New interpretable machine-learning method for single-cell data reveals correlates of clinical response to cancer immunotherapy

Patterns (N Y)

2021 Evan Greene; Paul Nghiem; Raphael Gottardo; Leonard D'Amico; Chihiro Moroshima; Nirasha Ramchurren; Martin Cheever; Greg Finak; Steven Fling

We introduce a new method for single-cell cytometry studies, FAUST, which performs unbiased cell population discovery and annotation. FAUST processes experimental data on a per-sample basis and returns biologically interpretable cell phenotypes, making it well suited for the analysis of complex datasets. We provide simulation studies that compare FAUST with existing methodology, exemplifying its strength. We apply FAUST to data from a Merkel cell carcinoma anti-PD-1 trial and discover pre-treatment effector memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. Using FAUST, we then validate these correlates in cryopreserved peripheral blood mononuclear cell samples from the same study, as well as an independent CyTOF dataset from a published metastatic melanoma trial. Finally, we show how FAUST's phenotypes can be used to perform cross-study data integration in the presence of diverse staining panels. Together, these results establish FAUST as a powerful new approach for unbiased discovery in single-cell cytometry.

Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma

Nat Commun

2021 Martin Cheever; Steven Fling; Robert Pierce; Nirasha Ramchurren; Kimberly Smythe

Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1+ CD4+ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies.

Haematological Care Priorities in sub-Saharan Africa 3 Haematological malignancies in sub-Saharan Africa: east Africa as an example for improving care

Lancet Haematol

2021 Nixon Niyonzima; Katherine Tarlock; Thomas Uldrick; Henry Ddungu; Clement Okello; Jackson Orem; Abrahams Omoding

Haematological malignancies account for almost 10% of all cancers diagnosed in sub-Saharan Africa, although the exact incidences and treatment outcomes are difficult to discern because population-based cancer registries in the region are still underdeveloped. More research on haematological malignancies in sub-Saharan Africa is required to establish whether these cancers have a natural history similar to those diagnosed in high-income countries, about which more is known. Several factors negatively affect the outcome of haematological malignancies in sub-Saharan Africa, showcasing a need for improved understanding of the clinicobiological profile of these cancers to facilitate prevention, early detection, diagnosis, and appropriate treatment through increased capacity building, infrastructure, community awareness, coordinated resource mobilisation, and collaboration across the world. The east African governments have pooled resources for common investments to tackle non-communicable diseases, developing the East Africa's Centres of Excellence for Skills and Tertiary Education project funded by the African Development Bank, an initiative that could be replicated for the care of haematological malignancies in other countries in sub-Saharan Africa. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

CD4+T cell and M2 macrophage infiltration predict dedifferentiated liposarcoma patient outcomes

J Immunother Cancer

2021 Laura Riolobos; Seth Pollack; Lee Cranmer; Robert Pierce; Brett Schroeder; Michael Wagner; Shihong Zhang; Eleanor Chen; Teresa Kim; Kimberly Smythe

BACKGROUND: Dedifferentiated liposarcoma (DDLPS) is one of the most common soft tissue sarcoma subtypes and is devastating in the advanced/metastatic stage. Despite the observation of clinical responses to PD-1 inhibitors, little is known about the immune microenvironment in relation to patient prognosis. METHODS: We performed a retrospective study of 61 patients with DDLPS. We completed deep sequencing of the T-cell receptor (TCR) -chain and RNA sequencing for predictive modeling, evaluating both immune markers and tumor escape genes. Hierarchical clustering and recursive partitioning were employed to elucidate relationships of cellular infiltrates within the tumor microenvironment, while an immune score for single markers was created as a predictive tool. RESULTS: Although many DDLPS samples had low TCR clonality, high TCR clonality combined with low T-cell fraction predicted lower 3-year overall survival (p=0.05). Higher levels of CD14+ monocytes (p=0.02) inversely correlated with 3-year recurrence-free survival (RFS), while CD4+ T-cell infiltration (p=0.05) was associated with a higher RFS. Genes associated with longer RFS included PD-1 (p=0.003), ICOS (p=0.006), BTLA (p=0.033), and CTLA4 (p=0.02). In a composite immune score, CD4+ Tcells had the strongest positive predictive value, while CD14+ monocytesand M2 macrophages had the strongest negative predictive values. CONCLUSIONS: Immune cell infiltration predicts clinical outcome in DDLPS, with CD4+ cells associated with better outcomes; CD14+ cellsand M2 macrophages are associated with worse outcomes. Future checkpoint inhibitor studies in DDLPS should incorporate immunosequencing and gene expression profiling techniques that can generate immune landscape profiles.

IL-7 expands lymphocyte populations and enhances immune responses to sipuleucel-T in patients with metastatic castration-resistant prostate cancer (mCRPC)

J Immunother Cancer

2021 Nirasha Ramchurren; Judith Kaiser; Steven Fling; Evan Yu; Chihiro Moroshima; Andreanne Lacroix; Martin Cheever; Leonard D'Amico

BACKGROUND: Sipuleucel-T (sip-T) is a Food and Drug Administration (FDA)-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). We hypothesized that combining sip-T with interleukin (IL)-7, a homeostatic cytokine that enhances both B and T cell development and proliferation, would augment and prolong antigen-specific immune responses against both PA2024 (the immunogen for sip-T) and prostatic acid phosphatase (PAP). METHODS: Fifty-four patients with mCRPC treated with sip-T were subsequently enrolled and randomized 1:1 into observation (n=26) or IL-7 (n=28) arms of a phase II clinical trial (NCT01881867). Recombinant human (rh) IL-7 (CYT107) was given weekly4. Immune responses were evaluated using flow cytometry, mass cytometry (CyTOF), interferon (IFN)- ELISpot, 3H-thymidine incorporation, and ELISA. RESULTS: Treatment with rhIL-7 was well tolerated. For the rhIL-7-treated, but not observation group, statistically significant lymphocyte subset expansion was found, with 2.3-2.6-fold increases in CD4+T, CD8+T, and CD56bright NK cells at week 6 compared with baseline. No significant differences in PA2024 or PAP-specific T cell responses measured by IFN- ELISpot assay were found between rhIL-7 and observation groups. However, antigen-specific T cell proliferative responses and humoral IgG and IgG/IgM responses significantly increased over time in the rhIL-7-treated group only. CyTOF analyses revealed pleiotropic effects of rhIL-7 on lymphocyte subsets, including increases in CD137 and intracellular IL-2 and IFN- expression. While not powered to detect clinical outcomes, we found that 31% of patients in the rhIL-7 group had prostate specific antigen (PSA) doubling times of >6 months, compared with 14% in the observation group. CONCLUSIONS: Treatment with rhIL-7 led to a significant expansion of CD4+ and CD8+ T cells, and CD56bright natural killer (NK) cells compared with observation after treatment with sip-T. The rhIL-7 treatment also led to improved antigen-specific humoral and T cell proliferative responses over time as well as to increased expression of activation markers and beneficial cytokines. This is the first study to evaluate the use of rhIL-7 after sip-T in patients with mCRPC and demonstrates encouraging results for combination approaches to augment beneficial immune responses.

Neoadjuvant selicrelumab, an agonist CD40 antibody, induces changes in the tumor microenvironment in patients with resectable pancreatic cancer

Clin Cancer Res

2021 Elena Chiorean; Judith Kaiser; Andreanne Lacroix; Martin Cheever

PURPOSE: CD40 activation is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological effects and treatment-related modulation of the tumor microenvironment (TME) remain poorly understood. EXPERIMENTAL DESIGN: Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy to 16 patients with resectable pancreatic ductal adenocarcinoma (PDAC) prior to surgery followed by adjuvant chemotherapy and CD40 mAb. RESULTS: The toxicity profile was acceptable and overall survival was 23.4 months (95% CI 18.0 - 28.8). Based on a novel multiplexed immunohistochemistry platform, we report evidence that neoadjuvant selicrelumab leads to major differences in the TME compared to resection specimens from treatment-naive PDAC patients or patients given neoadjuvant chemotherapy/chemoradiotherapy only. For selicrelumab-treated tumors, 82% were T cell-enriched, compared to 37% of untreated tumors (p=0.004) and 23% of chemotherapy/chemoradiation-treated tumors (p=0.012). T cells in both the TME and circulation were more active and proliferative after selicrelumab. Tumor fibrosis was reduced, M2-like tumor-associated macrophages were fewer, and intratumoral dendritic cells were more mature. Inflammatory cytokines CXCL10 and CCL22 increased systemically after selicrelumab. CONCLUSIONS: This unparalleled examination of CD40 therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting this pathway.

COVID-19 in immunocompromised populations: implications for prognosis and repurposing of immunotherapies

J Immunother Cancer

2021 Jason Goldman; Thomas Uldrick

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic. COVID-19 has highly variable disease severity and a bimodal course characterized by acute respiratory viral infection followed by hyperinflammation in a subset of patients with severe disease. This immune dysregulation is characterized by lymphocytopenia, elevated levels of plasma cytokines and proliferative and exhausted T cells, among other dysfunctional cell types. Immunocompromised persons often fare worse in the context of acute respiratory infections, but preliminary data suggest this may not hold true for COVID-19. In this review, we explore the effect of SARS-CoV-2 infection on mortality in four populations with distinct forms of immunocompromise: (1) persons with hematological malignancies (HM) and hematopoietic stem cell transplant (HCT) recipients; (2) solid organ transplant recipients (SOTRs); (3) persons with rheumatological diseases; and (4) persons living with HIV (PLWH). For each population, key immunological defects are described and how these relate to the immune dysregulation in COVID-19. Next, outcomes including mortality after SARS-CoV-2 infection are described for each population, giving comparisons to the general population of age-matched and comorbidity-matched controls. In these four populations, iatrogenic or disease-related immunosuppression is not clearly associated with poor prognosis in HM, HCT, SOTR, rheumatological diseases, or HIV. However, certain individual immunosuppressants or disease states may be associated with harmful or beneficial effects, including harm from severe CD4 lymphocytopenia in PLWH and possible benefit to the calcineurin inhibitor ciclosporin in SOTRs, or tumor necrosis factor-α inhibitors in persons with rheumatic diseases. Lastly, insights gained from clinical and translational studies are explored as to the relevance for repurposing of immunosuppressive host-directed therapies for the treatment of hyperinflammation in COVID-19 in the general population.

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy

J Immunother Cancer

2021 Edward Kim; Jean Campbell; Jianhong Cao; Ralph Black; Lee Cranmer; Robert Pierce; Venu Pillarisetty; Stanley Riddell; Michael Wagner; Shihong Zhang; Seth Pollack; Douglas Hawkins; Karan Kohli; Brett Schroeder

BACKGROUND: Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. METHOD: We performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. RESULTS: Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers. CONCLUSIONS: ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

Three-year survival, correlates and salvage therapies in patients receiving first-line pembrolizumab for advanced Merkel cell carcinoma

J Immunother Cancer

2021 Shailender Bhatia; Nirasha Ramchurren; Michi Shinohara; Martin Cheever; Paul Nghiem; Steven Fling

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer associated with poor survival. Programmed cell death-1 (PD-1) pathway inhibitors have shown high rates of durable tumor regression compared with chemotherapy for MCC. The current study was undertaken to assess baseline and on-treatment factors associated with MCC regression and 3-year survival, and to explore the effects of salvage therapies in patients experiencing initial non-response or tumor progression after response or stable disease following first-line pembrolizumab therapy on Cancer Immunotherapy Trials Network-09/KEYNOTE-017. METHODS: In this multicenter phase II trial, 50 patients with advanced unresectable MCC received pembrolizumab 2 mg/kg every 3 weeks for 2 years. Patients were followed for a median of 31.8 months. RESULTS: Overall response rate to pembrolizumab was 58% (complete response 30%+partial response 28%; 95%CI 43.2 to 71.8). Among 29 responders, the median response duration was not reached (NR) at 3 years (range 1.0+ to 51.8+ months). Median progression-free survival (PFS) was 16.8 months (95%CI 4.6 to 43.4) and the 3-year PFS was 39.1%. Median OS was NR; the 3-year OS was 59.4% for all patients and 89.5% for responders. Baseline Eastern Cooperative Oncology Group performance status of 0, greater per cent tumor reduction, completion of 2 years of treatment and low neutrophil-to-lymphocyte ratio were associated with response and longer survival. Among patients with initial disease progression or those who developed progression after response or stable disease, some had extended survival with subsequent treatments including chemotherapies and immunotherapies. CONCLUSIONS: This study represents the longest available follow-up from any first-line anti-programmed death-(ligand) 1 (anti-PD-(L)1) therapy in MCC, confirming durable PFS and OS in a proportion of patients. After initial tumor progression or relapse following response, some patients receiving salvage therapies survived. Improving the management of anti-PD-(L)1-refractory MCC remains a challenge and a high priority. TRIAL REGISTRATION NUMBER: NCT02267603.

Characteristics and outcomes of KSHV-associated multicentric Castleman disease with or without other KSHV diseases

Blood Adv

2021 Thomas Uldrick

Kaposi sarcoma (KS)-associated herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a relapsing and remitting systemic lymphoproliferative disorder characterized by severe inflammatory symptoms most common among people living with HIV (PLWH). Patients with KSHV-MCD may present with concurrent KSHV-associated diseases, such as KS and/or primary effusion lymphoma (PEL). We evaluated clinical and immunologic characteristics, the effects of concurrent KSHV malignancies, and treatments from the largest prospective natural history study of participants with KSHV-MCD within the United States. Treatment options administered at investigator discretion included high-dose zidovudine with valganciclovir (AZT/VGC), rituximab, or rituximab with liposomal doxorubicin (R-Dox) during KSHV-MCD flares. Survival analyses and prognostic factors were explored for all participants. Sixty-two participants with HIV were enrolled, including 20 with KSHV-MCD alone, 34 with KSHV-MCD and KS, 1 with KSHV-MCD and PEL, and 7 with all KSHV-associated diseases. Forty-four percent of KSHV-MCD diagnoses were made at our institution. Forty-four participants received rituximab-based therapies, 20 of whom had maintenance AZT/VGC or interferon. Participants receiving R-Dox and then maintenance AZT/VGC had the highest 5-year progression-free survival (89%). Cytokine profiles during KSHV-MCD flares did not differ by the presence of concurrent KSHV-associated diseases. The 10-year survival was 71% (95% confidence interval [CI], 56% to 82%) for all participants. A concurrent diagnosis of PEL negatively impacted survival (PEL hazard ratio, 5.4; 95% CI, 1.8 to 16.8). KSHV-MCD is an underdiagnosed condition among PLWH, including those with KS. KSHV-MCD has an excellent prognosis with appropriate treatment. Physicians should be alert for patients with multiple KSHV diseases, which impact optimal treatment and survival outcomes. This study was registered at as #NCT00099073.