveal melanoma; MET inhibitors demonstrated early clinical signals of efficacy in slowing uveal melanoma growth. The primary objective of our study was to compare the progression-free survival rate at 4 months (PFS4) of patients with uveal melanoma treated with cabozantinib or chemotherapy.

Patients And Methods: Patients with metastatic uveal melanoma and RECIST measurable disease were randomized 2:1 to receive either cabozantinib (arm 1) versus temozolomide or dacarbazine (arm 2) with restaging imaging every two cycles. Cross-over from arm 2 to cabozantinib after progression was allowed (arm 2X). Available tumor specimens were analyzed by whole-exome sequencing (WES) and results were correlated with outcome.

Results: Forty-six eligible patients were accrued with 31, 15, and 9 in arms 1, 2, and 2X, respectively. Median lines of prior therapy, including hepatic embolization, were two. Rates of PFS4 in arm 1 and arm 2 were 32.3% and 26.7% ( = 0.35), respectively, with median PFS time of 60 and 59 days ( = 0.964; HR = 0.99). Median overall survival (OS) was 6.4 months and 7.3 months ( = 0.580; HR = 1.21), respectively. Grade 3-4 Common Terminology Criteria for Adverse Events were present in 61.3%, 46.7%, and 37.5% in arms 1, 2, and 2X, respectively. WES demonstrated a mean tumor mutational burden of 1.53 mutations/Mb and did not separate OS ≤ or >1 year ( = 0.14). Known mutations were identified by WES and novel mutations were nominated.

Conclusions: MET/VEGFR blockade with cabozantinib demonstrated no improvement in PFS but an increase in toxicity relative to temozolomide/dacarbazine in metastatic uveal melanoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055933PMC
February 2020

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP.

J Autoimmun 2019 08 8;102:114-125. Epub 2019 May 8.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, M5G 2M9, Canada; Department of Immunology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. Electronic address:

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPβ chain (DP). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4 T cells.
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http://dx.doi.org/10.1016/j.jaut.2019.04.023DOI Listing
August 2019

An interim report on the investigator-initiated phase 2 study of pembrolizumab immunological response evaluation (INSPIRE).

J Immunother Cancer 2019 03 13;7(1):72. Epub 2019 Mar 13.

Princess Margaret Cancer Centre, University Health Network, Toronto, Canada.

Background: Immune checkpoint inhibitors (ICIs) demonstrate unprecedented efficacy in multiple malignancies; however, the mechanisms of sensitivity and resistance are poorly understood and predictive biomarkers are scarce. INSPIRE is a phase 2 basket study to evaluate the genomic and immune landscapes of peripheral blood and tumors following pembrolizumab treatment.

Methods: Patients with incurable, locally advanced or metastatic solid tumors that have progressed on standard therapy, or for whom no standard therapy exists or standard therapy was not deemed appropriate, received 200 mg pembrolizumab intravenously every three weeks. Blood and tissue samples were collected at baseline, during treatment, and at progression. One core biopsy was used for immunohistochemistry and the remaining cores were pooled and divided for genomic and immune analyses. Univariable analysis of clinical, genomic, and immunophenotyping parameters was conducted to evaluate associations with treatment response in this exploratory analysis.

Results: Eighty patients were enrolled from March 21, 2016 to June 1, 2017, and 129 tumor and 382 blood samples were collected. Immune biomarkers were significantly different between the blood and tissue. T cell PD-1 was blocked (≥98%) in the blood of all patients by the third week of treatment. In the tumor, 5/11 (45%) and 11/14 (79%) patients had T cell surface PD-1 occupance at weeks six and nine, respectively. The proportion of genome copy number alterations and abundance of intratumoral 4-1BB+ PD-1+ CD8 T cells at baseline (P < 0.05), and fold-expansion of intratumoral CD8 T cells from baseline to cycle 2-3 (P < 0.05) were associated with treatment response.

Conclusion: This study provides technical feasibility data for correlative studies. Tissue biopsies provide distinct data from the blood and may predict response to pembrolizumab.
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http://dx.doi.org/10.1186/s40425-019-0541-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417194PMC
March 2019

Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities.

Cancer 2019 04 15;125(8):1341-1349. Epub 2019 Feb 15.

Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Background: A subset of patients treated with immune checkpoint inhibitors experience an accelerated tumor growth rate (TGR) in comparison with pretreatment kinetics; this is known as hyperprogression. This study assessed the relation between hyperprogressive disease (HPD) and treatment-related toxicity and clinical factors.

Methods: This study reviewed patients with solid tumors who were enrolled in early-phase immunotherapy trials at Princess Margaret Cancer Centre between August 2012 and September 2016 and had computed tomography scans in the pre-immunotherapy (reference) and on-immunotherapy (experimental) periods. HPD was defined as progression according to Response Evaluation Criteria in Solid Tumors 1.1 at the first on-treatment scan and a ≥2-fold increase in TGR between the reference and experimental periods. Treatment-related toxicities requiring systemic therapy, drug delays, or discontinuation were considered clinically significant adverse events (CSAEs).

Results: Of 352 patients, 182 were eligible for analysis. The median age was 60 years, and 54% were male. The Eastern Cooperative Oncology Group performance status was 0 (32%) or 1 (68%). The Royal Marsden Hospital (RMH) prognostic score was 0/1 in 59%. Single-agent immunotherapy was given to 80% of the patients. Most patients (89%) received anti-programmed death (ligand) 1 antibodies alone or in combination with other therapies. HPD occurred in 12 of 182 patients (7%). A higher proportion of females was seen among HPD patients (P = .01), but no association with age, performance status, tumor type, RMH prognostic score, combination immunotherapy, or CSAEs was found. The 1-year overall survival rate was 28% for HPD patients and 53% for non-HPD patients (hazard ratio, 1.7; 95% confidence interval, 0.9-3.3; P = .11).

Conclusions: HPD was observed in 7% of patients with solid tumors treated with immunotherapy. HPD was not associated with CSAEs, age, tumor type, or the type of immunotherapy but was more common in females.
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http://dx.doi.org/10.1002/cncr.31999DOI Listing
April 2019

Phase II clinical trial of adoptive cell therapy for patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and low-dose interleukin-2.

Cancer Immunol Immunother 2019 May 11;68(5):773-785. Epub 2019 Feb 11.

Tumor Immunotherapy Program, Princess Margaret Cancer Centre, Toronto, Canada.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown significant clinical benefit, but is limited by toxicities due to a requirement for post-infusion interleukin-2 (IL-2), for which high dose is standard. To assess a modified TIL protocol using lower dose IL-2, we performed a single institution phase II protocol in unresectable, metastatic melanoma. The primary endpoint was response rate. Secondary endpoints were safety and assessment of immune correlates following TIL infusion. Twelve metastatic melanoma patients were treated with non-myeloablative lymphodepleting chemotherapy, TIL, and low-dose subcutaneous IL-2 (125,000 IU/kg/day, maximum 9-10 doses over 2 weeks). All but one patient had previously progressed after treatment with immune checkpoint inhibitors. No unexpected adverse events were observed, and patients received an average of 6.8 doses of IL-2. By RECIST v1.1, two patients experienced a partial response, one patient had an unconfirmed partial response, and six had stable disease. Biomarker assessment confirmed an increase in IL-15 levels following lymphodepleting chemotherapy as expected and a lack of peripheral regulatory T-cell expansion following protocol treatment. Interrogation of the TIL infusion product and monitoring of the peripheral blood following infusion suggested engraftment of TIL. In one responding patient, a population of T cells expressing a T-cell receptor Vβ chain that was dominant in the infusion product was present at a high percentage in peripheral blood more than 2 years after TIL infusion. This study shows that this protocol of low-dose IL-2 following adoptive cell transfer of TIL is feasible and clinically active. (ClinicalTrials.gov identifier NCT01883323.).
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http://dx.doi.org/10.1007/s00262-019-02307-xDOI Listing
May 2019

mutations in high grade serous ovarian cancer and impact on clinical outcomes: a comparison of next generation sequencing and bioinformatics analyses.

Int J Gynecol Cancer 2019 Feb 18;29(2):346-352. Epub 2019 Jan 18.

Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Ontario, Canada

Objective: Mutations in are found in the majority of high grade serous ovarian cancers, leading to gain of function or loss of function of its protein product, p53, involved in oncogenesis. There have been conflicting reports as to the impact of the type of these on prognosis. We aim to further elucidate this relationship in our cohort of patients.

Methods: 229 patients with high grade serous ovarian cancer underwent tumor profiling through an institutional molecular screening program with targeted next generation sequencing. mutations were classified using methods previously described in the literature. Immunohistochemistry on formalin-fixed paraffin embedded tissue was used to assess for mutation. Using divisive hierarchal clustering, we generated patient clusters with similar clinicopathologic characteristics to investigate differences in outcomes.

Results: Six different classification schemes of mutations were studied. These did not show an association with first platinum-free interval or overall survival. Next generation sequencing reliably predicted mutation in 80% of cases, similar to the proportion detected by immunohistochemistry. Divisive hierarchical clustering generated four main clusters, with cluster 3 having a significantly worse prognosis (p<0.0001; log-rank test). This cluster had a higher concentration of gain of function mutations and these patients were less likely to have undergone optimal debulking surgery.

Conclusions: Different classifications of mutations did not show an impact on outcomes in this study. Immunohistochemistry was a good predictor for mutation. Cluster analysis showed that a subgroup of patients with gain of function mutations (cluster 3) had a worse prognosis.
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http://dx.doi.org/10.1136/ijgc-2018-000087DOI Listing
February 2019

CapTCR-seq: hybrid capture for T-cell receptor repertoire profiling.

Blood Adv 2018 12;2(23):3506-3514

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

Mature T-cell lymphomas consisting of an expanded clonal population of T cells that possess common rearrangements of the T-cell receptor (TCR) encoding genes can be identified and monitored using molecular methods of T-cell repertoire analysis. We have developed a hybrid-capture method that enriches DNA sequencing libraries for fragments encoding rearranged TCR genes from all 4 loci in a single reaction. We use this method to describe the TCR repertoires of 63 putative lymphoma clinical isolates, 7 peripheral blood mononuclear cell (PBMC) populations, and a collection of tumor infiltrating lymphocytes. Dominant Variable (V) and Joining (J) gene pair rearrangements in cancer cells were confirmed by polymerase chain reaction (PCR) amplification and Sanger sequencing; clonality assessment of clinical isolates using BIOMED-2 methods showed agreement for 73% and 77% of samples at the β and γ loci, respectively, whereas β locus V and J allele prevalence in PBMCs were well correlated with results from commercial PCR-based DNA sequencing assays ( = 0.94 with Adaptive ImmunoSEQ, 0.77-0.83 with Invivoscribe LymphoTrack TRB Assay). CapTCR-seq allows for rapid, high-throughput and flexible characterization of dominant clones within TCR repertoire that will facilitate quantitative analysis of patient samples and enhance sensitivity of tumor surveillance over time.
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http://dx.doi.org/10.1182/bloodadvances.2017014639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290103PMC
December 2018

Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells.

J Autoimmun 2019 02 11;97:10-21. Epub 2018 Oct 11.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, M5G 2M9, Canada; Department of Immunology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. Electronic address:

Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3 T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions.
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http://dx.doi.org/10.1016/j.jaut.2018.09.011DOI Listing
February 2019

A Clinical and Molecular Phase II Trial of Oral ENMD-2076 in Ovarian Clear Cell Carcinoma (OCCC): A Study of the Princess Margaret Phase II Consortium.

Clin Cancer Res 2018 12 14;24(24):6168-6174. Epub 2018 Aug 14.

Princess Margaret Cancer Centre, Division of Medical Oncology and Hematology, Toronto, Ontario, Canada.

Purpose: Patients with recurrent ovarian clear cell carcinoma (OCCC) have limited effective options due to chemoresistance. A phase II study was designed to assess the activity of ENMD-2076, an oral multitarget kinase selective against Aurora A and VEGFR.

Patients And Methods: This multicenter phase II study included patients with recurrent OCCC who received prior platinum-based chemotherapy. Primary endpoints were objective response and 6-month progression-free survival (PFS) rates. Correlative analyses include ARID1A and PTEN expression by IHC and gene sequencing with a targeted custom capture next-generation sequencing panel.

Results: Forty patients were enrolled with a median age of 54, of which 38 patients were evaluable. ENMD-2076 was well tolerated with main related grade 3 toxicities being hypertension (28%), proteinuria (10%), and diarrhea (10%). Best response was partial response for 3 patients (1 unconfirmed) and stable disease for 26 patients. The overall 6-month PFS rate was 22% and differed according to ARID1A expression (ARIDIA vs. ARID1A; 33% vs. 12%, = 0.023). PTEN-positive expression was observed in 20 of 36 patients, and there was no correlation with outcome. Median PFS in patients with wild-type versus -mutated group was 5 versus 3.7 months ( = 0.049). Molecular profiling showed variants in (27%), (26%), and (7%). The patient with the longest treatment duration (22 months) was wild-type, diploid PTEN with putative biallelic inactivation of .

Conclusions: Single-agent ENMD-2076 did not meet the preset bar for efficacy. Loss of ARID1A correlated with better PFS on ENMD-2076 and warrants further investigation as a potential predictive biomarker.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1244DOI Listing
December 2018

Response to Immune Checkpoint Inhibition in Two Patients with Alveolar Soft-Part Sarcoma.

Cancer Immunol Res 2018 09 17;6(9):1001-1007. Epub 2018 Jul 17.

Department of Medical Oncology and Hematology, Princess Margaret Cancer Centre, Toronto, Canada.

Alveolar soft-part sarcoma (ASPS) is a morphologically distinctive mesenchymal tumor characterized by a canonical ASPL-TFE3 fusion product. In the metastatic setting, standard cytotoxic chemotherapies are typically ineffective. Studies have suggested modest clinical response to multitargeted receptor tyrosine kinase inhibitors. Here, we report sustained partial responses in two patients with immune checkpoint inhibition treated with either durvalumab (anti-PD-L1) alone or in combination with tremelimumab (anti-CTLA-4), which appeared unrelated to tumor immune infiltrates or mutational burden. Genomic analysis of these patients, and other cases of ASPS, demonstrated molecular mismatch-repair deficiency signatures. These findings suggest that immune checkpoint blockade may be a useful therapeutic strategy for ASPS. .
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0037DOI Listing
September 2018

DOT1L inhibition attenuates graft-versus-host disease by allogeneic T cells in adoptive immunotherapy models.

Nat Commun 2018 05 15;9(1):1915. Epub 2018 May 15.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 2M9, Canada.

Adoptive T-cell therapy is a promising therapeutic approach for cancer patients. The use of allogeneic T-cell grafts will improve its applicability and versatility provided that inherent allogeneic responses are controlled. T-cell activation is finely regulated by multiple signaling molecules that are transcriptionally controlled by epigenetic mechanisms. Here we report that inhibiting DOT1L, a histone H3-lysine 79 methyltransferase, alleviates allogeneic T-cell responses. DOT1L inhibition reduces miR-181a expression, which in turn increases the ERK phosphatase DUSP6 expression and selectively ameliorates low-avidity T-cell responses through globally suppressing T-cell activation-induced gene expression alterations. The inhibition of DOT1L or DUSP6 overexpression in T cells attenuates the development of graft-versus-host disease, while retaining potent antitumor activity in xenogeneic and allogeneic adoptive immunotherapy models. These results suggest that DOT1L inhibition may enable the safe and effective use of allogeneic antitumor T cells by suppressing unwanted immunological reactions in adoptive immunotherapy.
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http://dx.doi.org/10.1038/s41467-018-04262-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954061PMC
May 2018

Mechanisms underlying the lack of endogenous processing and CLIP-mediated binding of the invariant chain by HLA-DP.

Sci Rep 2018 03 19;8(1):4804. Epub 2018 Mar 19.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, M5G 2M9, Canada.

While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPβ (DP) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP and DP as clinically relevant in human disease.
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http://dx.doi.org/10.1038/s41598-018-22931-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859192PMC
March 2018

A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects.

Nat Med 2018 03 5;24(3):352-359. Epub 2018 Feb 5.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

The adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) (hereafter referred to as CAR-T cells) specific for the B lymphocyte antigen CD19 has shown impressive clinical responses in patients with refractory B cell malignancies. However, the therapeutic effects of CAR-T cells that target other malignancies have not yet resulted in significant clinical benefit. Although inefficient tumor trafficking and various immunosuppressive mechanisms can impede CAR-T cell effector responses, the signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals, including T cell receptor (TCR) engagement (signal 1), co-stimulation (signal 2) and cytokine engagement (signal 3). However, CAR constructs currently being tested in the clinic contain a CD3z (TCR signaling) domain and co-stimulatory domain(s) but not a domain that transmits signal 3 (refs. 13, 14, 15, 16, 17, 18). Here we have developed a novel CAR construct capable of inducing cytokine signaling after antigen stimulation. This new-generation CD19 CAR encodes a truncated cytoplasmic domain from the interleukin (IL)-2 receptor β-chain (IL-2Rβ) and a STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif, together with the TCR signaling (CD3z) and co-stimulatory (CD28) domains (hereafter referred to as 28-ΔIL2RB-z(YXXQ)). The 28-ΔIL2RB-z(YXXQ) CAR-T cells showed antigen-dependent activation of the JAK kinase and of the STAT3 and STAT5 transcription factors signaling pathways, which promoted their proliferation and prevented terminal differentiation in vitro. The 28-ΔIL2RB-z(YXXQ) CAR-T cells demonstrated superior in vivo persistence and antitumor effects in models of liquid and solid tumors as compared with CAR-T cells expressing a CD28 or 4-1BB co-stimulatory domain alone. Taken together, these results suggest that our new-generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicity in the clinic and that clinical translation of this novel CAR is warranted.
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http://dx.doi.org/10.1038/nm.4478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839992PMC
March 2018

A Subset of Human Autoreactive CD1c-Restricted T Cells Preferentially Expresses TRBV4-1 TCRs.

J Immunol 2018 01 13;200(2):500-511. Epub 2017 Dec 13.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5G 2M9, Canada;

In humans, a substantial portion of T cells recognize lipids presented by the monomorphic CD1 proteins. Recent studies have revealed the molecular basis of mycobacterial lipid recognition by CD1c-restricted T cells. Subsets of CD1c-restricted T cells recognize self-lipids in addition to foreign lipids, which may have implications in human diseases involving autoimmunity and malignancy. However, the molecular identity of these self-reactive T cells remains largely elusive. In this study, using a novel CD1c artificial APC (aAPC)-based system, we isolated human CD1c-restricted autoreactive T cells and characterized them at the molecular level. By using the human cell line K562, which is deficient in MHC class I/II and CD1 expression, we generated an aAPC expressing CD1c as the sole Ag-presenting molecule. When stimulated with this CD1c aAPC presenting endogenous lipids, a subpopulation of primary CD4 T cells from multiple donors was consistently activated, as measured by CD154 upregulation and cytokine production in a CD1c-specific manner. These activated CD4 T cells preferentially expressed TRBV4-1 TCRs. Clonotypic analyses of the reconstituted TRBV4-1 TCR genes confirmed CD1c-restricted autoreactivity of this repertoire, and the strength of CD1c reactivity was influenced by the diversity of CDR3β sequences. Finally, alanine scanning of CDR1 and CDR2 sequences of TRBV4-1 revealed two unique residues, Arg and Tyr, as critical in conferring CD1c-restricted autoreactivity, thus elucidating the molecular basis of the observed V gene bias. These data provide new insights into the molecular identity of human autoreactive CD1c-restricted T cells.
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http://dx.doi.org/10.4049/jimmunol.1700677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760327PMC
January 2018

Association of Ipilimumab With Safety and Antitumor Activity in Women With Metastatic or Recurrent Human Papillomavirus-Related Cervical Carcinoma.

JAMA Oncol 2018 07 12;4(7):e173776. Epub 2018 Jul 12.

Drug Development Program, Division of Medical Oncology, Princess Margaret Cancer Centre, Toronto, Ontario, Canada.

Importance: Based on evidence of human papillomavirus (HPV)-induced immune evasion, immunotherapy may be an attractive strategy in cervical cancer. Ipilimumab is a fully humanized monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4), which acts to downregulate the T-cell immune response.

Objective: To assess the safety and antitumor activity of ipilimumab in recurrent cervical cancer.

Design, Setting, And Participants: A multicenter trial was designed for patients with metastatic cervical cancer (squamous cell carcinoma or adenocarcinoma) with measurable disease and progression after at least 1 line of platinum chemotherapy. A run-in safety cohort using ipilimumab, 3 mg/kg, every 21 days for 4 cycles in 6 patients was followed by a phase II cohort of ipilimumab, 10 mg/kg, every 21 days for 4 cycles and then 4 cycles of maintenance therapy every 12 weeks for patients demonstrating radiologic response or stabilization. Immune correlative studies were performed on peripheral blood before and after therapy on archival tissue and fresh tumor obtained prior to registration and 7 days after cycle 2. The study was conducted from December 3, 2012, to September 15, 2014. The data were analyzed from April 2016 to June 2016 and in July 2017.

Main Outcomes And Measures: The primary end points were safety and objective response rate. Immune analyses were performed on blood and tumor tissue.

Results: A total of 42 women (median age, 49 years; range, 23-78 years) were enrolled (29 [69%] squamous cell cervical cancer and 13 [31%] adenocarcinoma; 37 [93%] of 40 patients with tissue available for analysis had HPV-positive confirmation; there was no archival tissue for 2 women). Grade 3 toxic effects included diarrhea in 4 patients, 3 of whom had colitis. Of 34 patients evaluated for best response (Response Evaluation Criteria in Solid Tumors, version 1.1), 1 patient had partial response and 10 had stable disease. The median progression-free survival and overall survival were 2.5 months (95% CI, 2.1-3.2 months) and 8.5 months (95% CI, 3.6-not reached; 1 patient was still alive), respectively. Intratumoral pretreatment CD3, CD4, CD8, FoxP3, indoleamine 2,3-dioxygenase, and programmed cell death ligand 1 (PD-L1) expression was not predictive of benefit and did not significantly change with treatment. Multicolor flow cytometry on peripheral lymphocytes revealed a treatment-dependent increase of inducible T-cell costimulator, human leukocyte antigen-antigen D related, and PD-1 during initial treatment, which returned to baseline during maintenance.

Conclusions And Relevance: Ipilimumab was tolerable in this population but did not show significant single-agent activity. Immune changes were induced by anti-CTLA-4 therapy but did not correlate with clinical activity. Changes in these markers may guide further treatment strategies.
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http://dx.doi.org/10.1001/jamaoncol.2017.3776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145732PMC
July 2018