Background Despite major advancements, effective treatment for patients with SMARCB1-deficient cancers has remained elusive. Here, we report the first case of a SMARCB1-deficient undifferentiated carcinoma in the rectum expressing high PD-L1 and responding to a PD-1 inhibitor, as well as with low tumor mutation burden (TMB), proficient mismatch repair (MMR) and BRAF V600E mutation. Case presentation A 35-year-old man visited our hospital complaining of increased defecation frequency, bloody stools and weight loss of 3 kg for one month. Colonoscopy revealed an ulcerated and irregular mass approximately 8–12 cm from the anus. Surgical resection was performed. Histopathological findings revealed that the tumor cells had poor connectivity with each other; each cell had eosinophilic cytoplasm and a polymorphic nucleus. Brisk mitotic activity and necrosis were frequently observed in the tumor cells. Immunohistochemical examination showed that the tumor cells were negative for SMARCB1. The tumor proportion score (TPS) of PD-L1 (22C3) expression was 95%, and the combined positive score (CPS) was 100; the tumor was mismatch repair (MMR) proficient. Next-generation sequencing showed a low tumor mutation burden (TMB), as well as the BRAF V600E mutation. The final diagnosis was SMARCB1-deficient undifferentiated carcinoma. Chemotherapy was useless in this case. His tumor recurred during chemotherapy, and he then received targeted therapy with tirelizumab, an inhibitor of PD-1. At present, his general condition is good. A recent computed tomography (CT) scan showed that the tumor had disappeared, indicating that the immunotherapy was effective. Astonishingly, his most recent follow-up was in August, and his condition continued to improve with the tumor has disappeared. Conclusion SMARCB1‑deficient undifferentiated carcinoma in the rectum is extremely rare, and it has aggressive histological malignancy and poor progression. The observed response to PD-1 inhibitors suggests a role for prospective use of SMARCB1 alterations as a predictive marker for immune checkpoint blockade.

Summary of 29 MET FISH positive NSCLC and their co-mutation status

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer‐associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF‐secreted insulin‐like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF‐derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD‐1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K–AKT pathway, RNA‐seq analysis revealed that the YAP1‐target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2–IGF1R. By single‐cell RNA sequencing (scRNA‐seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2–IGF1R–YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

Esophageal basaloid squamous cell carcinoma (bSCC) is a subtype of squamous cell carcinoma (SCC) with a different behavior and poor prognosis. Exploring bSCC's molecular characteristics and treatment strategies are of great clinical significance. We performed multi-omics analysis of paired bSCC and common SCC (cSCC) using whole exome sequencing and a NanoString nCounter gene expression panel. Immunohistochemistry was used for verification of candidate biomarkers. Different treatment response was analyzed on both patients receiving neoadjuvant treatment and late-stage patients. The common genetically-clonal origin of bSCC and cSCC was confirmed. No significant differences between their genetic alterations or mutation spectra were observed. Mutation signature 15 (associated with defective DNA damage repair) was less prominent, and tumor mutational burden (TMB) was lower in bSCC. bSCC with an RNA expression pattern resembling cSCC had a better survival than other bSCCs. Moreover, bSCC showed significant upregulation of expression of genes associated with angiogenesis response, basement membranes, and epithelial-mesenchymal transition, and downregulation of KRT14 (squamous differentiation) and CCL21 (associated with immune response). Immunohistochemistry for SFRP1 was shown to be highly sensitive and specific for bSCC diagnosis (p < 0.001). In addition, bSCC receiving neoadjuvant immuno-chemotherapy had a worse pathological response than bSCC receiving neoadjuvant chemotherapy (but without statistical significance), even in bSCC positive for PD-L1. Our results demonstrated the molecular characteristics of esophageal bSCC as a subtype with a distinct RNA expression pattern and immune characteristics, but no specific genetic mutations. We provided a useful biomarker, SFRP1, for diagnosis. After outcome analysis for six bSCCs with neoadjuvant immunotherapy treatment and four late-stage bSCCs with immunotherapy, we found that immunotherapy may not be an effective treatment option for most bSCCs. This may also provide a clue for the same subtypes of lung and head and neck cancer. Our study highlighted the heterogeneity among bSCC patients, and might explain the conflicting results of bSCC outcomes in existing studies. © 2022 The Pathological Society of Great Britain and Ireland.

e16062 Background: Esophageal squamous cell carcinoma (SCC) is one of the most common cancers and the leading cause of cancer-related death in China, and esophageal basaloid squamous cell carcinoma (BSC) is a rare variant (2.2%-11.3%) of SCC with highly aggressive behavior due to its propensity for vessel invasion and distant metastasis. Most reports stated that BSCs had poorer prognosis than conventional esophageal SCC. However, because of the histological morphologic similarity and lacking of diagnostic approaches, esophageal BSC can easily result in misdiagnosis as SCC. Methods: To deepen our understanding of esophageal BSC and seek for diagnostic biomarkers, we analyze the difference of mRNA expression between esophageal BSC and conventional SCC. Nanostring nCounter PanCancer Progression gene expression panel was used on different tumor components from 36 patients diagnosed with esophageal BSC component (Ba) coexisting with conventional SCC component (Co), who underwent esophagectomy at our Hospital (Cancer Hospital, Chinese Academy of Medical Sciences, China). Immunohistochemistry (IHC) was used for verification of candidate biomarkers sFRP1 concluded from mRNA expression analysis. The expression of sFRP1 was evaluated by H-score = % cells staining (0-100%) * intensity (1 to 3). Results: Among 770 RNAs analyzed in the study, 22.9% (176/770) showed downregulation (FDR-adjusted p < 0.05) in Ba than Co, while 5.3% (41/770) showed upregulation. A set of significantly upregulated RNAs (FDR-adjusted p < 0.01, log2(fold change) > 2) were observed in Ba, including sFRP1, FGFR1, ITGA9, VIT, VWA2 and PROM1, which were associated with angiogenesis response, basement membrane, and epithelial-mesenchymal transition (EMT). Besides, significant downregulation (FDR-adjusted p < 0.01, log2(fold change) < -2) of KRT14 was observed in Ba, which was well known as a biomarker for squamous differentiation. Clustering analysis with different pathway scores according to mRNA expression results of Ba yielded two major clusters: Cluster1 with higher pathway scores with resembled to Co, and Cluster2 with lower pathway scores. Survival analysis revealed that patients in Cluster1 seemed to have a longer RFS and a longer OS than those in Cluster2 (Figure 4C&D), although with no statistically significance (RFS: p = 0.322, median RFS = 23.7 vs 10.8 months; OS: p = 0.217, median OS = 51.6 vs 26.5 months), which was probably due to the limited number of patients evolved (10 patients in Cluster1 and 26 in Cluster2). The most upregulated mRNA, sFRP1, was validated by IHC. Receiver operating characteristic (ROC) curve revealed that sFRP1 IHC could be a remarkable diagnosis biomarker to distinguish BSC from conventional SCC (area under ROC = 0.935, p < 0.001 95%CI = 0.871-1.000). Conclusions: Our results revealed BSC as a subtype with distinct RNA expression pattern compared with conventional SCC, and provided a remarkable biomarker, sFRP1, for diagnosis.

Serrated polyps are a clinically and molecularly heterogeneous group of lesions that can contribute to the development of colorectal cancers (CRCs). However, the molecular mechanism underlying the development of serrated lesions is still not well understood. Here, we combined multiple approaches to analyze the genetic alterations in 86 colorectal adenomas (including 35 sessile serrated lesions, 15 traditional adenomas, and 36 conventional adenomatous polyps). We also investigated the in vitro and in vivo oncogenic properties of a novel variant of the NCOA4-RET fusion gene. Molecular profiling revealed that sessile serrated lesions and traditional serrated adenomas have distinct clinicopathological and molecular features. Moreover, we identified receptor tyrosine kinase translocations exclusively in sessile serrated lesions (17%), and the observation was validated in a separate cohort of 34 sessile serrated lesions (15%). The kinase fusions as well as the BRAF and KRAS mutations were mutually exclusive to each other. Ectopic expression of a novel variant of the NCOA4-RET fusion gene promoted cell proliferation in vitro and in vivo, and the proliferation was significantly suppressed by RET kinase inhibitors. All of these underscored the importance of mitogen-activated protein kinase (MAPK) pathway activation in the serrated pathway of colorectal tumorigenesis. In addition, we demonstrated that the kinase fusion may occur early in the precursor lesion and subsequent loss of TP53 may drives the transformation to carcinoma during serrated tumorigenesis. In conclusion, we identified kinase fusions as a significant alternative driver of the serrated pathway in colorectal cancer development, and detecting their presence may serve as a biomarker for the diagnosis of sessile serrated lesions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

AbstractEsophageal squamous cell carcinoma (ESCC) is more prevalent than esophageal adenocarcinoma in Asia, especially in China, where more than half of ESCC cases occur worldwide. Many studies have reported that the automatic detection of lymph node metastasis using semantic segmentation shows good performance in breast cancer and other adenocarcinomas. However, the detection of squamous cell carcinoma metastasis in hematoxylin‐eosin (H&E)‐stained slides has never been reported. We collected a training set of 110 esophageal lymph node slides with metastasis and 132 lymph node slides without metastasis. An iPad‐based annotation system was used to draw the contours of the cancer metastasis region. A DeepLab v3 model was trained to achieve the best fit with the training data. The learned model could estimate the probability of metastasis. To evaluate the effectiveness of the detection model of learned metastasis, we used another large cohort of clinical H&E‐stained esophageal lymph node slides containing 795 esophageal lymph nodes from 154 esophageal cancer patients. The basic authenticity label for each slide was confirmed by experienced pathologists. After filtering isolated noise in the prediction, we obtained an accuracy of 94%. Furthermore, we applied the learned model to throat and lung lymph node squamous cell carcinoma metastases and achieved the following promising results: an accuracy of 96.7% in throat cancer and an accuracy of 90% in lung cancer. In this work, we organized an annotated dataset of H&E‐stained esophageal lymph node and trained a deep neural network to detect lymph node metastasis in H&E‐stained slides of squamous cell carcinoma automatically. Moreover, it is possible to use this model to detect lymph nodes metastasis in squamous cell carcinoma from other organs. This study directly demonstrates the potential for determining the localization of squamous cell carcinoma metastases in lymph node and assisting in pathological diagnosis.

BACKGROUND:Granulin epithelin precursor (GEP) is reported to function as a growth factor stimulating proliferation and migration, and conferring chemoresistance in many cancer types. However, the expression and functional roles of GEP in colorectal cancer (CRC) remain elusive. The aim of this study was thus to investigate the clinical significance of GEP in CRC and reveal the molecular mechanism of GEP in CRC initiation and progression.METHODS:The mRNA expression of GEP in CRC cell lines were detected by qRT-PCR. The GEP protein expression was validated by immunohistochemistry in tissue microarray (TMA) including 190 CRC patient samples. The clinicopathological correlation analysis were achieved by GEP expression on TMA. Functional roles of GEP were determined by MTT proliferation, monolayer colony formation, cell invasion and migration and in vivo studies through siRNA/shRNA mediated knockdown assays. The cancer signaling pathway identification was acquired by flow cytometry, western blot and luciferase activity assays.RESULTS:The mRNA expression of GEP in CRC was significantly higher than it in normal colon tissues. GEP protein was predominantly localized in the cytoplasm and most of the CRC cases demonstrated abundant GEP protein compared with non-tumorous tissues. GEP overexpression was associated with non-rectal location, advanced AJCC stage, regional lymph node and distant metastasis. By Kaplan-Meier survival analysis, GEP abundance served as a prognostic marker for worse survival in CRC patients. GEP knockdown exhibited anti-cancer effect such as inhibiting cell proliferation, monolayer colony formation, cell invasion and migration in DLD-1 and HCT 116 cells and decelerating xenograft formation in nude mice. siGEP also induced G1 cell cycle arrest and apoptosis. Luciferase activity assays further demonstrated GEP activation was involved in MAPK/ERK signaling pathway.CONCLUSION:In summary, we compressively delineate the oncogenic role of GEP in colorectal tumorigenesis by activating MAPK/ERK signaling pathway. GEP might serve as a useful prognostic biomarker and therapeutic target for CRC.

Fibroblast growth factors (FGFs) and their receptors are significant components during fundamental cellular processes. FGF18 plays a distinctive role in modulating the activity of both tumor cells and tumor microenvironment. This study aims to comprehensively investigate the expression and functional role of FGF18 in gastric cancer (GC) and elucidate its regulatory mechanisms. The upregulation of FGF18 was detected in seven out of eleven (63.6%) GC cell lines. In primary GC samples, FGF18 was overexpressed in genomically stable and chromosomal instability subtypes of GC and its overexpression was associated with poor survival. Knocking down FGF18 inhibited tumor formation abilities, induced G1 phase cell cycle arrest and enhanced anti-cancer drug sensitivity. Expression microarray profiling revealed that silencing of FGF18 activated ATM pathway but quenched TGF-β pathway. The key factors that altered in the related signaling were validated by western blot and immunofluorescence. Meanwhile, treating GC cells with human recombinant FGF18 or FGF18-conditioned medium accelerated tumor growth through activation of ERK-MAPK signaling. FGF18 was further confirmed to be a direct target of tumor suppressor, miR-590-5p. Their expressions showed a negative correlation in primary GC samples and more importantly, re-overexpression of FGF18 partly abolished the tumor-suppressive effect of miR-590-5p. Our study not only identified that FGF18 serves as a novel prognostic marker and a therapeutic target in GC but also enriched the knowledge of FGF-FGFR signaling during gastric tumorigenesis.

Colorectal cancer (CRC) is a heterogeneous disease with complex mechanisms of pathogenesis. Classification systems have been proposed based on molecular features of tumors in clinical practice. Thus, more molecular markers associated with development and progression of CRC might serve as useful tools for early diagnosis even for providing more accurate molecular classification. Frequent gain of chromosome 8q was detected in CRC by array‐CGH and overexpression of exosome component 4 (EXOSC4) in this region was revealed by expression microarray analysis. Through qRT‐PCR and immunohistochemistry (IHC) analysis, EXOSC4 showed increased expression in CRC cell lines and clinical specimens. Higher expression of EXOSC4 was more often detected in left side, and correlated with BRAF wild type, MSI‐low or MSS, CIMP‐low, and MLH1‐no‐silence CRC patients. Functionally, EXOSC4 overexpression increased early tumorigenic capacity by promoting cell proliferation and monolayer colony formation, enhancing cell invasion and migration study and accelerating xenograft formation in nude mice. While EXOSC4 knockdown exhibited anti‐oncogenic role such as inhibiting cell proliferation and invasion. EXOSC4 inhibition also resulted in G1 phase cell cycle arrest. For the downstream signaling analysis, EXOSC4 was found to be involved in multiple signaling pathways such as cell cycle, p53 pathway and Wnt pathway. In summary, our findings demonstrated the oncogenic role of EXOSC4 in development and progression of CRC. Deep understanding of EXOSC4 as a potential diagnostic molecular biomarker will provide clinical translational potential for intervention therapy.

BACKGROUND:Patients with colorectal cancer (CRC) have a high incidence of regional and distant metastases. Although metastasis is the main cause of CRC-related death, its molecular mechanisms remain largely unknown.METHODS:Using array-CGH and expression microarray analyses, changes in DNA copy number and mRNA expression levels were investigated in human CRC samples. The mRNA expression level of RASAL2 was validated by qRT-PCR, and the protein expression was evaluated by western blot as well as immunohistochemistry in CRC cell lines and primary tumors. The functional role of RASAL2 in CRC was determined by MTT proliferation assay, monolayer and soft agar colony formation assays, cell cycle analysis, cell invasion and migration and in vivo study through siRNA/shRNA mediated knockdown and overexpression assays. Identification of RASAL2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays.RESULTS:Integrated genomic analysis identified copy number gains and upregulation of RASAL2 in metastatic CRC. RASAL2 encodes a RAS-GTPase-activating protein (RAS-GAP) and showed increased expression in CRC cell lines and clinical specimens. Higher RASAL2 expression was significantly correlated with lymph node involvement and distant metastasis in CRC patients. Moreover, we found that RASAL2 serves as an independent prognostic marker of overall survival in CRC patients. In vitro and in vivo functional studies revealed that RASAL2 promoted tumor progression in both KRAS/NRAS mutant and wild-type CRC cells. Knockdown of RASAL2 promoted YAP1 phosphorylation, cytoplasm retention and ubiquitination, therefore activating the hippo pathway through the LATS2/YAP1 axis.CONCLUSIONS:Our findings demonstrated the roles of RASAL2 in CRC tumorigenesis as well as metastasis, and RASAL2 exerts its oncogenic property through LATS2/YAP1 axis of hippo signaling pathway in CRC.

miR-375 is a tumor-suppressive microRNA (miRNA) in gastric cancer (GC). However, its molecular mechanism remains unclear. The aim of this study is to comprehensively investigate how miR-375 is involved in Hippo pathway by targeting multiple oncogenes. miR-375 expression in gastric cancer cell lines and primary GC was investigated by qRT-PCR. The regulation of YAP1, TEAD4, and CTGF expression by miR-375 was evaluated by qRT-PCR, western blot, and luciferase reporter assays, respectively. The functional roles of the related genes were examined by siRNA-mediated knockdown or ectopic expression assays. The clinical significance and expression correlation analysis of miR-375, YAP1, and CTGF were performed in primary GCs. TCGA cohort was also used to analyze the expression correlation of YAP1, TEAD4, CTGF, and miR-375 in primary GCs. miR-375 was down-regulated in GC due to promoter methylation and histone deacetylation. miR-375 downregulation was associated with unfavorable outcome and lymph node metastasis. Ectopic expression of miR-375 inhibited tumor growth in vitro and in vivo. Three components of Hippo pathway, YAP1, TEAD4 and CTGF, were revealed to be direct targets of miR-375. The expression of three genes showed a negative correlation with miR-375 expression and YAP1 re-expression partly abolished the tumor-suppressive effect of miR-375. Furthermore, CTGF was confirmed to be the key downstream of Hippo-YAP1 cascade and its knockdown phenocopied siYAP1 or miR-375 overexpression. YAP1 nuclear accumulation was positively correlated with CTGF cytoplasmic expression in primary GC tissues. Verteporfin exerted an anti-oncogenic effect in GC cell lines by quenching CTGF expression through YAP1 degradation. In short, miR-375 was involved in the Hippo pathway by targeting YAP1-TEAD4-CTGF axis and enriched our knowledge on the miRNA dysregulation in gastric tumorigenesis.

Abstract Background: The diversity of the genomic landscape in colorectal cancer (CRC) is under active investigation. Concordance in molecular events between primary CRC and metastatic tumors is a subject of debate. Understanding the genetic background of metastatic CRC facilitates personalized therapies that target specific molecular events associated with the tumor. Methods: We performed whole genome sequencing in matched primary, metastasis and normal tissues from 3 patients with RAS/NRAS/BRAF wild types and microsatellite stable CRC. We implemented an analysis pipeline that integrated the genetic lesions to characterize somatic single nucleotide variations (SNVs), short insertions and deletions (indels), structural variations including fusions and copy number alterations. Results: Both primary and metastatic tumors showed a predominant mutational signature of C: G →T: A transition. A total of 282 non-silent mutations were identified in 3 pairs of CRC (median 63 per tumor, range 42 to 98). There were 148 (52.5%) variants shared in matched primary and metastatic tumors, 63 (22.3%) were found unique to primary and 71 (25.2%) were unique to the metastases. Only common well known genes (APC and TP53) and TTN mutations were found to be recurrent in the 3 patients. A total of 609 (range 33-171 per tumor) structural variants were discovered with 44.1% concordance between primary and metastasis pairs. We also identified 6 novel candidate fusions shared in primary and metastases and 2 private to the primary tumors. Consistent with the result of previous studies, copy loss at chromosome 17p and 18, and gain at chromosome 5, 8 and 13 were the common events both in the primary and metastatic tumor. Discussion and conclusions: Our data demonstrated a considerable difference in mutational landscape between primary CRCs and their matching metastatic tumors. The result suggested that the metastasis might originate from a distinct subclone from a heterogeneous primary tumor, or additional gain or loss of function via mutation to permit tumor invasion and spreading is necessary to facilitate metastasis. Each of the two models is complicated by the possibility of tumor heterogeneity within the primary tumor. In conclusion, comprehensive analysis and assessment for the genomic events is a powerful approach to delve deeper into the internal relations and contradictions between primary and paired metastases, helping to identify novel genomic events associated with metastasis of CRC. Citation Format: Yi Pan, Joanna Tong, Johnny Kwan, Kwok Wai Lo, Anthony Chan, Ka Fai To. Genomic events associated with metastasis of colorectal cancer by whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2017-4382

Abstract Metastatic colorectal cancer (CRC) is a fatal disease with a poor prognosis. It is crucial to understanding the underlying pathogenesis to battle this dismal condition. We previously identified that AK4, a gene on chromosome 1p31.3 encoding a member of adenylate kinase family enzymes, was progressively up-regulated from normal colon, primary CRC to metastasis by mRNA gene expression microarray of paired CRC samples. This study was aimed to validate the overexpression of AK4 and explore in-vitro effects in CRC. We firstly examined the mRNA expression level of AK4 in a panel of CRC cell lines. AK4 mRNA expression was higher in CRC cell lines (n=8) than normal colon cell line, and significantly higher in advanced stage cell lines (n=4, Dukes’ C and D) than early stage ones (n=4, Dukes’ A and B) (P=0.03). We further evaluated expression level of AK4 in two different cohorts of clinical samples. The first cohort consisted of 18 paired primary CRCs and its corresponding normal colonic mucosa, and 27 metastatic CRC (including 15 cases with paired primary CRC and normal colonic mucosa). AK4 mRNA level was significantly higher in metastatic CRC than normal colon (P<0.001), and metastatic CRC than primary CRC (P<0.001). Among 15 cases with paired primary and metastatic tumors, 14 (93.3%) demonstrated higher AK4 in metastatic tumors than their primary counterparts(P<0.05). The second cohort composed of 190 consecutive primary CRCs. Upregulation of AK4 protein expression evaluated by immunohistochemistry was found in 68.4% of CRC, and associated with advanced AJCC stage (P<0.001), poorer overall survival (P=0.002) and worse disease-free survival (P<0.001). To evaluate functional properties of AK4 in CRC, we knocked down AK4 on a CRC cell lines with high endogenous AK4 expression (DLD1, HCT116 and SW620) by siRNA. knockdown of AK4 significantly reduced anchorage-dependent colony forming ability (P<0.05) and eliminated migratory (P<0.05) and invasive (P<0.05) abilities. In conclusion, AK4 is a potential oncogene upregulated in metastatic CRC and exhibiting in-vitro oncogenic properties. AK4 may play an important role in tumor progression and metastasis. Citation Format: Yau Hei Yu, Joanna Hung Man Tong, Yi Pan, Raymond Wai Ming Lung, Ka Fai To, Anthony Wing Hung Chan. AK4 promotes colorectal cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4858. doi:10.1158/1538-7445.AM2017-4858

Abstract Background: Metastatic colorectal cancer (mCRC) is a heterogeneous disease with a poor prognosis. By using high resolution array comparative genomic hybridization (aCGH) and mRNA gene expression microarray of mCRC samples, we found up-regulation of RASAL2, a RAS GAP gene that is located on chromosome 1q, in colorectal tumors. Oncogenic RAS represents one of the most common molecular changes in human colorectal adenocarcinoma. However, the role of RASAL2 in colorectal adenocarcinoma metastasis and the related mechanisms are still unclear. We analyzed its aberrant expression, clinical significance and biological effects in colorectal cancer. Methods: Expression of RASAL2 was examined by QRT-PCR, western blot and immunohistochemistry in CRC cell lines, primary and metastatic CRC and the corresponding normal colonic mucosa. The biological effects of RASAL2 on proliferation, apoptosis, cell cycle, and cell motility and invasiveness were evaluated by siRNA knock down and RASAL2 reconstitution in CRC cell lines. Results: Up-regulation of RASAL2 mRNA was observed in CRC cell lines (n = 9) than normal colon mucosal tissues. Interestingly, significantly higher RASAL2 mRNA was found in cell lines derived from advanced stage tumors (n = 4, Dukes’ C and D) than those from early stage tumors (n = 5, Dukes’ A and B) (P = 0.0317). Up-regulation of RASAL2 mRNA was also found in primary CRCs (n = 77) compared with normal colon mucosa (n = 18, P<0.0001). In 15 cases that paired primary and metastatic tumors were available, 12 (80%) demonstrated higher RASAL2 in metastatic tumors than their primary counterparts. RASAL2 protein was detected in 37% (81 of 219) of CRC by immunohistochemistry. Whereas in the paired normal colonic mucosa, the positive rate is 14% (20 of 142, P<0.0001). We knocked down RASAL2 by siRNA in 2 CRC cell lines (DLD1 and HCT116) with high endogenous RASALs level. Successful knockdown was demonstrated by western blot analysis. SiRASAL2 significantly inhibited cell proliferation (P<0.05), reduced colony formation (P<0.05) and repressed cell invasion and migration ability (P<0.05) in both cell lines. Flow cytometry analysis revealed G1 arrest in cells treated with siRASAL2. Ectopic expression of RASAL2 was performed in 2 CRC cell lines with low endogenous RASAL2 (SW480 and LoVo). Over expression of RASAL2 did not change the cell proliferation and anchorage-dependent growth of these cell lines but effectively enhanced cell invasiveness by transwell invasion assay. Conclusions: Up-regulation of RASAL2 was frequently found in CRC cell lines and primary and metastatic tumors. Our experimental findings suggested that RASAL2 might play an oncogenetic role in CRC and promotes tumor invasion and metastasis. A better understanding of the molecular mechanism of RASAL2 in promoting CRC cell metastasis may lead to a more effective management of mCRC patients. Citation Format: Yi PAN, Joanna Hung Man TONG, Raymond Wai Ming LUNG, Samantha Wei Man LUN, Kwok Wai LO, Anthony Wing Hung CHAN, Ka Fai TO. RASAL2 promotes tumor progression and metastasis in colorectal cancer. [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 1529.

Abstract Purpose: Activation of MET oncogene as the result of amplification or activation mutation represents an emerging molecular target for cancer treatment. We comprehensively studied MET alterations and the clinicopathologic correlations in a large cohort of treatment-naïve non–small cell lung carcinoma (NSCLC). Experimental Design: Six hundred eighty-seven NSCLCs were tested for MET exon 14 splicing site mutation (METΔ14), DNA copy number alterations, and protein expression by Sanger sequencing, FISH, and IHC, respectively. Results: METΔ14 mutation was detected in 2.62% (18/687) of NSCLC. The mutation rates were 2.6% in adenocarcinoma, 4.8% in adenosquamous carcinoma, and 31.8% in sarcomatoid carcinoma. METΔ14 mutation was not detected in squamous cell carcinoma, large cell carcinoma, and lymphoepithelioma-like carcinoma but significantly enriched in sarcomatoid carcinoma (P < 0.001). METΔ14 occurred mutually exclusively with known driver mutations but tended to coexist with MET amplification or copy number gain (P < 0.001). Low-level MET amplification and polysomy might occur in the background of EGFR or KRAS mutation whereas high-level amplification (MET/CEP7 ratio ≥5) was mutually exclusive to the major driver genes except METΔ14. Oncogenic METΔ14 mutation and/or high-level amplification occurred in a total of 3.3% (23/687) of NSCLC and associated with higher MET protein expression. METΔ14 occurred more frequently in older patients whereas amplification was more common in ever-smokers. Both METΔ14 and high-level amplification were independent prognostic factors that predicted poorer survival by multivariable analysis. Conclusions: The high incidence of METΔ14 mutation in sarcomatoid carcinoma suggested that MET inhibition might benefit this specific subgroup of patients. Clin Cancer Res; 22(12); 3048–56. ©2016 AACR. See related commentary by Drilon, p. 2832

BACKGROUND:IDH1/2 mutation, 1p/19q-codeletion and MGMT hypermethylation are well known molecular markers for gliomas. ATRX and p53 alterations are two lineage-specific genetic aberrations in diffuse astrocytic tumors. The aim of the present study is to clarify the significance of ATRX loss and its correlation with p53 overexpression, IDH1/2 mutations, 1p/19q-codeletion and MGMT hypermethylation in supertentorial astrocytoma, and to determine the prognostic value of these factors in Chinese patients.METHODS AND RESULTS:A total of 135 adult supertentorial astrocytomas were evaluated. ATRX loss was detected by immunohistochemistry (IHC) and was shown to be much less frequent in pGBs (3.5%) than in grade II, III astrocytomas and IV sGBs (31%). Direct sequencing and/or IHC analysis of the IDH1R132H gene mutation and p53 accumulation demonstrated correlation with age. Strong correlations were found between ATRX loss and IDH1R132H mutation, p53 overexpression as well as MGMT hypermethylation. 1p/19q-codeletion detected by fluorescence in situ hybridization (FISH) showed mutually exclusive with ATRX loss and p53 accumulation. In addition, patients with p53 overexpression combined with ATRX alterations demonstrated substantially longer survival than patients with wild-type ATRX.CONCLUSIONS:There may be interactions among these distinct molecules in astrocytoma development. ATRX loss may predict better clinical outcome in astrocytoma patients with p53 overexpression as compared to patients with wild-type ATRX. Tumors with astrocytoma phenotype accompanied by 1p/19q-codeletion and IDH1R132H mutation are mutually exclusive with ATRX and p53 alterations. Routine IHC can be used for evaluation of ATRX loss, p53 protein accumulation and IDH1R132H mutation, which may allow a means of classification of astrocytoma outcome.

Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is an uncommon variant of HCC with only 22 cases reported in the literature. To better determine the incidence, clinicopathologic features, prognostic significance, and molecular pathogenesis of LEL-HCC, we presented the largest series of LEL-HCC from a 9-year retrospective cohort of patients with HCC undergoing surgical resection. LEL-HCC was identified in 20 patients (4.9%). Compared with patients having HCC without significant tumor-infiltrating lymphocyte (TIL), patients with LEL-HCC had a relatively lower frequency of male sex (P=0.022), tended to present at early-stage disease (80.0% vs. 56.3% as AJCC stage I, P=0.037; 100% vs. 77.3% as BCLC stage 0/A, P=0.010), and all harbored a solitary tumor only (P=0.006). There was no significant difference in the age at presentation, underlying chronic liver disease, cirrhotic background, serum α-fetoprotein level, tumor size, histologic grade, and frequencies of vascular invasion. Most of the TILs in LEL-HCC were cytotoxic T lymphocytes. None of the LEL-HCCs was associated with Epstein-Barr virus. LEL-HCC was associated with better overall (5-y survival: 94.1% vs. 63.9%; P=0.007) and progression-free (5-y survival: 87.8% vs. 46.6%; P=0.002) survivals compared with HCC without significant TIL. The multivariate analysis revealed that LEL-HCC was an independent prognostic factor for overall and progression-free survivals. The adjusted hazard ratio of cancer death and tumor progression for LEL-HCC was 0.12 (P=0.037) and 0.14 (P=0.002), respectively. LEL-HCC did not differ in frequencies of microsatellite instability, BRAF mutation, and DNA hypermethylation. In brief, LEL-HCC is a distinct uncommon variant of HCC characterized by dense cytotoxic T-cell infiltration and favorable prognosis.