Surgical timing after preoperative chemotherapy is associated with oncologic outcomes in resectable colorectal liver metastases.

INTRODUCTION: Preoperative chemotherapy (POC) is often employed for patients with resectable colorectal liver metastasis (CRLM). The time to resection (TTR) following the end of chemotherapy may impact oncologic outcomes; this phenomenon has not been studied in CRLM. METHODS: We queried our institutional cancer database for patients with resected CRLM after POC from 2003 to 2019. TTR was calculated from date of last cytotoxic chemotherapy. Kaplan-Meier analysis and multivariable Cox proportional hazards modeling were used to analyze recurrence-free survival (RFS) and overall survival (OS). RESULTS: We identified n = 187 patients. One hundred twenty-four (66%) patients had a TTR of <2 months, while 63 (33%) had a TTR of ≥2 months. Median follow-up was 36 months. On Kaplan-Meier analysis, patients with TTR ≥ 2 months had shorter RFS (median 11 vs. 17 months, p = 0.002) and OS (median 44 vs. 62 months, p < 0.001). On multivariable analysis, TTR ≥ 2 months was independently associated with worse RFS (hazard ratio [HR] = 1.54, 95% confidence interval [CI] = 1.06-2.22, p = 0.02) and OS (HR = 1.75, 95% CI = 1.11-2.77, p = 0.01). CONCLUSION: TTR ≥ 2 months following POC is independently associated with worse oncologic outcomes in patients with resectable CRLM. We therefore recommend consideration for hepatic resection of CRLM within this window whenever feasible.

CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function.

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

Conventional hepatic arterial anatomy? Novel findings and insights of a multi-disciplinary hepatic arterial infusion pump program.

INTRODUCTION: Variant hepatic arterial anatomy (vHAA) is thought to occur in 20-30% of patients. Hepatic arterial infusion (HAI) pump placement for liver cancers requires thorough hepatic artery dissection; we sought to compare vHAA identified during pump placement with established dogma. METHODS: Between 2016 and 2020, n = 30 patients received a HAI pump. Intra-operatively identified vHAA was characterized and compared with published data. RESULTS: vHAA was identified in 60% (n = 18) of patients, significantly higher than 19% (3671 of 19013) in the largest published series (P < 0.001). The most common variations were accessory left (n = 12; 40%) and replaced right (n = 6; 20%) hepatic arteries; six (20%) had ≥2 variants. Pre-operative imaging correctly identified 67% of variant hepatic arteries. DISCUSSION: Meticulous operative dissection of the hepatic arterial tree reveals vHAA not captured by imaging or cadaveric dissection. vHAA likely has a higher prevalence than previously reported and should be addressed to optimize therapeutic efficacy of HAI pump therapy.

Surgical and oncologic outcomes following repeat hepatic resection of colorectal liver metastasis: Who benefits?

BACKGROUND: Resected colorectal liver metastases (CRLM) frequently recur intrahepatically. Selection criteria for repeat hepatectomy of recurrent CRLM are ill-defined. METHODS: We performed an institutional review of patients with recurrent CRLM undergoing repeat hepatectomy from 2003 to 19. Post-recurrence overall (rOS) and recurrence-free survival (RFS) were analyzed with Cox proportional hazards modeling. RESULTS: n = 147 experienced recurrent CRLM; 11% (n = 38) received repeat hepatectomy of which there was one Clavien-Dindo IIIa complication. Median rOS was 41 months; median RFS was 9 months. Improved rOS and RFS were independently associated with additional post-operative chemotherapy after repeat hepatectomy (HR 0.35 and 0.34, respectively); poor rOS with recurrent CRLM >3 cm (HR 4.4) and <12 months from first hepatectomy to recurrence (HR 4.8); poor RFS with ≥3 recurrence liver metastases (HR 2.8) (All P < 0.05). DISCUSSION: Repeat hepatectomy for recurrent CRLM can be performed safely. Worse survival following repeat hepatectomy is independently associated with >3 cm and ≥3 liver lesions at recurrence, and <12 months to recurrence. Additional post-operative chemotherapy after repeat hepatectomy is associated with improved outcomes.

Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy.

Adoptive immunotherapies using T cells genetically redirected with a chimeric antigen receptor (CAR) or T cell receptor (TCR) are entering mainstream clinical practice. Despite encouraging results, some patients do not respond to current therapies. In part, this phenomenon has been associated with infusion of reduced numbers of early memory T cells. Herein, we report that AKT signaling inhibition is compatible with CAR and TCR retroviral transduction of human T cells while promoting a CD62L-expressing central memory phenotype. Critically, this intervention did not compromise cell yield. Mechanistically, disruption of AKT signaling preserved MAPK activation and promoted the intranuclear localization of FOXO1, a transcriptional regulator of T cell memory. Consequently, AKT signaling inhibition synchronized the transcriptional profile for FOXO1-dependent target genes across multiple donors. Expression of an AKT-resistant FOXO1 mutant phenocopied the influence of AKT signaling inhibition, while addition of AKT signaling inhibition to T cells expressing mutant FOXO1 failed to further augment the frequency of CD62L-expressing cells. Finally, treatment of established B cell acute lymphoblastic leukemia was superior using anti-CD19 CAR-modified T cells transduced and expanded in the presence of an AKT inhibitor compared with conventionally grown T cells. Thus, inhibition of signaling along the PI3K/AKT axis represents a generalizable strategy to generate large numbers of receptor-modified T cells with an early memory phenotype and superior antitumor efficacy.

Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche.

Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T-cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4(+)-regulatory T (Treg) cell induction, and restrain CD8(+) T cell effector function. Tumor colonization is accompanied by PHD-protein-dependent induction of pulmonary Treg cells and suppression of IFN-γ-dependent tumor clearance. T-cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. PAPERCLIP.

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

The transcription factor BACH2 promotes tumor immunosuppression.

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance.

Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8(+) T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8(+) T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8(+) T cells and can be manipulated to improve adoptive cancer immunotherapy.

The interplay of effector and regulatory T cells in cancer.

Regulatory T (Treg) cells suppress effector T (Teff) cells and prevent immune-mediated rejection of cancer. Much less appreciated are mechanisms by which Teff cells antagonize Treg cells. Herein, we consider how complex reciprocal interactions between Teff and Treg cells shape their population dynamics within tumors. Under states of tolerance, including during tumor escape, suppressed Teff cells support Treg cell populations through antigen-dependent provision of interleukin (IL)-2. During immune activation, Teff cells can lose this supportive capacity and directly antagonize Treg cell populations to neutralize their immunosuppressive function. While this latter state is rarely achieved spontaneously within tumors, we propose that therapeutic induction of immune activation has the potential to stably disrupt immunosuppressive population states resulting in durable cancer regression.

Akt inhibition enhances expansion of potent tumor-specific lymphocytes with memory cell characteristics.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer.