Monocyte-derived macrophages (mo-macs) and polymorphonuclear leukocytes (PMNs) are abundant in non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC), limiting response to immune checkpoint blockade (ICB) by promoting an immunosuppressive tumor microenvironment (TME). Preclinical work shows that tumor-derived CCR2 ligands and IL8 play a key role in recruiting mo-macs and PMNs, respectively, to the TME. Disrupting these signaling pathways augments ICB in mouse models, but clinical benefit has yet to be observed. This phase IIa trial assessed the efficacy of BMS-813160 (CCR2/5i) or BMS-986253 (anti-IL8) administered with nivolumab (NIVO) over 4 weeks prior to resection. NSCLC patients were treated with NIVO and CCR2/5i (arm A) or NIVO and anti-IL8 (arm B). HCC patients were treated with NIVO (arm C), NIVO and CCR2/5i (arm D), or NIVO and anti-IL8 (arm E). Primary endpoints were major pathologic response (≤10% viable tumor) for NSCLC and significant tumor necrosis (>70% necrosis) for HCC. Secondary endpoints were safety/tolerability, time to surgery (TTS), and radiographic response. 36 patients were enrolled from March 2020-August 2023; 14 were treated with NIVO and CCR2/5i, 16 with NIVO and anti-IL8, and 6 with NIVO. CCR2/5i and anti-IL8 were safe/tolerated (dose-limiting toxicities or grade 3/4 treatment-related adverse events). 32 of 36 patients underwent resection (mean TTS of 34.8 days). 3 patients met the primary endpoints: 2 in arm B and 1 in arm D. In patients treated with CCR2/5i, serum concentration of CCR2/5 ligands increased, and number of circulating monocytes decreased, after treatment. In patients treated with anti-IL8, serum concentration of IL8 decreased after treatment; however, number of circulating PMNs was not affected. Tissue analysis with scRNA-seq and multiplex imaging is ongoing to elucidate the biologic effects of these agents. Although CCR2/5i and anti-IL8 appear to be biologically active and exert effects on chemokine levels, they fail to significantly augment the role of ICB in the preoperative setting, contrary to preclinical evidence.

Immune checkpoint inhibitors (ICIs) are the standard of care for advanced hepatocellular carcinoma (HCC). Small trials in HCC and other tumours have evaluated the addition of stereotactic body radiotherapy (SBRT) to induce immunogenic cell death and augment ICI activity. Early-stage HCC is often surgically resected but recurrence occurs in ∼70% of patients. We previously presented the first study of perioperative ICI using cemiplimab in resectable HCC and showed ≥50% necrosis in 35% of patients after 2 doses of cemiplimab. Here we present the first trial to investigate low-dose SBRT prior to ICIs in patients with early-stage HCC. In this single-arm, open-label phase 2 trial (NCT03916627), adults with resectable HCC tumours received SBRT (8Gy x3 fractions), then 2 cycles of neoadjuvant cemiplimab 350 mg every 3 weeks before surgical resection and 8 cycles of adjuvant cemiplimab. The primary endpoint was significant tumour necrosis (STN; >70% necrosis of the resected tumour). Secondary endpoints included overall response rate, incidence of adverse events (AEs) and change in lymphocyte infiltration. Patients had pre-treatment biopsies and serial blood collection during treatment for exploratory analyses, including multiplex immunohistochemistry and single-cell proteomic and transcriptomic analysis. Data cutoff was 15 Sep 2023; surgical results obtained from 1 patient after the data cutoff are included. 20 patients were enrolled from Dec 2021 to Aug 2023: median age was 65 years, 80% were male, 50% were Asian, and 85% had a history of viral hepatitis. Of 16 patients who underwent surgical resection 3 (19%) achieved STN, 2 (13%) of which had complete tumour necrosis; in total, 6 (38%) had ≥50% tumour necrosis. Most common AEs were anaemia (35%), elevated transaminases (30%) and hyperglycemia (30%). No Grade ≥3 treatment-related AEs occurred during neoadjuvant therapy. This is the first clinical trial to report efficacy of SBRT + ICIs in patients with resectable HCC. Pathologic response rates were similar to those observed with cemiplimab alone. Planned deep tissue and blood analyses will be used to define the immunodynamic effects of this combination compared to cemiplimab alone.

When treating patients (pts) with GIC who underwent neoadjuvant chemotherapy (NAC) and resection, the decision to change to a different AC regimen based on PR remains controversial. We aimed to understand factors associated with changes in AC and whether these impacted patient outcomes. Medical records of GIC pts treated with NAC at a single institution between 1/2012-12/2018 were reviewed. Favorable PR (FPR) was defined as College of American Pathologists (CAP) score 0/1 and unfavorable PR (UPR) as score 2/3. RECIST 1.1 radiologic responses and serum tumor markers pre and post NAC were recorded. Univariable and multivariable logistic regression and survival analyses were performed. Kaplan-Meier method was used to estimate recurrence-free and overall survival (RFS, OS). 155 pts (58% male, median age 64 [27-84] years) were identified. Primary tumor sites were: 43 (27.7%) pancreas, 62 (40%) esophagogastric, and 50 (32.3%) colorectal. After NAC, 31 (20%) pts had FPR, and 124 (80%) had UPR. Of 106 pts with radiological data, 59 (55.7%) demonstrated partial or complete response after NAC. Of 112 pts with serological data, 61 (54.5%) demonstrated >50% tumor marker reduction after NAC. FPR was associated with reduced odds of changing AC (OR 0.05; CI, 0.01 - 0.39); radiographic (OR 0.92; CI, 0.38 - 2.26) and serologic (OR 1.18; CI, 0.49 - 2.87) responses were not. RFS in pts with UPR did not differ between those who changed vs. did not change AC (HR 1.01; CI, 0.55 - 1.86) nor those who received vs. did not receive AC (HR 1.96; CI, 0.97 - 3.96) after adjusting for pathological stage, surgical margin, and cancer type. Though OS in pts with UPR did not differ between those who changed vs. did not change AC (HR 1.30; CI, 0.48 - 3.51), survival was decreased in those who did not receive AC vs. those who did (HR 3.34; CI, 1.3 - 8.53). We found that UPR, but not serologic or radiographic response, was associated with a change in AC in GIC pts who received NAC. In patients with UPR, changing AC did not impact RFS and OS, though not receiving AC was associated with decreased survival. Prospective studies are needed to better understand the role of PR in AC decisions and patient outcomes.

Abstract Introduction/Objective Variable histologic findings that may be seen in porto-sinusoidal vascular disease (PSVD) liver biopsies are subject to high interobserver variability, requiring correlation with clinical history of portal hypertension (traditionally interpreted as non-cirrhotic portal hypertension NCPH). We investigated which histologic features are reproducible in PSVD biopsies. Methods Archived liver biopsies (n=38) from patients with NCPH (n=14) and without NCPH (n=21) were reviewed. Static H&E images of lobules (L, x100, NCPH=27, non-NCPH=23) and portal tracts (P, x200, NCPH=23, non- NCPH=27) were distributed among 9 gastrointestinal pathologists blinded to clinical history. Each pathologist answered multiple choice questions based on the presence (Q2) or absence (Q1) of portal hypertension clinically. The choice selected by 6 pathologists or more was considered consensus answer for the image. The interpretation of the image was considered reproducible when consensus was reached on both Q1 and Q2. Results The interpretations of 27 (54%; 17L, 10P) images from NCPH and 21 (42%; 10L, 11P) from non-NCPH were reproducible. In NCPH, the interpretations of normal (n=10, 4L, 6P), sinusoidal dilatation (n=7), and increased parenchymal draining vessels (n=3) were reproducible, while there was no consensus on the diagnoses of nodular regeneration and increased number of portal vessels. In non-NCPH, the interpretations of normal (n=8, 2L, 6P), sinusoidal dilatation (n=6), and paraportal shunting vessel(s) (n=4) were reproducible, whereas no consensus was reached on the diagnoses of nodular regeneration, incomplete fibrous septa, and increased number of portal vessels. Conclusion Histologic assessment of normal L and P as well as sinusoidal dilatation appears to be reproducible independent of clinical history. The findings of increased parenchymal draining vessels in NCPH group and paraportal shunting vessels in non-NCPH group may be consistently diagnosed to a certain extent. The assessment for nodular regeneration without reticulin stain, incomplete fibrous septa, or increased number of portal vessels appears to be unreliable.

Abstract Background and Aims: Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy. Based on its anatomical location, CCA can be divided into intrahepatic (iCCA) or extrahepatic (eCCA), with differences in etiology, pathogenesis and clinical management. Few studies have focused on the molecular profiling of eCCA as a single entity, even though it accounts for the most prevalent subtype. Thus, integrative genomic analysis of eCCA would provide critical understanding for the biological traits of this tumor. Methods: 189 FFPE primary eCCA treated by resection were collected at seven international centers from 1995 to 2015. Median survival of the cohort was of 48.5mo. Whole gene-expression profiles were submitted to unsupervised clustering by NMF consensus. Clusters were characterized by Gene Set Enrichment Analysis and Ingenuity Pathway Analysis. Activation of signaling pathways (mTOR/pRPS6 and HER2) was assessed by immunohistochemistry (IHC). Molecular features were correlated with clinico-pathological data. Screening of most prevalent somatic mutations and copy number aberrations is ongoing. Results: We have identified four distinct molecular subtypes of eCCA (cophenetic coefficient=0.995). Tumors classified within the metabolic class (18.7%) were enriched by gene signatures defining bile and fatty acid metabolism (p<0.001) and presented overexpression of classic hepatocyte markers, with HNF4A as the major activated upstream transcription factor (p<0.001). The proliferation class (22.5%) was associated with papillary histology (p=0.004) and presented enrichment of MYC (p<0.001), mTOR (p=0.018) and HER2 (p=0.024) signaling. Subclass mapping identified similarity with the iCCA proliferation subclass (p<0.001). The mesenchymal class (47.3%) was associated with signatures defining epithelial-mesenchymal transition (p<0.001) as well as stromal activation (p<0.001), which was in accordance with TGFB1 as the major activated upstream regulator (p<0.001) and the presence of higher desmoplasia at pathological analysis (p=0.046). Moreover, mesenchymal tumors were significantly associated with poor prognosis in terms of OS (33.2 vs 55.5mo, HR=2.02, p=0.022). Finally, tumors classified as immune class (11.5%) presented increased tumor-infiltrating lymphocytes (p=0.001) and were characterized by enrichment of IFNγ signaling (p<0.001). Conclusions: Transcriptome-based subtyping of eCCA identifies four distinct molecular classes (metabolic, proliferation, mesenchymal and immune) that correlate with clinical-pathological characteristics. These findings enhance the opportunities for therapeutic development in this tumor with dismal prognosis and without approved molecular treatments. Citation Format: Robert Montal, Wei Qiang Leow, Carla Montironi, Laia Bassaganyas, Agrin Moeini, Daniela Sia, Roser Pinyol, Laia Cabellos, Judit Peix, Miho Maeda, Carlos Villacorta, Parissa Tabrizian, Christine Sempoux, Beatriz Minguez, Tim Pawlik, Ismail Labgaa, Lewis Roberts, Manel Sole, Maria Isabel Fiel, Swan Thung, Sasan Roayaie, Augusto Villanueva, Myron Schwartz, Josep Maria Llovet. Integrative molecular classification of extrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4618.

AFFILIATIONS: 1. Liver Cancer Translational Research Laboratory, Liver Unit, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, CIBERehd, Universitat de Barcelona, Barcelona, Catalonia, Spain. 2. Mount Sinai Liver Cancer Program, (Divisions of Liver Diseases, Hematology and Medical Oncology, Department of Medicine, Department of Pathology, Recanati Miller Transplantation Institute), Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA. 3. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, USA 4. Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, USA. 5. Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain.

Objective: α-fetoprotein (AFP) is utilized as a biomarker in patients with hepatocellular carcinoma (HCC) to predict disease recurrence after curative resection. It is not clear whether a peri-operative drop of AFP suggests any prognostic correlation. This study aims to review the specific peri-operative kinetics of serum AFP in patients with hepatitis B-associated hepatocellular carcinoma (HBV-HCC) and their outcomes. Methods: Data was analyzed from 175 HBV-HCC patients who underwent liver resection with curative intent at a single institution. Patients had pre-operative serum AFP measurement and paired post-operative AFP measurement within 8-24 weeks of surgery. Quintiles of AFP were used to categorize AFP levels, and the impact on outcome was analyzed by univariate and multivariate analysis. Results: Sixty percent of all patients (quintiles 1–3) had post-operative AFP lower than 5.8 ng/ml within 8–24 weeks of surgery. These patients did not differ significantly in overall survival or disease-free survival when stratified based on pre-operative AFP levels. 35 patients (quintile 5) had post-operative AFP greater than 30.4 ng/ml, and these patients had significantly lower 5 year overall survival (31%, p = 0.008) compared to patients with post-operative AFP <30.4 ng/ml (OS = 82%). 83% of patients in Q5 had HCC recurrence, all of which occurred within 24 months of surgery. Multi-variate analysis showed that post-operative AFP greater than 30.4 ng/ml was significantly associated with poor overall survival independent of other variables (HR 5.1, p < 0.001). Conclusion: Post-operative AFP within 8–24 month of surgery identifies a subset of patients with HBV-HCC at high risk of recurrence, regardless of pre-operative AFP levels.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Background and aims: Molecular heterogeneity in hepatocellular carcinoma (HCC) is ill-defined since trunk drivers (early events; common to all cells), branch drivers (later events; present in a subset of cells) and passenger mutations (not relevant), have not been thoroughly described. Most FDA/EMA approved molecular drugs target trunk drivers. We explored heterogeneity by analyzing trunk vs branch mutations in different HCC regions within single and multinodular tumours. Methods: Intra-tumoral heterogeneity was assessed in 21 patients with single HCCs (size > 4cm; 2 regions/tumour: 42 samples) and inter-tumoral heterogeneity was studied in 17 patients with multinodular HCCs (2-3 nodules/patient; total: 39 samples). Gene expression profiling, SNP array and deep-sequencing (coverage ∼850x) assessing 6 oncodrivers (TERT promoter, TP53, CTNNB1, ARID1A, AXIN1-2 by TruSeqAmplicon, validated by sanger) were explored. Clonality differentiating metastatic (clonal) vs synchronic (non-clonal) tumours was defined by SNP profiles. Trunk mutations were defined as present in a) all regions of a given tumour, or b) in all nodules of metastatic-clonal tumours; all other were considered as branch. Results: Intra-tumoral heterogeneity assessed by sequencing identified at least 1 oncodriver in 19/21 patients with single tumours. Among those, trunk mutations accounted for 17/19 (90%), and branch for 2/19 cases. Overall 63 mutations were identified, 56 (90%) were identical in different tumoral regions (i.e. truncal; TERT promoter most prevalent). Inter-tumoral heterogeneity explored by SNP profiles defined metastases in 35% (6/17 multinodular cases) and synchronous tumors in 65% (11/17 cases). Genetic proximity confirmed clonality in all metastatic nodules. Regarding molecular subclasses, half of clonal tumours retained identical molecular fingerprint, but the other half switched to more aggressive subclass. All non-clonal tumours belonged to distinct molecular subclasses. Driver oncogenes were explored in 9 patients (5 metastasis and 4 synchronic). Metastatic tumours showed 13 mutations, among which 11 (85%) were truncal. Mutations in non-clonal synchronic tumours were distinct. Conclusions: Single large HCCs shared common trunk drivers at distinct regions (90%). Similarly, 40% of multinodular tumours were clonal (metastasis) and shared common trunk oncodrivers, while 60% were synchronic, with distinct genomic profile/oncodrivers. Further studies at single-cell sequencing level are recommended. Citation Format: Daniela Sia, Andrew Neelis Harrington, Sara Torrecilla, Zhongyang Zhang, Genis Camprecios, Agrin Moeini, Sara Toffanin, Maria Isabel Fiel, Ke Hao, Monica Higuera, Laia Cabellos, Helena Cornella, Milind Mahajan, Yujin Hoshida, Augusto Villanueva, Sander Florman, Myron Schwartz, Josep Maria Llovet. Molecular heterogeneity and trunk driver mutations in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2388.

Purpose: To quantify baseline relaxation rates R-2(*) and R-1 in the abdomen, their changes after respiratory challenges, and their reproducibility in healthy volunteers and patients with hepatocellular carcinoma (HCC) at 1.5T and 3.0T.Materials and Methods: R-2(*) measurements were acquired in the liver in 8 volunteers and 27 patients with 34 HCCs using multiecho T-2(*) at baseline and after respiratory challenges with 100% oxygen (O-2) and carbogen (CB=95%O-2/5%CO2). R-1 was measured at 1.5T in one volunteer and 21 patients with 23 HCCs. Test-retest coefficient of variation (CV) was assessed in 10 subjects. Intra- and interobserver variability of R-2(*) and R-1 measurements was assessed in 12 and 10 patients, respectively. Parameters for HCC, liver, and muscle were compared between baseline and after gas challenges.Results: We observed that R-2(*) and R-1 imaging of HCCs with O-2 and CB is feasible and reproducible (test-retest CV R-2(*)<15%/R-1<5%; intra- and interobserver intraclass correlation coefficient R-2(*)>0.88/R-1>0.7 and CV R-2(*)<7%/R-1<3% at 1.5T). measurements were observed to be less reproducible at 3.0T (CV<35%). There was a statistically significant decrease in R-2(*) values in HCC before and after O-2 (P=0.02) and increase in R-1 after O-2 (P=0.004). CB had no significant effect (P R-2(*)=0.47/R-1=0.278).Conclusion: R-2(*) measurements in HCC and liver parenchyma are more reproducible at 1.5T than at 3.0T, and with O-2 than with CB challenge. We observed a decrease in R-2(*) and an increase in R-1 of HCCs from baseline in response to O-2 challenge, as expected with increased tissue and blood oxygenation.