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The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8+ T cells (TRMs), resident natural killer cells (NKRs), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3+CD4+ and PD-1+ CD8+ T cells. Interestingly, Tregs and TRMs isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and TRMs isolated from the NTME. We found PD-1+ TRMs were the predominant T-cell subset responsive to anti-PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8+ T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NKRs via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed. [ABSTRACT FROM AUTHOR]

Purpose: The recent discovery and characterization of an oncogenic ROS1 gene rearrangement has raised significant interest because small molecule inhibitors are effective in these tumors. The aim of this study was to determine frequency and clinicopathological features associated with ROS1 rearrangement in patients with cholangiocarcinoma (CCA). Materials and Methods: A total of 261 patients who underwent surgery for CCA between October 1997 and August 2013 were identified from an international, multi-institutional database. ROS1 rearrangement was evaluated by break-apart fluorescence in situ hybridization using tissue microarrays of these patients. Results: Of 261 CCA evaluated, three cases (1.1%) showed ROS1 rearrangement by fluorescence in situ hybridization (FISH), all of which were derived from intrahepatic origin. ROS1 protein expression was observed in 38 samples (19.1%). Significantly larger tumor size was observed in ROS1 immunohistochemistry (IHC)-negative patients compared with ROS1 IHC-positive patients. ROS1 FISH-positive patients had a single tumor with a median size of 4 cm and well-to-moderate differentiation. Overall, there was no difference in terms of baseline characteristics, overall survival, and recurrence-free survival between ROS1-positive and -negative patients. Conclusion: ROS1 rearrangement was detected in 1.1% of CCA patients. Although rare, conduct of clinical trials using ROS1 inhibitors in these genetically unique patients is warranted. [ABSTRACT FROM AUTHOR]

Background: Carcinoma of the oral tongue (OTSCC) is the most common malignancy of the oral cavity, characterized by frequent recurrence and poor survival. The last three decades has witnessed a change in the OTSCC epidemiological profile, with increasing incidence in younger patients, females and never-smokers. Here, we sought to characterize the OTSCC genomic landscape and to determine factors that may delineate the genetic basis of this disease, inform prognosis and identify targets for therapeutic intervention. Methods: Seventy-eight cases were subjected to whole-exome (n = 18) and targeted deep sequencing (n = 60). Results: While the most common mutation was in TP53, the OTSCC genetic landscape differed from previously described cohorts of patients with head and neck tumors: OTSCCs demonstrated frequent mutations in DST and RNF213, while alterations in CDKN2A and NOTCH1 were significantly less frequent. Despite a lack of previously reported NOTCH1 mutations, integrated analysis showed enrichments of alterations affecting Notch signaling in OTSCC. Importantly, these Notch pathway alterations were prognostic on multivariate analyses. A high proportion of OTSCCs also presented with alterations in drug targetable and chromatin remodeling genes. Patients harboring mutations in actionable pathways were more likely to succumb from recurrent disease compared with those who did not, suggesting that the former should be considered for treatment with targeted compounds in future trials. Conclusions: Our study defines the Asian OTSCC mutational landscape, highlighting the key role of Notch signaling in oral tongue tumorigenesis. We also observed somatic mutations in multiple therapeutically relevant genes, which may represent candidate drug targets in this highly lethal tumor type. [ABSTRACT FROM AUTHOR]

Objective Gastric cancer (GC) is a deadly malignancy for which new therapeutic strategies are needed. Three transcription factors, KLF5, GATA4 and GATA6, have been previously reported to exhibit genomic amplification in GC. We sought to validate these findings, investigate how these factors function to promote GC, and identify potential treatment strategies for GCs harbouring these amplifications. Design KLF5, GATA4 and GATA6 copy number and gene expression was examined in multiple GC cohorts. Chromatin immunoprecipitation with DNA sequencing was used to identify KLF5/GATA4/GATA6 genomic binding sites in GC cell lines, and integrated with transcriptomics to highlight direct target genes. Phenotypical assays were conducted to assess the function of these factors in GC cell lines and xenografts in nude mice. Results KLF5, GATA4 and GATA6 amplifications were confirmed in independent GC cohorts. Although factor amplifications occurred in distinct sets of GCs, they exhibited significant mRNA coexpression in primary GCs, consistent with KLF5/GATA4/GATA6 cross-regulation. Chromatin immunoprecipitation with DNA sequencing revealed a large number of genomic sites co-occupied by KLF5 and GATA4/GATA6, primarily located at gene promoters and exhibiting higher binding strengths. KLF5 physically interacted with GATA factors, supporting KLF5/ GATA4/GATA6 cooperative regulation on co-occupied genes. Depletion and overexpression of these factors, singly or in combination, reduced and promoted cancer proliferation, respectively, in vitro and in vivo. Among the KLF5/GATA4/GATA6 direct target genes relevant for cancer development, one target gene, HNF4a, was also required for GC proliferation and could be targeted by the antidiabetic drug metformin, revealing a therapeutic opportunity for KLF5/GATA4/GATA6 amplified GCs. Conclusions KLF5/GATA4/GATA6 may promote GC development by engaging in mutual crosstalk, collaborating to maintain a pro-oncogenic transcriptional regulatory network in GC cells. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is the second leading cause of global cancer mortality worldwide. However, the molecular mechanism underlying its carcinogenesis and drug resistance is not well understood. To identify novel functionally important genes that were differentially expressed due to combinations of genetic and epigenetic changes, we analyzed datasets containing genome-wide mRNA expression, DNA copy number alterations and DNA methylation status from 154 primary GC samples and 47 matched non-neoplastic mucosa tissues from Asian patients. We used concepts of 'within' and 'between' statistical analysis to compare the difference between tumors and controls within each platform, and assessed the correlations between platforms. This 'multi-regulated gene (MRG)' analysis identified 126 differentially expressed genes that underwent a combination of copy number and DNA methylation changes. Most genes were located at genomic loci associated with GC. Statistical enrichment analysis showed that MRGs were enriched for cancer, GC and drug response. We analysed several MRGs that previously had not been associated with GC. Knockdown of DDX27, TH1L or IDH3G sensitized cells to epirubicin or cisplatin, and knockdown of RAI14 reduced cell proliferation. Further studies showed that overexpression of DDX27 reduced epirubicin-induced DNA damage and apoptosis. Levels of DDX27 mRNA and protein were increased in early-stage gastric tumors, and may be a potential diagnostic and prognostic marker for GC. In summary, we used an integrative bioinformatics strategy to identify novel genes that are altered in GC and regulate resistance of GC cells to drugs in vitro. [ABSTRACT FROM AUTHOR]

The article presents a case study of a middle aged female who presented to physicians with raised liver enzymes and antibodies against the hepatitis B core antigen which were otherwise negative for hepatitis B surface antigen as well as antibodies against the hepatitis B surface antigen. A discussion of testing which was conducted on the patient, and resulted in her receiving a diagnosis of a focal nodular hyperplasia-like lesion in her cirrhotic liver which resembled cholangiocarcinoma, is presented.

The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a nonreceptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in "normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches. [ABSTRACT FROM AUTHOR]

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and cooccurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies. [ABSTRACT FROM AUTHOR]

Introduction: Conflicting data on the clinicopathological characteristics as well as prognosis and survival of signet ring cell (SRC) and mucinous adenocarcinomas (MA) of the colorectum persist. Methods: Consecutive patients (2,764) with sporadic colorectal cancer from 1999 to 2005 were evaluated. The clinicopathological characteristics of these patients were reviewed. Univariate analysis was performed, and survival curves were constructed using the Kaplan–Meier method. Multivariate analysis assessed independent prognostic factors. Results: The incidence of MA and SRC is 6% and 1.1%, respectively. MA and SRC tend to occur in patients aged ≤50 years (SRC 33%, MA 22%, ordinary adenocarcinomas (OA) 14%, p = 0.001) and are more commonly right-sided (MA 30%, SRC 27%, OA 19%, p = 0.001) than OA. SRC tend to be poorly differentiated (SRC 77%, MA 26%, OA 6%, p < 0.0001) and have a higher risk of recurrence (SRC 40%, MA 26%, OA 6%, p < 0.0001). SRC and MA are more likely to have locally advanced lesions (T3/T4; SRC 100%, MA 90%, OA 83%, p = 0.002) and lymph node metastases (SRC 89%, MA 61%, OA 52%, p < 0.0001) and present with an advanced stage at diagnosis (stage III/IV SRC 94%, MA 67%, OA 56%, p < 0.0001). SRC has poorer 5-year cancer-specific survival (CSS; 11.1%, 95% confidence interval (CI) 0–22.9%) compared with MA (46.8%, 95% CI 38.6–55.0%) and OA (58.7%, 95% CI 56.5–60.9%, p < 0.001). In a multivariate analysis, SRC is an independent poor prognostic factor (HR 1.9, 95% CI 1.1–3.0) but MA is not. Conclusion: SRC and MA demonstrate clinicopathologic characteristics suggestive of a different biology compared with OA. In our dataset, SRC has a significantly poorer CSS whereas survival rates for MA are similar to OA. These characteristics are similar in both Asian and Western studies reported. Asian reports however suggest a lower incidence of MA. [ABSTRACT FROM AUTHOR]

Background/Aims: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the two most common primary malignant liver tumours in children. Hepatic progenitor cells have been described and can be stained with K19, EpCAM and CD117. We investigated the morphology and staining patterns of primary liver tumours in Asian children. Methods: Four pathologists studied slides from 39 paediatric patients from Vietnam and Korea aged 8 months to 16 years. We performed immunohistochemical stains for K19, EpCAM and CD117, and polymerase chain reaction for tissue hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. Results: There was agreement on the diagnosis of 24 cases of HCC and 10 cases of HB (one recurrent case). The diagnosis was split for six cases (HCC/HB). All 20 cases of HCC tested were HBV DNA+ and HCV RNA−. All nine cases of HB tested were HBV DNA−, while one was HCV RNA+. Of four HCC/HB cases tested, three were HBV DNA+ and all were HCV RNA−. By immunohistochemistry, 8/24 (33%) cases of HCC were K19+ and 18/24 (75%) were EpCAM+, 5/10 (50%) cases of HB were K19+ and 7/10 (70%) were EpCAM+ and 3/6 (50%) cases of HCC/HB were K19+ and 5/6 (83%) were EpCAM+. CD117 was negative in all 38 cases tested. Paediatric HCC has a morphology different from adult HCC, sometimes resembling HB, and a larger proportion of paediatric tumours have progenitor cell features. Conclusions: HB and HCC in children may represent malignant transformation at an early stage in the cellular lineage and often arise from hepatic progenitor cells. [ABSTRACT FROM AUTHOR]

OBJECTIVES:Hereditary mixed polyposis syndrome (HMPS) is characterized by polyps of mixed adenomatous/hyperplastic/atypical juvenile histology that are autosomal dominantly inherited and that eventually lead to colorectal cancer (CRC). Although CRC with adenomatous polyps is initiated by inactivating adenomatous polyposis coli (APC), the initiating event of CRC with mixed polyps remains unclear. We aimed to identify the underlying germline defect in HMPS.METHODS:We screened for bone morphogenesis protein receptor 1A (BMPR1A) mutation by exonic sequencing, reverse-transcriptase polymerase chain reaction (PCR) followed by cDNA sequencing, and multiplex ligation-dependent probe amplification (MLPA) analysis in eight Singapore Chinese HMPS families.RESULTS:Germline BMPR1A defects were found in four (50%) families. In two families, it is shown to co-segregate with the disease phenotype in all affected members over three generations, indicating that it is the disease-causing mutation. CRC incidence is 75%. The most defining characteristic is the presence of mixed hyperplastic-adenomatous polyps. Juvenile polyps are rarely reported, and if present, are usually of mixed components. Detailed histology of the polyps from one patient over 11 years distinguishes HMPS from juvenile polyposis syndrome (JPS). We report further the first cases of Wilms' tumor and papillary thyroid carcinoma associated with BMPR1A germline defect.CONCLUSIONS:Germline BMPR1A defect is the disease-causing mutation in 50% of the HMPS families. If patients present with mixed morphology polyps in the large bowel that are autosomal dominantly inherited and corresponding absence of upper gastrointestinal abnormalities, the gene to begin mutation screening should be BMPR1A rather than APC. [ABSTRACT FROM AUTHOR]

In this paper, we report a case of bleeding solitary jejunal Dieulafoy pseudopolyp that was detected on capsule endoscopy and treated with a laparoscopic-assisted transumbilical polypectomy procedure. This case illustrates an innovative, tailored application of minimal invasive techniques in the management of a relatively uncommon lesion. To our knowledge, this is the first case report of the combination of capsule endoscopy and laparoscopic-assisted transumbilical resection for a bleeding jejunal Dieulafoy pseudopolyp in children. [ABSTRACT FROM AUTHOR]

The article presents the case of a 65-year old Chinese man who had prostatic ductal adenocarcinoma which expressed thyroid transcription factor-1. A nodule and extracellular mucin was found in his prostate biopsy. He underwent a radical prostatectomy to remove the nodular lesion in his left prostate lobe. The patient had a nodule between the bladder and rectum which represents a hematoma after the surgery.

Abstract Peritoneal metastasis (PM), the regional progression of intra-abdominal malignancies, is a common sequelae of colorectal cancer (CRC). Immunotherapy is slated to be effective in generating long-lasting anti-tumour response as it utilizes the specificity and memory of the immune system. In the tumour microenvironment, tumour associated macrophages (TAMs) are posited to create an anti-inflammatory pro-tumorigenic environment. In this paper, we aimed to identify immunomodulatory factors associated with colorectal PM (CPM). A publicly available colorectal single cell database (GSE183916) was analysed to identify possible immunological markers that are associated with the activation of macrophages in cancers. Immunohistochemical analysis for V-set and immunoglobin containing domain 4 (VSIG4) expression was performed on tumour microarrays (TMAs) of tumours of colorectal origin (n = 211). Expression of VSIG4 in cell-free ascites obtained from CPM patients (n = 39) was determined using enzyme-linked immunosorbent assay (ELISA). CD163-positive TAMs cluster expression was extracted from a publicly available single cell database and evaluated for the top 100 genes. From these macrophage-expressed genes, VSIG4, a membrane protein produced by the M2 macrophages, mediates the up-regulation of anti-inflammatory and down-regulation of pro-inflammatory macrophages, contributing to an overall anti-inflammatory state. CRC TMA IHC staining showed that low expression of VSIG4 in stromal tissues of primary CRC are associated with poor prognosis (p = 0.0226). CPM ascites also contained varying concentrations of VSIG4, which points to a possible role of VSIG4 in the ascites. The contribution of VSIG4 to CPM development can be further evaluated for its potential as an immunotherapeutic agent.

IntroductionDeep learning (DL) models predicting biomarker expression in images of hematoxylin and eosin (H&E)-stained tissues can improve access to multi-marker immunophenotyping, crucial for therapeutic monitoring, biomarker discovery, and personalized treatment development. Conventionally, these models are trained on ground truth cell labels derived from IHC-stained tissue sections adjacent to H&E-stained ones, which might be less accurate than labels from the same section. Although many such DL models have been developed, the impact of ground truth cell label derivation methods on their performance has not been studied.MethodologyIn this study, we assess the impact of cell label derivation on H&E model performance, with CD3+ T-cells in lung cancer tissues as a proof-of-concept. We compare two Pix2Pix generative adversarial network (P2P-GAN)-based virtual staining models: one trained with cell labels obtained from the same tissue section as the H&E-stained section (the ‘same-section’ model) and one trained on cell labels from an adjacent tissue section (the ‘serial-section’ model).ResultsWe show that the same-section model exhibited significantly improved prediction performance compared to the ‘serial-section’ model. Furthermore, the same-section model outperformed the serial-section model in stratifying lung cancer patients within a public lung cancer cohort based on survival outcomes, demonstrating its potential clinical utility.DiscussionCollectively, our findings suggest that employing ground truth cell labels obtained through the same-section approach boosts immunophenotyping DL solutions.

The article presents a case study of a Chinese male who was referred for dysphagia and regurgitation. Using Barium swallows and gastroscopy, a presumptive diagnosis of achalasia was made. After serial esophageal dilatations, he experienced intermittent episodes of abdominal bloating, weight loss and reduced bowel movements which clinical investigations diagnosed to be small-bowel intestinal obstruction (IO) without a mechanical cause called chronic intestinal pseudo-obstruction (CIPO).

Several photographs related to pneumoperitoneum resulting from pneumatosis cystoides intestinalis, including those on erect chest X-ray, high-power microscopic view, and low-power microscopic view, are presented.

Abstract Background Conventional differential expression (DE) testing compares the grouped mean value of tumour samples to the grouped mean value of the normal samples, and may miss out dysregulated genes in small subgroup of patients. This is especially so for highly heterogeneous cancer like Hepatocellular Carcinoma (HCC). Methods Using multi-region sampled RNA-seq data of 90 patients, we performed patient-specific differential expression testing, together with the patients’ matched adjacent normal samples. Results Comparing the results from conventional DE analysis and patient-specific DE analyses, we show that the conventional DE analysis omits some genes due to high inter-individual variability present in both tumour and normal tissues. Dysregulated genes shared in small subgroup of patients were useful in stratifying patients, and presented differential prognosis. We also showed that the target genes of some of the current targeted agents used in HCC exhibited highly individualistic dysregulation pattern, which may explain the poor response rate. Discussion/conclusion Our results highlight the importance of identifying patient-specific DE genes, with its potential to provide clinically valuable insights into patient subgroups for applications in precision medicine.

Background Combination therapy with radioembolization (yttrium-90)-resin microspheres) followed by nivolumab has shown a promising response rate of 30.6% in a Phase II trial (CA209-678) for advanced hepatocellular carcinoma (HCC); however, the response mechanisms and relevant biomarkers remain unknown.Methods By collecting both pretreatment and on-treatment samples, we performed multimodal profiling of tissue and blood samples and investigated molecular changes associated with favorable responses in 33 patients from the trial.Results We found that higher tumor mutation burden, NCOR1 mutations and higher expression of interferon gamma pathways occurred more frequently in responders. Meanwhile, non-responders tended to be enriched for a novel Asian-specific transcriptomic subtype (Kaya_P2) with a high frequency of chromosome 16 deletions and upregulated cell cycle pathways. Strikingly, unlike other cancer types, we did not observe any association between T-cell populations and treatment response, but tumors from responders had a higher proportion of CXCL9+/CXCR3+ macrophages. Moreover, biomarkers discovered in previous immunotherapy trials were not predictive in the current cohort, suggesting a distinctive molecular landscape associated with differential responses to the combination therapy.Conclusions This study unraveled extensive molecular changes underlying distinctive responses to the novel treatment and pinpointed new directions for harnessing combination therapy in patients with advanced HCC.

IntroductionImmune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC.MethodsHCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses.ResultsImmunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status.ConclusionImmunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.

Background & Aims: Lifestyle and environmental-related exposures are important risk factors for hepatocellular carcinoma (HCC), suggesting that epigenetic dysregulation significantly underpins HCC. We profiled 30 surgically resected tumours and the matched adjacent normal tissues to understand the aberrant epigenetic events associated with HCC. Methods: We identified tumour differential enhancers and the associated genes by analysing H3K27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C/HiChIP data from the resected tumour samples of 30 patients with early-stage HCC. This epigenome dataset was analysed with previously reported genome and transcriptome data of the overlapping group of patients from the same cohort. We performed patient-specific differential expression testing using multiregion sequencing data to identify genes that undergo both enhancer and gene expression changes. Based on the genes selected, we identified two patient groups and performed a recurrence-free survival analysis. Results: We observed large-scale changes in the enhancer distribution between HCC tumours and the adjacent normal samples. Many of the gain-in-tumour enhancers showed corresponding upregulation of the associated genes and vice versa, but much of the enhancer and gene expression changes were patient-specific. A subset of the upregulated genes was activated in a subgroup of patients’ tumours. Recurrence-free survival analysis revealed that the patients with a more robust upregulation of those genes showed a worse prognosis. Conclusions: We report the genomic enhancer signature associated with differential prognosis in HCC. Findings that cohere with oncofoetal reprogramming in HCC were underpinned by genome-wide enhancer rewiring. Our results present the epigenetic changes in HCC that offer the rational selection of epigenetic-driven gene targets for therapeutic intervention or disease prognostication in HCC. Impact and Implications: Lifestyle and environmental-related exposures are the important risk factors of hepatocellular carcinoma (HCC), suggesting that tumour-associated epigenetic dysregulations may significantly underpin HCC. We profiled tumour tissues and their matched normal from 30 patients with early-stage HCC to study the dysregulated epigenetic changes associated with HCC. By also analysing the patients’ RNA-seq and clinical data, we found the signature genes – with epigenetic and transcriptomic dysregulation – associated with worse prognosis. Our findings suggest that systemic approaches are needed to consider the surrounding cellular environmental and epigenetic changes in HCC tumours.

Abstract Colorectal cancer is one of the most common cancers worldwide, accounting for an annual estimated 1.8 million incident cases. With the increasing number of colonoscopies being performed, colorectal biopsies make up a large proportion of any histopathology laboratory workload. We trained and validated a unique artificial intelligence (AI) deep learning model as an assistive tool to screen for colonic malignancies in colorectal specimens, in order to improve cancer detection and classification; enabling busy pathologists to focus on higher order decision-making tasks. The study cohort consists of Whole Slide Images (WSI) obtained from 294 colorectal specimens. Qritive’s unique composite algorithm comprises both a deep learning model based on a Faster Region Based Convolutional Neural Network (Faster-RCNN) architecture for instance segmentation with a ResNet-101 feature extraction backbone that provides glandular segmentation, and a classical machine learning classifier. The initial training used pathologists’ annotations on a cohort of 66,191 image tiles extracted from 39 WSIs. A subsequent application of a classical machine learning-based slide classifier sorted the WSIs into ‘low risk’ (benign, inflammation) and ‘high risk’ (dysplasia, malignancy) categories. We further trained the composite AI-model’s performance on a larger cohort of 105 resections WSIs and then validated our findings on a cohort of 150 biopsies WSIs against the classifications of two independently blinded pathologists. We evaluated the area under the receiver-operator characteristic curve (AUC) and other performance metrics. The AI model achieved an AUC of 0.917 in the validation cohort, with excellent sensitivity (97.4%) in detection of high risk features of dysplasia and malignancy. We demonstrate an unique composite AI-model incorporating both a glandular segmentation deep learning model and a classical machine learning classifier, with excellent sensitivity in picking up high risk colorectal features. As such, AI plays a role as a potential screening tool in assisting busy pathologists by outlining the dysplastic and malignant glands.

IntroductionCirculating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together.MethodsConcurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients.ResultsHigher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression.ConclusionsA standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.