Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Clinical and biological landscape of constitutional mismatch-repair deficiency syndrome: an International Replication Repair Deficiency Consortium cohort study.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD. METHODS: In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions. FINDINGS: We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions. INTERPRETATION: The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD. FUNDING: The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center.

LOGGIC/FIREFLY-2: a phase 3, randomized trial of tovorafenib vs. chemotherapy in pediatric and young adult patients with newly diagnosed low-grade glioma harboring an activating RAF alteration.

BACKGROUND: Pediatric low-grade glioma (pLGG) is essentially a single pathway disease, with most tumors driven by genomic alterations affecting the mitogen-activated protein kinase/ERK (MAPK) pathway, predominantly KIAA1549::BRAF fusions and BRAF V600E mutations. This makes pLGG an ideal candidate for MAPK pathway-targeted treatments. The type I BRAF inhibitor, dabrafenib, in combination with the MEK inhibitor, trametinib, has been approved by the United States Food and Drug Administration for the systemic treatment of BRAF V600E-mutated pLGG. However, this combination is not approved for the treatment of patients with tumors harboring BRAF fusions as type I RAF inhibitors are ineffective in this setting and may paradoxically enhance tumor growth. The type II RAF inhibitor, tovorafenib (formerly DAY101, TAK-580, MLN2480), has shown promising activity and good tolerability in patients with BRAF-altered pLGG in the phase 2 FIREFLY-1 study, with an objective response rate (ORR) per Response Assessment in Neuro-Oncology high-grade glioma (RANO-HGG) criteria of 67%. Tumor response was independent of histologic subtype, BRAF alteration type (fusion vs. mutation), number of prior lines of therapy, and prior MAPK-pathway inhibitor use. METHODS: LOGGIC/FIREFLY-2 is a two-arm, randomized, open-label, multicenter, global, phase 3 trial to evaluate the efficacy, safety, and tolerability of tovorafenib monotherapy vs. current standard of care (SoC) chemotherapy in patients < 25 years of age with pLGG harboring an activating RAF alteration who require first-line systemic therapy. Patients are randomized 1:1 to either tovorafenib, administered once weekly at 420 mg/m2 (not to exceed 600 mg), or investigator's choice of prespecified SoC chemotherapy regimens. The primary objective is to compare ORR between the two treatment arms, as assessed by independent review per RANO-LGG criteria. Secondary objectives include comparisons of progression-free survival, duration of response, safety, neurologic function, and clinical benefit rate. DISCUSSION: The promising tovorafenib activity data, CNS-penetration properties, strong scientific rationale combined with the manageable tolerability and safety profile seen in patients with pLGG led to the SIOPe-BTG-LGG working group to nominate tovorafenib for comparison with SoC chemotherapy in this first-line phase 3 trial. The efficacy, safety, and functional response data generated from the trial may define a new SoC treatment for newly diagnosed pLGG. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05566795. Registered on October 4, 2022.

Correlation Between Indocyanine Green Fluorescence Patterns and Grade of Differentiation of Hepatocellular Carcinoma: A Western Prospective Cohort Study.

Background. Most of the available evidence on the use of indocyanine green (ICG) fluorescence in clinical practice consists of articles published by surgeons of the Asian-Pacific area. We performed a prospective cohort study to assess the patterns of ICG fluorescence in Western hepatocellular carcinoma (HCC) counterparts.Methods. From April 2019 to January 2022, a total of 31 consecutive patients who underwent laparoscopic liver resection (LLR) for superficial HCC were enrolled in this prospective study. All patients underwent laparoscopic staging with both laparoscopic ultrasound (LUS) and ICG fluorescence imaging.Results. A total of 38 hepatocellular carcinomas (HCCs) were enrolled: 23 superficial (surfacing at the liver's Glissonian capsule), 5 exophytic, 5 shallow (<8 mm from the hepatic surface) and 5 deep (>10 mm from the hepatic surface). The detection rate with preoperative imaging (abdominal CT/MRI), LUS, ICG fluorescence and combined modalities (ICG and LUS) was 97.4%, 94.9%, 89.7% and 100%, respectively. The five deep seated lesions underwent ultrasound-guided laparoscopic thermal ablation. The other 33 HCCs were treated with minimally invasive liver resection. Intraoperative ultrasound patterns were registered for each single nodule resected. The ICG fluorescence pattern was classified in two types: total fluorescence (all the tumoral tissue showed strong and homogeneous fluorescence), n = 9/33 (27.3%), and non-total fluorescence (partial and rim fluorescence), n = 24/33 (72.7%). There was a statistical correlation between ICG patterns and grade of differentiation. Almost all lesions with uniform fluorescence pattern were well-differentiated HCCs (G1-G2), while partial and rim-type fluorescence pattern were more common among moderately and poorly differentiated HCCs (G3-G4) (88.9% vs 11.1%, 37.5% vs 62.5%, P = .025, respectively).Conclusions. ICG fluorescence imaging could be used to identify early the grade of HCC, ie intraoperatively, thus influencing the intraoperative treatment.

Adjuvant therapy of histopathological risk factors of retinoblastoma in Europe: A survey by the European Retinoblastoma Group (EURbG).

INTRODUCTION: Advanced intraocular retinoblastoma can be cured by enucleation, but spread of retinoblastoma cells beyond the natural limits of the eye is related to a high mortality. Adjuvant therapy after enucleation has been shown to prevent metastasis in children with risk factors for extraocular retinoblastoma. However, histological criteria and adjuvant treatment regimens vary and there is no unifying consensus on the optimal choice of treatment. METHOD: Data on guidelines for adjuvant treatment in European retinoblastoma referral centres were collected in an online survey among all members of the European Retinoblastoma Group (EURbG) network. Extended information was gathered via personal email communication. RESULTS: Data were collected from 26 centres in 17 countries. Guidelines for adjuvant treatment were in place at 92.3% of retinoblastoma centres. There was a consensus on indication for and intensity of adjuvant treatment among more than 80% of all centres. The majority of centres use no adjuvant treatment for isolated focal choroidal invasion or prelaminar optic nerve invasion. Patients with massive choroidal invasion or postlaminar optic nerve invasion receive adjuvant chemotherapy, while microscopic invasion of the resection margin of the optic nerve or extension through the sclera are treated with combined chemo- and radiotherapy. CONCLUSION: Indications and adjuvant treatment regimens in European retinoblastoma referral centres are similar but not uniform. Further biomarkers in addition to histopathological risk factors could improve treatment stratification. The high consensus in European centres is an excellent foundation for a common European study with prospective validation of new biomarkers.

COVID-19 outbreak and acute cholecystitis in a Hub Hospital in Milan: wider indications for percutaneous cholecystostomy.

BACKGROUND: COVID-19 pandemic has impacted the Italian National Health Care system at many different levels, causing a complete reorganization of surgical wards. In this context, our study retrospectively analysed the management strategy for patients with acute cholecystitis. METHODS: We analysed all patients admitted to our Emergency Department for acute cholecystitis between February and April 2020 and we graded each case according to 2018 Tokyo Guidelines. All patients were tested for positivity to SARS-CoV-2 and received an initial conservative treatment. We focused on patients submitted to cholecystostomy during the acute phase of pandemic and their subsequent disease evolution. RESULTS: Thirty-seven patients were admitted for acute cholecystitis (13 grade I, 16 grade II, 8 grade III). According to Tokyo Guidelines (2018), patients were successfully treated with antibiotic only, bedside percutaneous transhepatic gallbladder drainage (PC) and laparoscopic cholecystectomy (LC) in 29.7%, 21.6% and 48.7% of cases respectively. Therapeutic strategy of three out of 8 cases, otherwise fit for surgery, submitted to bedside percutaneous transhepatic gallbladder drainage (37.5%), were directly modified by COVID-19 pandemic: one due to the SARS-CoV-2 positivity, while two others due to unavailability of operating room and intensive care unit for post-operative monitoring respectively. Overall success rate of percutaneous cholecystostomy was of 87.5%. The mean post-procedural hospitalization length was 9 days, and no related adverse events were observed apart from transient parietal bleeding, conservatively treated. Once discharged, two patients required readmission because of acute biliary symptoms. Median time of drainage removal was 43 days and only 50% patients thereafter underwent cholecystectomy. CONCLUSIONS: Percutaneous cholecystostomy has shown to be an effective and safe treatment thus acquiring an increased relevance in the first phase of the pandemic. Nowadays, considering we are forced to live with the SARS-CoV-2 virus, PC should be considered as a virtuous, alternative tool for potentially all COVID-19 positive patients and selectively for negative cases unresponsive to conservative therapy and unfit for surgery.

LI-RADS to categorize liver nodules in patients at risk of HCC: tool or a gadget in daily practice?

PURPOSE: To determine the effectiveness of liver reporting and data system (LI-RADS) to diagnose hepatocellular carcinoma (HCC) and to retrospectively evaluate its impact on the adopted therapeutic strategy. MATERIALS AND METHODS: Preoperative imaging of 40 of 350 patients (median age 66, 31 M/9 F) submitted to liver resection for suspected HCC, between January 2008 and August 2019, has been retrospectively analyzed by two radiologists with different expertise, according to CT/MRI LI-RADS® v2018, both blinded to clinical and pathological results and untrained to using aforementioned scoring system. RESULTS: The perfect agreement between the readers was about 62.5% (25/40) (Cohen k: 0.41), better for LR-5 category (16/25) and higher in magnetic resonance imaging (MRI) investigations (68%; 13/19), which has been demonstrated the modality of choice for diagnosis of high probable and certain HCC, with arterial phase hyperenhancement as the most sensitive and accurate major feature. Compared to final histology, LR4 and LR5 scores assigned by senior radiologist reached sensitivity, specificity, positive and negative predictive values (PPV, PNV) and diagnostic accuracy of 90,9%, 29,0%, 93,8%, 62,5% and 87,5%, respectively, slightly higher than junior's ones. Misdiagnosis of HCC was done by both radiologists in the same two patients: 1 primary hepatic lymphoma (PHL) and 1 regenerative liver nodule (RLN). If LI-RADS would have been applied at the time of pre-surgical imaging, treatment planning would be modified in 10% of patients (4/40); the patient scheduled as LR-3 and finally resulted a focal nodular hyperplasia would have avoided liver resection. CONCLUSIONS: Application of LI-RADS, especially on MRI, may provide a more accurate evaluation of suspected HCC. PHL and RLN are the Achille's heels according to our experience.

Deficient Natural Killer Cell NKp30-Mediated Function and Altered NCR3 Splice Variants in Hepatocellular Carcinoma.

The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor-3 (NCR3) gene is transcribed into several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7-H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T-cell immunoglobulin and mucin-domain containing-3. Moreover, NKp30-positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30-mediated functionality. Tumor-infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30-mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non-neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7-H6-expressing HCC cells significantly down-modulated NKp30, that was prevented by small interfering RNA-mediated knockdown, suggesting a role for this ligand in inhibiting NKp30-mediated responses. Interestingly, B7-H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.