Engaging stimulatory immune checkpoint interactions in the tumour immune microenvironment of primary liver cancers - how to push the gas after having released the brake.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the first and second most common primary liver cancer (PLC). For decades, systemic therapies consisting of tyrosine kinase inhibitors (TKIs) or chemotherapy have formed the cornerstone of treating advanced-stage HCC and CCA, respectively. More recently, immunotherapy using immune checkpoint inhibition (ICI) has shown anti-tumour reactivity in some patients. The combination regimen of anti-PD-L1 and anti-VEGF antibodies has been approved as new first-line treatment of advanced-stage HCC. Furthermore, gemcibatine plus cisplatin (GEMCIS) with an anti-PD-L1 antibody is awaiting global approval for the treatment of advanced-stage CCA. As effective anti-tumour reactivity using ICI is achieved in a minor subset of both HCC and CCA patients only, alternative immune strategies to sensitise the tumour microenvironment of PLC are waited for. Here we discuss immune checkpoint stimulation (ICS) as additional tool to enhance anti-tumour reactivity. Up-to-date information on the clinical application of ICS in onco-immunology is provided. This review provides a rationale of the application of next-generation ICS either alone or in combination regimen to potentially enhance anti-tumour reactivity in PLC patients.

Cryptogenic non-cirrhotic HCC: Clinical, prognostic and immunologic aspects of an emerging HCC etiology.

The incidence of hepatocellular carcinoma (HCC) in non-cirrhotic livers is rising significantly, but clear risk factors for screening remain elusive. This study sought to characterize non-cirrhotic HCC etiologies. HCC cases from 2009 to 2020 in a Dutch referral center were examined, revealing 371 out of 1654 cases (22%) as non-cirrhotic. Notably, the incidence of non-cirrhotic HCC increased by 61% in the time frame between 2009 and 2020. Interestingly 39% of non-cirrhotic HCC cases had cryptogenic origins. Cryptogenic non-cirrhotic HCC exhibited similarities with non-cirrhotic NAFLD HCC, but displayed advanced tumor stages, lower surgical rates, and a more frequent presence of symptoms, which substantiated in poor survival rates. Advanced cryptogenic non-cirrhotic HCC stages exhibited elevated serum interleukin-6 levels compared to non-cirrhotic HCC with defined etiologies. Comparative analysis encompassing cryptogenic and NAFLD non-cirrhotic HCC cohorts and controls unveiled comparable circulating immune biomarker profiles and PNPLA3 polymorphisms. To conclude, the primary etiology of non-cirrhotic HCC in our cohort has not defined risk factors. This cryptogenic variant exhibits distinct traits, such as advanced tumors and increased symptoms, and most resemble burned-out NAFLD. Understanding this HCC variant is crucial for improving screening and management strategies.

The Yield of Routine Post-Operative Doppler Ultrasound to Detect Early Post-Liver Transplantation Vascular Complications.

Early detection of liver transplantation (LT) vascular complications enables timely management. Our aim was to assess if routine Doppler ultrasound (rDUS) improves the detection of hepatic artery thrombosis (HAT), portal vein thrombosis (PVT) and hepatic venous outflow obstruction (HVOO). We retrospectively analysed timing and outcomes, number needed to diagnose one complication (NND) and positive predictive value (PPV) of rDUS on post-operative day (POD) 0,1 and 7 in 708 adult patients who underwent primary LT between 2010-2022. We showed that HAT developed in 7.1%, PVT in 8.2% and HVOO in 3.1% of patients. Most early complications were diagnosed on POD 0 (26.9%), 1 (17.3%) and 5 (17.3%). rDUS correctly detected 21 out of 26 vascular events during the protocol days. PPV of rDUS was 53.8%, detection rate 1.1% and NND was 90.5. Median time to diagnosis was 4 days for HAT and 47 days for PVT and 21 days for HVOO. After intervention, liver grafts were preserved in 57.1%. In conclusion, rDUS protocol helps to detect first week's vascular events, but with low PPV and a high number of ultrasounds needed.

Benefit and harm of waiting time in liver transplantation for HCC.

Liver transplantation is the most successful treatment for limited-stage HCC. The waiting time for liver transplantation (LT) can be a critical factor affecting the oncological prognosis and outcome of patients with HCC. Efficient strategies to optimize waiting time are essential to maximize the benefits of LT and to reduce the harm of delay in transplantation. The ever-increasing demand for donor livers emphasizes the need to improve the organization of the waiting list for transplantation and to optimize organ availability for patients with and without HCC. Current progress in innovations to expand the donor pool includes the implementation of living donor LT and the use of grafts from extended donors. By expanding selection criteria, an increased number of patients are eligible for transplantation, which necessitates criteria to prevent futile transplantations. Thus, the selection criteria for LT have evolved to include not only tumor characteristics but biomarkers as well. Enhancing our understanding of HCC tumor biology through the analysis of subtypes and molecular genetics holds significant promise in advancing the personalized approach for patients. In this review, the effect of waiting time duration on outcome in patients with HCC enlisted for LT is discussed.

Transarterial Chemoembolization With Drug-Eluting Beads Versus Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma: Outcomes From a Multicenter, Randomized, Phase 2 Trial (the TRENDY Trial).

PURPOSE: To compare transarterial chemoembolization delivered with drug eluting beads (TACE-DEB) with stereotactioc body radiation therapy (SBRT) in patients with hepatocellular carcinoma (HCC) in a multicenter randomized trial. METHODS AND MATERIALS: Patients were included if they were eligible for TACE. They could also be recruited if they required treatment prior to liver transplantation. A maximum of four TACE-DEB procedures and ablation after incomplete TACE-DEB were both allowed. SBRT was delivered in six fractions of 8-9Gy. Primary end point was time to progression (TTP). Secondary endpoints were local control (LC), overall survival (OS), response rate (RR), toxicity, and quality of life (QoL). The calculated sample size was 100 patients. RESULTS: Between May 2015 and April 2020, 30 patients were randomized to the study. Due to slow accrual the trial was closed prematurely. Two patients in the SBRT arm were considered ineligible leaving 16 patients in the TACE-DEB arm and 12 in the SBRT arm. Median follow-up was 28.1 months. Median TTP was 12 months for TACEDEB and 19 months for SBRT (p=0.15). Median LC was 12 months for TACE-DEB and >40 months (not reached) for SBRT (p=0.075). Median OS was 36.8 months for TACEDEB and 44.1 months for SBRT (p=0.36). A post-hoc analysis showed 100% for SBRT 1- and 2-year LC, and 54.4% and 43.6% for TACE-DEB (p=0.019). Both treatments resulted in RR>80%. Three episodes of possibly related toxicity grade ≥3 were observed after TACE-DEB. No episodes were observed after SBRT. QoL remained stable after both treatment arms. CONCLUSIONS: In this trial, TTP after TACE-DEB was not significantly improved by SBRT, while SBRT showed higher local antitumoral activity than TACE-DEB, without detrimental effects on OS, toxicity and QoL. To overcome poor accrual in randomized trials that include SBRT, and to generate evidence for including SBRT in treatment guidelines, international cooperation is needed.

GITR Ligation Improves Anti-PD1-Mediated Restoration of Human MMR-Proficient Colorectal Carcinoma Tumor-Derived T Cells.

BACKGROUND & AIMS: In contrast to mismatch repair deficient colorectal carcinoma (CRC), MMR proficient (pMMR) CRC does not respond to immune checkpoint blockade. We studied immune checkpoint stimulation via glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) on ex vivo functionality of human tumor-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM). METHODS: Using lymphocytes from resected tumor, adjacent tissues, and peripheral blood mononuclear cells (PBMC) of 132 pMMR primary CRC or CRLM patients, we determined GITR expression and the in vitro T-cell agonistic activity of recombinant GITR ligation. RESULTS: Here, we show that GITR was overexpressed on TIL when compared with other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced in TIL compared with PBMC and adjacent tissues. Among CD4+ TIL, GITR expression was primarily expressed by CD45RA- FoxP3hi activated regulatory T cells. Within CD8+ TIL, GITR was predominantly expressed on functionally exhausted and putative tumor-reactive CD103+ CD39+ TIL. Strikingly, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers. Dual treatment with GITRL and nivolumab (anti-PD1) enhanced CD8+ TIL expansion compared with GITRL monotherapy. Moreover, GITRL/anti-PD1 dual therapy further improved anti-PD1-mediated reinvigoration of interferon gamma secretion by exhausted CD8 TIL from primary CRC. CONCLUSIONS: GITR is overexpressed on CD4+ and CD8+ TIL from pMMR CRC and CRLM. Agonistic targeting of GITR enhances ex vivo human TIL functionality and may therefore be a promising approach for novel monotherapy or combined immunotherapies in primary pMRR CRC and CRLM.

Systemic T-cell and humoral responses against cancer testis antigens in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide due to high recurrence rates after curative treatment and being frequently diagnosed at an advanced stage. Immune-checkpoint inhibition (ICPI) has yielded impressive clinical successes in a variety of solid cancers, however results in treatment of HCC have been modest. Vaccination could be a promising treatment to synergize with ICPI and enhance response rates. Cancer testis antigens (CTAs) were recently discovered to be widely expressed in HCC and expression in macroscopically tumor-free tissues correlated with recurrence, implying the presence of micro-satellites. To determine whether CTAs are immunogenic in HCC patients, we analyzed systemic T-cell and humoral responses against seven CTAs in 38 HCC patients using a multitude of techniques; flowcytometry, ELISA and whole antigen and peptide stimulation assays. CTA-specific T-cells were detected in all (25/25) analyzed patients, of which most had a memory phenotype but did not exhibit unequivocal signs of chronic stimulation or recent antigen encounter. Proliferative CD4+ and CD8+ T-cell responses against these CTAs were found in 14/16 analyzed HCC patients. CTA-peptide stimulation-induced granzyme B, IL2, and TNFa in 8/8 analyzed patients, including two MAGEA1 peptides included based on in silico prediction. Finally, IgG responses were observed in 13/32 patients, albeit with low titers. The presence of CD4+ and CD8+ T-cells and IgG responses shows the immunogenicity of these CTAs in HCC-patients. We hypothesize that vaccines based on these tumor-specific antigens may boost preexisting CTA-specific immunity and could enhance therapeutic efficacy of ICPI in advanced HCC.

Safety and Efficacy of 166Ho Radioembolization in Hepatocellular Carcinoma: The HEPAR Primary Study.

The safety and efficacy of 166Ho radioembolization was first determined in the HEPAR and HEPAR II studies, which, however, excluded patients with hepatocellular carcinoma (HCC). The aim of this prospective clinical early phase II study was to establish the toxicity profile of 166Ho radioembolization in patients with measurable, liver-dominant HCC; Barcelona clinic liver cancer stage B or C; a Child-Pugh score of no more than B7; and an Eastern Cooperative Oncology Group performance status of 0-1 without curative treatment options. Methods: The primary endpoint was a rate of unacceptable toxicity defined as grade 3 hyperbilirubinemia (Common Terminology Cancer Adverse Events, version 4.03) in combination with a low albumin or ascites level in the absence of disease progression or treatment-related serious adverse events. Secondary endpoints included overall toxicity, response, survival, change in α-fetoprotein, and quality of life. Thirty-one patients with Barcelona clinic liver cancer stage B (71%) or C (29%) HCC were included, mostly multifocal (87%) or bilobar (55%) disease. Results: Common grade 1 or 2 clinical toxicity included fatigue (71%), back pain (55%), ascites (32%), dyspnea (23%), nausea (23%), and abdominal pain (23%), with no more than 10% grade 3-5 toxicity. Grade 3 laboratory toxicity (>10%) included an aspartate transaminase and γ-glutamyltransferase increase (16%), hyperglycemia (19%), and lymphopenia (29%). Treatment-related unacceptable toxicity occurred in 3 of 31 patients. At 3 mo, 54% of target lesions showed a complete or partial response according to modified RECIST. Median overall survival was 14.9 mo (95% CI, 10.4-24.9 mo). No significant changes in quality of life or pain were observed. Conclusion: The safety of 166Ho radioembolization was confirmed in HCC, with less than 10% unacceptable toxicity. Efficacy data support further evaluation.

Modelling immune cytotoxicity for cholangiocarcinoma with tumour-derived organoids and effector T cells.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. METHODS: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. RESULTS: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. CONCLUSIONS: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.

External Validation of the RETREAT Score for Prediction of Hepatocellular Carcinoma Recurrence after Liver Transplantation.

BACKGROUND: We aimed to externally validate the performance of the RETREAT score in a European population. METHODS: This single center retrospective cohort study enrolled all consecutive patients with HCC who underwent LT between 1989 and 2019. The performance of RETREAT was assessed in the overall population and after stratification between being within or beyond the Milan criteria based on the explant pathology report. Recurrence probabilities were estimated by using the Kaplan-Meier method and compared by log-rank test. RESULTS: We studied 203 patients; 42 patients were beyond the Milan criteria based on explant pathology. The median follow-up was 26.8 months (IQR 7.2-60.7). Overall cumulative HCC recurrence rates were 10.6%, 21.3%, and 23.0% at 2, 5, and 10 years, with the majority of recurrences extrahepatic and at multiple sites. Higher RETREAT scores were associated with higher recurrence rates, with a 10-year recurrence rate of 60.5% in patients with RETREAT ≥ 3 (n = 65), compared to 6.2% in those with RETREAT ≤2 (n = 138; p < 0.001). HCC recurrence rates were even lower in patients within the Milan criteria who also had a low RETREAT score (n = 122; 2.7% at 10 years). CONCLUSION: Low RETREAT scores identify patients at low risk of HCC recurrence after LT in patients within the Milan criteria based on explant pathology.

Histopathological growth patterns modify the prognostic impact of microvascular invasion in non-cirrhotic hepatocellular carcinoma.

BACKGROUND: Microvascular invasion (MVI) is an established prognosticator in hepatocellular carcinoma (HCC). Histopathological growth patterns (HGPs) classify the invasive margin of hepatic tumors, with superior survival observed for the desmoplastic HGP. Our aim was to investigate non-cirrhotic HCC in light of MVI and the HGP. METHODS: A retrospective cohort study was performed in resected non-cirrhotic HCC. MVI was assessed prospectively. The HGP was determined retrospectively, blinded, and according to guidelines. Overall and disease-free survival (OS, DFS) were evaluated by Kaplan-Meier and multivariable Cox regression. RESULTS: The HGP was determined in 155 eligible patients, 55 (35%) featured a desmoplastic HGP. MVI was observed in 92 (59%) and was uncorrelated with HGP (64% vs 57%, p = 0.42). On multivariable analysis, non-desmoplastic and MVI-positive were associated with an adjusted HR [95%CI] of 1.61 [0.98-2.65] and 3.22 [1.89-5.51] for OS, and 1.59 [1.05-2.41] and 2.30 [1.52-3.50] for DFS. Effect modification for OS existed between HGP and MVI (p < 0.01). Non-desmoplastic MVI-positive patients had a 5-year OS of 36% (HR: 5.21 [2.68-10.12]), compared to 60% for desmoplastic regardless of MVI (HR: 2.12 [1.08-4.18]), and 86% in non-desmoplastic MVI-negative. CONCLUSION: HCCs in non-cirrhotic livers display HGPs which may be of prognostic importance, especially when combined with MVI.

Immune suppressive checkpoint interactions in the tumour microenvironment of primary liver cancers.

Liver cancer is one of the most prevalent cancers, and the third most common cause of cancer-related mortality worldwide. The therapeutic options for the main types of primary liver cancer-hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA)-are very limited. HCC and CCA are immunogenic cancers, but effective immune-mediated tumour control is prevented by their immunosuppressive tumour microenvironment. Despite the critical involvement of key co-inhibitory immune checkpoint interactions in immunosuppression in liver cancer, only a minority of patients with HCC respond to monotherapy using approved checkpoint inhibitor antibodies. To develop effective (combinatorial) therapeutic immune checkpoint strategies for liver cancer, in-depth knowledge of the different mechanisms that contribute to intratumoral immunosuppression is needed. Here, we review the co-inhibitory pathways that are known to suppress intratumoral T cells in HCC and CCA. We provide a detailed description of insights from preclinical studies in cellular crosstalk within the tumour microenvironment that results in interactions between co-inhibitory receptors on different T-cell subsets and their ligands on other cell types, including tumour cells. We suggest alternative immune checkpoints as promising targets, and draw attention to the possibility of combined targeting of co-inhibitory and co-stimulatory pathways to abrogate immunosuppression.

Activated CD4+ T Cells and Highly Differentiated Alloreactive CD4+ T Cells Distinguish Operationally Tolerant Liver Transplantation Recipients.

Spontaneous operational tolerance to the allograft develops in a proportion of liver transplantation (LT) recipients weaned off immunosuppressive (IS) drugs. Several studies have investigated whether peripheral blood circulating T cells could play a role in the development or identify operational tolerance, but never characterized alloreactive T cells in detail due to the lack of a marker for these T cells. In this study, we comprehensively investigated phenotypic and functional characteristics of alloreactive circulating T cell subsets in tolerant LT recipients (n = 15) using multiparameter flow cytometry and compared these with LT recipients on IS drugs (n = 23) and healthy individuals (n = 16). Activation-induced CD137 was used as a marker for alloreactive T cells upon allogenic stimulation. We found that central and effector memory CD4+ T cells were hyporesponsive against donor and third-party splenocyte stimulation in tolerant LT recipients, whereas an overall hyperresponsiveness was observed in alloreactive terminally differentiated effector memory CD4+ T cells. In addition, elevated percentages of circulating activated T helper cells were observed in these recipients. Lastly, tolerant and control LT recipients did not differ in donor-specific antibody formation. In conclusion, a combination of circulating hyperresponsive highly differentiated alloreactive CD4+ T cells and circulating activated T helper cells could discriminate tolerant recipients from a larger group of LT recipients.

Real-life data on the impact of successful downstaging in patients with hepatocellular carcinoma: A Dutch Multicenter Study.

Patients with Barcelona Clinic Liver Cancer intermediate stage hepatocellular carcinoma (HCC) theoretically are an excellent group to consider downstaging using locoregional therapy (LRT) since they do not have extrahepatic spread or vascular invasion. Once successful, this can change the treatment strategy from palliative to curative intention. Although downstaging therapy is suggested in guidelines, it is still not widely accepted. Moreover, studies on downstaging are mainly performed in high-incidence HCC countries. Therefore, our aim was to gain insight in therapeutic strategies in patients with intermediate stage HCC and their impact on intention-to-treat survival in a real-life setting in a low-incidence HCC country. We retrospectively analyzed data from the national Dutch HCC registry. From this database, consisting of 1409 patients with a diagnosis of HCC between 2005-2013 in 5 Dutch tertiary referral centers, we identified 165 patients with intermediate stage HCC. Out of these patients, 63 (38%) were not offered LRT, whereas 102 (62%) did receive LRT. Subsequently, 50 (49%) of the 102 patients who received LRT were successfully downstaged. Eleven patients (22% of successfully downstaged patients) eventually underwent liver transplantation. Cox regression analysis showed that a lower MELD score, an AFP value <100 ng/ml, successful downstaging and liver transplantation (all ≤p = 0.01) were positively associated to overall survival. In conclusion, our results demonstrate that LRT is not routinely offered to intermediate stage HCC patients in the Netherlands. Nevertheless, we showed that patients with intermediate stage HCC who are successfully downstaged have a survival benefit compared to those who were not.

Liver Decompensation as Late Complication in HCC Patients with Long-Term Response following Selective Internal Radiation Therapy.

Selective internal radiation therapy (SIRT) is used as a treatment for hepatocellular carcinoma (HCC). The aim of this study was to assess long-term liver-related complications of SIRT in patients who had not developed radioembolization-induced liver disease (REILD). The primary outcome was the percentage of patients without REILD that developed Child-Pugh (CP) ≥ B7 liver decompensation after SIRT. The secondary outcomes were overall survival (OS) and tumor response. These data were compared with a matched cohort of patients treated with sorafenib. Eighty-five patients were included, of whom 16 developed REILD. Of the remaining 69 patients, 38 developed liver decompensation CP ≥ B7. The median OS was 18 months. In patients without REILD, the median OS in patients with CP ≥ B7 was significantly shorter compared to those without CP ≥ B7; 16 vs. 31 months. In the case-matched analysis, the median OS was significantly longer in SIRT-treated patients; 16 vs. 8 months in sorafenib. Liver decompensation CP ≥ B7 occurred significantly more in SIRT when compared to sorafenib; 62% vs. 27%. The ALBI score was an independent predictor of liver decompensation (OR 0.07) and OS (HR 2.83). After SIRT, liver decompensation CP ≥ B7 often developed as a late complication in HCC patients and was associated with a shorter OS. The ALBI score was predictive of CP ≥ B7 liver decompensation and the OS, and this may be a valuable marker for patient selection for SIRT.

TIGIT, the Next Step Towards Successful Combination Immune Checkpoint Therapy in Cancer.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on several types of lymphocytes. Efficacy of antibody blockade of TIGIT in cancer immunotherapy is currently widely being investigated in both pre-clinical and clinical studies. In multiple cancers TIGIT is expressed on tumor-infiltrating cytotoxic T cells, helper T cells, regulatory T cells and NK cells, and its main ligand CD155 is expressed on tumor-infiltrating myeloid cells and upregulated on cancer cells, which contributes to local suppression of immune-surveillance. While single TIGIT blockade has limited anti-tumor efficacy, pre-clinical studies indicate that co-blockade of TIGIT and PD-1/PD-L1 pathway leads to tumor rejection, notably even in anti-PD-1 resistant tumor models. Among inhibitory immune checkpoint molecules, a unique property of TIGIT blockade is that it enhances not only anti-tumor effector T-cell responses, but also NK-cell responses, and reduces the suppressive capacity of regulatory T cells. Numerous clinical trials on TIGIT-blockade in cancer have recently been initiated, predominantly combination treatments. The first interim results show promise for combined TIGIT and PD-L1 co-blockade in solid cancer patients. In this review, we summarize the current knowledge and identify the gaps in our current understanding of TIGIT's roles in cancer immunity, and provide, based on these insights, recommendations for its positioning in cancer immunotherapy.

An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.

Stereotactic Body Radiation Therapy after Chemotherapy for Unresectable Perihilar Cholangiocarcinoma: The STRONG Trial, a Phase I Safety and Feasibility Study.

BACKGROUND: In unresectable pCCA, the standard of care is palliative chemotherapy. We investigated the feasibility and safety of adding stereotactic body radiation therapy (SBRT) after chemotherapy. METHODS: Patients with unresectable pCCA, stage T1-T4N0-N1M0, ECOG 0-1, having finished 6-8 cycles of cisplatin and gemcitabine without disease progression were eligible. SBRT was planned in 15 fractions of 3.0-4.5 Gy. The primary endpoints were feasibility (defined as completing SBRT as planned) and toxicity, evaluated within 3 months after SBRT (CTCAE v4.03). A conventional "3 + 3" design was used, corresponding to a sample size of 6 patients. Dose-limiting toxicity (DLT) was defined as grade ≥ 4 hepatobiliary or grade ≥ 3 gastrointestinal toxicity. The secondary endpoints, measured from the start of radiotherapy, were local control, progression-free survival, overall survival, and quality of life (QoL). ClinicalTrials.gov identifier: NCT03307538. RESULTS: Six patients were enrolled between November 2017 and March 2020. SBRT was delivered as planned. All patients were treated with 60Gy (15 × 4.0Gy). No SBRT-related DLT was observed. The most common grade ≥ 3 toxicity was cholangitis (n = 5). The median follow-up was 14 months. The 12-month local control rate was 80%. We observed no substantial changes in QoL. CONCLUSION: In patients with unresectable pCCA with stable disease after palliative chemotherapy, adding SBRT is feasible and safe. The observed local control merits an additional evaluation of effectiveness.

Expression of Cancer Testis Antigens in Tumor-Adjacent Normal Liver Is Associated with Post-Resection Recurrence of Hepatocellular Carcinoma.

High recurrence rates after resection of hepatocellular carcinoma (HCC) with curative intent impair clinical outcomes of HCC. Cancer/testis antigens (CTAs) are suitable targets for cancer immunotherapy if selectively expressed in tumor cells. The aims were to identify CTAs that are frequently and selectively expressed in HCC-tumors, and to investigate whether CTAs could serve as biomarkers for occult metastasis. Tumor and paired tumor-free liver (TFL) tissues of HCC-patients and healthy tissues were assessed for mRNA expression of 49 CTAs by RT-qPCR and protein expression of five CTAs by immunohistochemistry. Twelve CTA-mRNAs were expressed in ≥10% of HCC-tumors and not in healthy tissues except testis. In tumors, mRNA and protein of ≥ 1 CTA was expressed in 78% and 71% of HCC-patients, respectively. In TFL, CTA mRNA and protein was found in 45% and 30% of HCC-patients, respectively. Interestingly, CTA-expression in TFL was an independent negative prognostic factor for post-resection HCC-recurrence and survival. We established a panel of 12 testis-restricted CTAs expressed in tumors of most HCC-patients. The increased risk of HCC-recurrence in patients with CTA expression in TFL, suggests that CTA-expressing (pre-)malignant cells may be a source of HCC-recurrence, reflecting the relevance of targeting these to prevent HCC-recurrence.

Cancer Cell B7-H3 Expression Is More Prevalent in the Pancreato-Biliary Subtype of Ampullary Cancer Than in Pancreatic Cancer.

B7-H3 is an immunomodulatory member of the B7-superfamily with limited expression in normal tissues, but overexpression in several types of cancer. Therefore it is currently being explored as a potential target for cancer immunotherapy. The biological relevance of B7-H3 expression in pancreatic cancer is unclear, while there are no data on B7-H3 expression in ampullary cancer. We aimed to compare intra-tumoral B7-H3 expression between these two closely related cancer types and analyze its association with post-surgical disease course. B7-H3 expression levels were determined by immunohistochemistry in tissue microarrays of resected tumors of 137 pancreatic cancer patients and 83 patients with ampullary cancer of the pancreato-biliary subtype. B7-H3 was more frequently expressed in cancer cells of ampullary cancer patients compared to pancreatic cancer patients (51% versus 21%; p< 0.001). In ampullary cancer patients, but not in pancreatic cancer patients, B7-H3 cancer cell expression was associated with longer disease-free survival and patient survival. However, the prognostic value of B7-H3 was lost upon adjustment for CA19-9 levels. The frequencies of B7-H3 expression in tumor stroma did not differ between the two types of cancer (66% versus 63%). In both cancer types, stromal B7-H3 expression was not associated with post-surgical disease course. Compared to pancreatic cancer, B7-H3 is more frequently expressed in cancer cells of patients with the pancreato-biliary subtype of ampullary cancer. These data suggest that B7-H3 may represent an interesting potential target for immunotherapy in ampullary cancer rather than in pancreatic cancer.