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Supplementary Table from Systematic Evaluation of the Immune Environment of Small Intestinal Neuroendocrine Tumors

Supplementary Data from Systematic Evaluation of the Immune Environment of Small Intestinal Neuroendocrine Tumors

Purpose:The immune tumor microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumors (siNET) have not been fully defined.Experimental Design:Herein, we studied 40 patients with primary and synchronous metastatic siNETs, and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. In addition, matched FFPE tissue was obtained for multi-parametric IHC to determine the relative abundance and distribution of T-cell infiltrate. Tumor mutational burden (TMB) was also assessed and correlated with immune infiltration.Results:Effector tumor-infiltrating lymphocytes (TIL) had a higher expression of PD-1 in the tumor microenvironment compared with the periphery. In addition, CD8+ TILs had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 (cytotoxic T lymphocyte antigen-4) and higher levels of PD-1 expression compared with normal tissue. IHC revealed that the majority of cases have ≤10% intra-tumoral T cells but a higher number of peri-tumoral T cells, demonstrating an “exclusion” phenotype. Finally, we confirmed that siNETs have a low TMB compared with other tumor types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio.Conclusions:Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit “excluded” T cells into the tumor microenvironment to treat patients with siNETs.

Supplementary figure S1 : Gene disruption analysis in HEK293 cells. Supplementary figure S2: Optimization of the generation of double KO cells Supplementary table S1: Deep sequencing analysis of TCR-negative cells Supplementary figure S3: Representative deletions found at disrupted TRAC gene Supplementary figure S4: Size distribution of the mutations found in TALEN-treated T cells Supplementary table S2: Size of insertions/deletions in mutated CD52/TRAC alleles Supplementary Table S3: NGS sequencing analysis of off target cleavage Supplementary figure S5: Gating strategy for definition of T-cellsubsets in UCART19

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator–like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αβ T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19+ tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an “off-the-shelf” manner akin to other biologic immunopharmaceuticals. Cancer Res; 75(18); 3853–64. ©2015 AACR.

Supplementary Methods from Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies

Purpose: The immune tumor microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumors (siNET) have not been fully defined. Experimental Design: Herein, we studied 40 patients with primary and synchronous metastatic siNETs, and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. In addition, matched FFPE tissue was obtained for multi-parametric IHC to determine the relative abundance and distribution of T-cell infiltrate. Tumor mutational burden (TMB) was also assessed and correlated with immune infiltration. Results: Effector tumor-infiltrating lymphocytes (TIL) had a higher expression of PD-1 in the tumor microenvironment compared with the periphery. In addition, CD8+ TILs had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 (cytotoxic T lymphocyte antigen-4) and higher levels of PD-1 expression compared with normal tissue. IHC revealed that the majority of cases have ≤10% intra-tumoral T cells but a higher number of peri-tumoral T cells, demonstrating an “exclusion” phenotype. Finally, we confirmed that siNETs have a low TMB compared with other tumor types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio. Conclusions: Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit “excluded” T cells into the tumor microenvironment to treat patients with siNETs.

Background: \ud Among patients with non–small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC.\ud \ud Methods: \ud In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival.\ud \ud Results: \ud We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10−4), which remained significant in multivariate analysis.\ud \ud Conclusions: \ud Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601.)

We performed a retrospective analysis of outcome in 45 patients with multiple myeloma receiving unrelated donor stem cell transplants (UD-SCT) in the UK between 1993 and 2002; 17 received myeloablative conditioning regimens and 28 received reduced intensity conditioning (RIC) protocols. Forty patients received pretransplant CAMPATH serotherapy. Forty-two of 45 patients had detectable disease at transplant, but 33 of 45 were chemoresponsive. Sixty per cent of patients had received a previous autograft. Myeloid engraftment was seen in 95% of recipients and was significantly faster in recipients receiving peripheral blood stem cells (P = 0.07) and RIC (P = 0.001). The incidence of severe (grade 3/4) acute graft versus host disease (aGvHD) was 5% (2/40). The 100-d non-relapse mortality was 18% (5/38) following RIC and 53% (9/17) following myeloablative regimens. Twenty-nine per cent of patients achieved a complete remission, 61% a partial remission, giving a 90% overall response rate. At median follow-up (513 d), overall survival was 40%: 54% in the RIC group (median follow-up: 489 d) and 18% in the myeloablative group (median follow-up: 560 d). In recipients of UD-SCT, RIC protocols that incorporated CAMPATH were associated with faster myeloid engraftment, less severe aGvHD and lower 100-d non-relapse mortality than myeloablative regimens, without a corresponding rise in relapse rate during the period of observation.

Mantle cell lymphoma (MCL) is a disease associated with a poor prognosis characterised by inevitable relapse following both conventional chemotherapy and autologous stem cell transplantation (SCT). The role of reduced intensity allogeneic stem cell transplantation (RIST) in MCL remains controversial. We have conducted a retrospective study of RIST in MCL as reported to the EBMT registry in an attempt to define factors predicting the outcome of this procedure. Between 1998 and 2006 279 patients with MCL received a RIST with 210 procedures performed after the year 2001. 219 (78%) were male and the median age at transplant was 55 years (range 30–71). At diagnosis 97% had stage IV disease and 39% had B symptoms. Patients had received a median of 3 lines (range 1–9) of prior therapy and 119 (43%) had undergone a previous autologous SCT. The median time from diagnosis to transplant was 30 months (range 3–161). The disease status at transplant was; complete remission 124, partial remission 95, refractory disease 32 and untested relapse 17. The performance status at transplant was poor in 15 patients. Conditioning for RIST was achieved with fludarabine+alkylating agent in 66%, fludarabine+TBI in 13%, and a variety of other reduced intensity regimens in 20%. Transplants were performed from 193 matched family donors, 60 matched unrelated donors and 22 mismatched donors who provided peripheral blood stem cells in 252 cases and bone marrow in 26. T cell depletion with either CAMPATH or ATG was performed in 85 transplants. 274 patients were evaluable for engraftment of whom 272 engrafted with 4 secondary graft failures. Acute graft versus host disease (GVHD) developed in 149 patients (grade I 45, grade II 52, grade III/IV 50, unknown extent 2). Of 214 patients at risk 30 developed limited chronic GVHD and 58 extensive chronic GVHD. There were 91 transplant related deaths with infection and GVHD accounting for 51 of these deaths. The resulting 100 day, 1 year and 3 year non-relapse mortality rates were 13, 32 and 41% respectively. NRM was associated with poor PS at transplant (RR=3.7 p=0.001) and transplantation prior to 2002 (RR= 1.7 p=0.02) but age, prior transplantation and donor relationship had no significant impact. Sixty one patients relapsed at a median time of 7 months (range 1–50 months) post transplant. The cumulative incidence of relapse at 1 years and 4 years was 19% and 31% respectively. Disease relapse was associated with refractory disease at transplant (RR=4.5 p