Evolution of clinical nature, treatment and survival of locally recurrent rectal cancer: Comparative analysis of two national cross-sectional cohorts.

BACKGROUND: In the Netherlands, use of neoadjuvant radiotherapy for rectal cancer declined after guideline revision in 2014. This decline is thought to affect the clinical nature and treatability of locally recurrent rectal cancer (LRRC). Therefore, this study compared two national cross-sectional cohorts before and after the guideline revision with the aim to determine the changes in treatment and survival of LRRC patients over time. METHODS: Patients who underwent resection of primary rectal cancer in 2011 (n = 2094) and 2016 (n = 2855) from two nationwide cohorts with a 4-year follow up were included. Main outcomes included time to LRRC, synchronous metastases at time of LRRC diagnosis, intention of treatment and 2-year overall survival after LRRC. RESULTS: Use of neoadjuvant (chemo)radiotherapy for the primary tumour decreased from 88.5% to 60.0% from 2011 to 2016. The 3-year LRRC rate was not significantly different with 5.1% in 2011 (n = 114, median time to LRRC 16 months) and 6.3% in 2016 (n = 202, median time to LRRC 16 months). Synchronous metastasis rate did not significantly differ (27.2% vs 33.7%, p = 0.257). Treatment intent of the LRRC shifted towards more curative treatment (30.4% vs. 47.0%, p = 0.009). In the curatively treated group, two-year overall survival after LRRC diagnoses increased from 47.5% to 78.7% (p = 0.013). CONCLUSION: Primary rectal cancer patients in 2016 were treated less often with neoadjuvant (chemo)radiotherapy, while LRRC rates remained similar. Those who developed LRRC were more often candidate for curative intent treatment compared to the 2011 cohort, and survival after curative intent treatment also improved substantially.

Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Assessing the learning curve of robot-assisted total mesorectal excision: a multicenter study considering procedural safety, pathological safety, and efficiency.

PURPOSE: Evidence regarding the learning curve of robot-assisted total mesorectal excision is scarce and of low quality. Case-mix is mostly not taken into account, and learning curves are based on operative time, while preferably clinical outcomes and literature-based limits should be used. Therefore, this study aims to assess the learning curve of robot-assisted total mesorectal excision. METHODS: A retrospective study was performed in four Dutch centers. The primary aim was to assess the safety of the individual and institutional learning curves using a RA-CUSUM analysis based on intraoperative complications, major postoperative complications, and compound pathological outcome (positive circumferential margin or incomplete TME specimen). The learning curve for efficiency was assessed using a LC-CUSUM analysis for operative time. Outcomes of patients before and after the learning curve were compared. RESULTS: In this study, seven participating surgeons performed robot-assisted total mesorectal excisions in 531 patients. Learning curves for intraoperative complications, postoperative complications, and compound pathological outcome did not exceed predefined literature-based limits. The LC-CUSUM for operative time showed lengths of the learning curve ranging from 12 to 35 cases. Intraoperative, postoperative, and pathological outcomes did not differ between patients operated during and after the learning curve. CONCLUSION: The learning curve of robot-assisted total mesorectal excision based on intraoperative complications, postoperative complications, and compound pathological outcome did not exceed predefined limits and is therefore suggested to be safe. Using operative time as a surrogate for efficiency, the learning curve is estimated to be between 12 and 35 procedures.

Neoadjuvant FOLFOXIRI prior to chemoradiotherapy for high-risk ("ugly") locally advanced rectal cancer: study protocol of a single-arm, multicentre, open-label, phase II trial (MEND-IT).

BACKGROUND: The presence of mesorectal fascia (MRF) invasion, grade 4 extramural venous invasion (EMVI), tumour deposits (TD) or extensive or bilateral extramesorectal (lateral) lymph nodes (LLN) on MRI has been suggested to identify patients with indisputable, extensive locally advanced rectal cancer (LARC), at high risk of treatment failure. The aim of this study is to evaluate whether or not intensified chemotherapy prior to neoadjuvant chemoradiotherapy improves the complete response (CR) rate in these patients. METHODS: This multicentre, single-arm, open-label, phase II trial will include 128 patients with non-metastatic high-risk LARC (hr-LARC), fit for triplet chemotherapy. To ensure a study population with indisputable, unfavourable prognostic characteristics, hr-LARC is defined as LARC with on baseline MRI at least one of the following characteristics; MRF invasion, EMVI grade 4, enlarged bilateral or extensive LLN at high risk of an incomplete resection, or TD. Exclusion criteria are the presence of a homozygous DPD deficiency, distant metastases, any chemotherapy within the past 6 months, previous radiotherapy within the pelvic area precluding standard chemoradiotherapy, and any contraindication for the planned treatment. All patients will be planned for six two-weekly cycles of FOLFOXIRI (5-fluorouracil, leucovorin, oxaliplatin and irinotecan) prior to chemoradiotherapy (25 × 2 Gy or 28 × 1.8 Gy with concomitant capecitabine). A resection will be performed following radiological confirmation of resectable disease after the completion of chemoradiotherapy. A watch and wait strategy is allowed in case of a clinical complete response. The primary endpoint is the CR rate, described as a pathological CR or a sustained clinical CR one year after chemoradiotherapy. The main secondary objectives are long-term oncological outcomes, radiological and pathological response, the number of resections with clear margins, treatment-related toxicity, perioperative complications, health-related costs, and quality of life. DISCUSSION: This trial protocol describes the MEND-IT study. The MEND-IT study aims to evaluate the CR rate after intensified chemotherapy prior to concomitant chemoradiotherapy in a homogeneous group of patients with locally advanced rectal cancer and indisputably unfavourable characteristics, defined as hr-LARC, in order to improve their prognosis. TRIAL REGISTRATION: Clinicaltrials.gov: NCT04838496 , registered on 02-04-2021 Netherlands Trial Register: NL9790. PROTOCOL VERSION: Version 3 dd 11-4-2022.

Changes in Bone Marrow Tumor and Immune Cells Correlate with Durability of Remissions Following BCMA CAR T Therapy in Myeloma.

Chimeric antigen-receptor (CAR) T cells lead to high response rates in myeloma, but most patients experience recurrent disease. We combined several high-dimensional approaches to study tumor/immune cells in the tumor microenvironment (TME) of myeloma patients pre- and post-B-cell maturation antigen (BCMA)-specific CAR T therapy. Lower diversity of pretherapy T-cell receptor (TCR) repertoire, presence of hyperexpanded clones with exhaustion phenotype, and BAFF+PD-L1+ myeloid cells in the marrow correlated with shorter progression-free survival (PFS) following CAR T therapy. In contrast, longer PFS was associated with an increased proportion of CLEC9A+ dendritic cells (DC), CD27+TCF1+ T cells with diverse T-cell receptors, and emergence of T cells expressing marrow-residence genes. Residual tumor cells at initial response express stemlike genes, and tumor recurrence was associated with the emergence of new dominant clones. These data illustrate a dynamic interplay between endogenous T, CAR T, myeloid/DC, and tumor compartments that affects the durability of response following CAR T therapy in myeloma. SIGNIFICANCE: There is an unmet need to identify determinants of durable responses following BCMA CAR T therapy of myeloma. High-dimensional analysis of the TME was performed to identify features of immune and tumor cells that correlate with survival and suggest several strategies to improve outcomes following CAR T therapy. See related commentary by Graham and Maus, p. 478. This article is highlighted in the In This Issue feature, p. 476.

Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL.

A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.

Local recurrence of robot-assisted total mesorectal excision: a multicentre cohort study evaluating the initial cases.

PURPOSE: Evidence regarding local recurrence rates in the initial cases after implementation of robot-assisted total mesorectal excision is limited. This study aims to describe local recurrence rates in four large Dutch centres during their initial cases. METHODS: Four large Dutch centres started with the implementation of robot-assisted total mesorectal excision in respectively 2011, 2012, 2015, and 2016. Patients who underwent robot-assisted total mesorectal excision with curative intent in an elective setting for rectal carcinoma defined according to the sigmoid take-off were included. Overall survival, disease-free survival, systemic recurrence, and local recurrence were assessed at 3 years postoperatively. Subsequently, outcomes between the initial 10 cases, cases 11-40, and the subsequent cases per surgeon were compared using Cox regression analysis. RESULTS: In total, 531 patients were included. Median follow-up time was 32 months (IQR: 19-50]. During the initial 10 cases, overall survival was 89.5%, disease-free survival was 73.1%, and local recurrence was 4.9%. During cases 11-40, this was 87.7%, 74.1%, and 6.6% respectively. Multivariable Cox regression did not reveal differences in local recurrence between the different case groups. CONCLUSION: Local recurrence rate during the initial phases of implantation of robot-assisted total mesorectal procedures is low. Implementation of the robot-assisted technique can safely be performed, without additional cases of local recurrence during the initial cases, if performed by surgeons experienced in laparoscopic rectal cancer surgery.

Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell-Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19.

PURPOSE: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment. EXPERIMENTAL DESIGN: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials. RESULTS: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS. CONCLUSIONS: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Anti-CD19 CAR T cells in combination with ibrutinib for the treatment of chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL) patients who achieve a complete remission (CR) to anti-CD19 chimeric antigen receptor T cells (CART-19), remissions are remarkably durable. Preclinical data suggesting synergy between CART-19 and the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib prompted us to conduct a prospective single-center phase 2 trial in which we added autologous anti-CD19 humanized binding domain T cells (huCART-19) to ibrutinib in patients with CLL not in CR despite ≥6 months of ibrutinib. The primary endpoints were safety, feasibility, and achievement of a CR within 3 months. Of 20 enrolled patients, 19 received huCART-19. The median follow-up for all infused patients was 41 months (range, 0.25-58 months). Eighteen patients developed cytokine release syndrome (CRS; grade 1-2 in 15 of 18 subjects), and 5 developed neurotoxicity (grade 1-2 in 4 patients, grade 4 in 1 patient). While the 3-month CR rate among International Working Group on CLL (iwCLL)-evaluable patients was 44% (90% confidence interval [CI], 23-67%), at 12 months, 72% of patients tested had no measurable residual disease (MRD). The estimated overall and progression-free survival at 48 months were 84% and 70%, respectively. Of 15 patients with undetectable MRD at 3 or 6 months, 13 remain in ongoing CR at the last follow-up. In patients with CLL not achieving a CR despite ≥6 months of ibrutinib, adding huCART-19 mediated a high rate of deep and durable remissions. ClinicalTrials.gov number, NCT02640209.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

Enhanced Costimulatory Signaling Improves CAR T-cell Effector Responses in CLL.

CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling. Significance: CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.

CAR T-cell immunotherapy: a powerful weapon for fighting hematological B-cell malignancies.

The current standard of care in hematological malignancies has brought considerable clinical benefits to patients. However, important bottlenecks still limit optimal achievements following a current medical practice. The genetic complexity of the diseases and the heterogeneity of tumor clones cause difficulty in ensuring long-term efficacy of conventional treatments for most hematological disorders. Consequently, new treatment strategies are necessary to improve clinical outcomes. Chimeric antigen receptor T-cell (CAR T) immunotherapy opens a new path for targeted therapy of hematological malignancies. In this review, through a representative case study, we summarize the current experience of CAR T-cell therapy, the management of common side effects, the causative mechanisms of therapy resistance, and new strategies to improve the efficacy of CAR T-cell therapy.

Engineering enhanced CAR T-cells for improved cancer therapy.

Chimeric antigen receptor (CAR) T-cell therapies have evolved from a research tool to a paradigm-shifting therapy with impressive responses in B cell malignancies. This review summarizes the current state of the CAR T-cell field, focusing on CD19- and B cell maturation antigen-directed CAR T-cells, the most developed of the CAR T-cell therapies. We discuss the many challenges to CAR-T therapeutic success and innovations in CAR design and T-cell engineering aimed at extending this therapeutic platform beyond hematologic malignancies.

The Safety of Bridging Radiation with Anti-BCMA CAR T-Cell Therapy for Multiple Myeloma.

PURPOSE: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cells (CART-BCMA) are a promising treatment for relapsed/refractory multiple myeloma (r/rMM). We evaluated the safety and feasibility of bridging radiation (RT) in subjects treated on a phase I trial of CART-BCMA. EXPERIMENTAL DESIGN: Twenty-five r/rMM subjects were treated in three cohorts with two doses of CART-BCMA cells ± cyclophosphamide. We retrospectively analyzed toxicity, response, and CART manufacturing data based on RT receipt. RESULTS: Thirteen subjects received no RT <1 year before CART infusion (Group A). Eight subjects received RT <1 year before CART infusion (Group B) with median time from RT to apheresis of 114 days (range 40-301). Four subjects received bridging-RT (Group C) with a median dose of 22 Gy and time from RT to infusion of 25 days (range 18-35). Group C had qualitatively lower rates of grade 4 (G4) hematologic toxicities (25%) versus A (61.5%) and B (62.5%). G3-4 neurotoxicity occurred in 7.7%, 25%, and 25% in Group A, B, and C, respectively. G3-4 cytokine release syndrome was observed in 38.5%, 25%, and 25% in Group A, B, and C, respectively. Partial response or better was observed in 54%, 38%, and 50% of Group A, B, and C, respectively. RT administered <1 year (P = 0.002) and <100 days (P = 0.069) before apheresis was associated with lower in vitro proliferation during manufacturing; however, in vivo CART-BCMA expansion appeared similar across groups. CONCLUSIONS: Bridging-RT appeared safe and feasible with CART-BCMA therapy in our r/rMM patients, though larger future studies are needed to draw definitive conclusions.

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.

The 2020 BMT CTN Myeloma Intergroup Workshop on Immune Profiling and Minimal Residual Disease Testing in Multiple Myeloma.

The fifth annual Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Immune Profiling and Minimal Residual Disease Testing in Multiple Myeloma was conducted as one of the American Society of Hematology Annual Meeting Scientific Workshops on Thursday December 3, 2020. This workshop focused on four main topics: (1) integrating minimal residual disease into clinical trial design and practice; (2) the molecular and immunobiology of disease evolution and progression in myeloma; (3) adaptation of next-generation sequencing, next-generation flow cytometry, and cytometry by time of flight techniques; and (4) chimeric antigen receptor T-cell and other cellular therapies for myeloma. In this report, we provide a summary of the workshop presentations and discuss future directions in the field.

Single-cell multiomics dissection of basal and antigen-specific activation states of CD19-targeted CAR T cells.

BACKGROUND: Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies. METHODS: Using single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio. RESULTS: We delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF(CSF2) expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation. CONCLUSIONS: Through integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation 'off- the-shelf' allogeneic products.