Ren-Chin Wu

Identification of molecular pathway aberrations in uterine serous carcinoma by genome-wide analyses.

BACKGROUND Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer. METHODS Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing. RESULTS We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification. CONCLUSION Molecular genetic aberrations involving the p53, cyclin E-FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.

The emerging roles of ARID1A in tumor suppression

ARID1A has emerged as a tumor suppressor gene, which is mutated in a broad spectrum of cancers, especially in those arising from ectopic or eutopic endometrium. As a subunit of SWI/SNF chromatin remodeler, ARID1A facilitates target-specific binding of SWI/SNF complexes to chromatin, thereby altering the accessibility of chromatin to a variety of nuclear factors. In human cancer, ARID1A possesses not only features of a gatekeeper, regulating cell cycle progression, but also features of a caretaker, preventing genomic instability. An increasing body of evidence suggests crosstalk between ARID1A and PI3K/Akt pathways, and between ARID1A and p53. In this review, we discuss the spectrum of ARID1A alterations in cancers, tumor suppression mechanisms of ARID1A, oncogenic pathways cooperating with ARID1A, and clinical implications of ARID1A mutation.

Cancer-Associated Mutations in Endometriosis without Cancer

Background Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive‐age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis. Methods We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer‐driver targeted sequencing (3 patients). Mutations were validated with the use of digital genomic methods in microdissected epithelium and stroma. Epithelial and stromal components of lesions from an additional 12 patients were analyzed by means of a droplet digital polymerase‐chain‐reaction (PCR) assay for recurrent activating KRAS mutations. Results Exome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients harbored known cancer driver mutations in ARID1A, PIK3CA, KRAS, or PPP2R1A, which were validated by Safe‐Sequencing System or immunohistochemical analysis. The likelihood of driver genes being affected at this rate in the absence of selection was estimated at P=0.001 (binomial test). Targeted sequencing and a droplet digital PCR assay identified KRAS mutations in 2 of 3 patients and 3 of 12 patients, respectively, with mutations in the epithelium but not the stroma. One patient harbored two different KRAS mutations, c.35G→T and c.35G→C, and another carried identical KRAS c.35G→A mutations in three distinct lesions. Conclusions We found that lesions in deep infiltrating endometriosis, which are associated with virtually no risk of malignant transformation, harbor somatic cancer driver mutations. Ten of 39 deep infiltrating lesions (26%) carried driver mutations; all the tested somatic mutations appeared to be confined to the epithelial compartment of endometriotic lesions.

Roles of Deletion of Arid1a, a Tumor Suppressor, in Mouse Ovarian Tumorigenesis

The chromatin remodeling gene, ARID1A, has been implied as a tumor suppressor, and its somatic inactivating mutations occur in a wide variety of human cancers, most frequently in ovarian and uterine endometrioid and ovarian clear cell carcinomas. Tumors with ARID1A mutations also frequently harbor PTEN or PIK3CA mutations, suggesting their collaboration in tumorigenesis. Here, we used a conditional knockout mouse model in which Arid1a and Pten were deleted either individually or in combination in the mouse ovarian surface epithelium. After 6 months, 59.1% of mice with Arid1a and Pten double knockout developed ovarian endometrioid or undifferentiated carcinoma, whereas the remaining mice showed hyperplasia of ovarian surface epithelium. In contrast, 52 mice with homozygous or heterozygous deletion in either Arid1a or Pten did not develop ovarian lesions. These results demonstrate that inactivation of Arid1a alone is insufficient for tumor initiation but it requires additional genetic alteration(s) such as Pten deletion to drive tumorigenesis.

ARID1A loss correlates with mismatch repair deficiency and intact p53 expression in high-grade endometrial carcinomas

BAF250a (ARID1A) loss is a frequent event in high-grade endometrial cancers. It has been proposed that ARID1A is a driver gene, with ARID1A mutations occurring secondary to deregulated mismatch repair mechanism in gastric cancers, representing an alternative oncogenic pathway to p53 alteration. The prognostic significance of ARID1A loss is controversial. In this study, we investigated the frequency of BAF250a immunohistochemical loss in a cohort of high-grade endometrial cancers (n=190) and correlated it with mismatch repair (hMLH1, hMSH2, hMSH6, and hPMS2) and p53 protein expression. The 190 cases consisted of 82 high-grade endometrioid, 88 serous, 10 clear cell, and 10 mixed (carcinosarcomas and mixed histology). There was BAF250a loss in 55/190 (29%) cancers, most commonly in high-grade endometrioid carcinomas (46 vs 9% in serous carcinomas, P<0.0001). Loss of any mismatch repair proteins was observed in 63/190 (33%) cancers, most commonly in high-grade endometrioid carcinomas (57 vs 10% in serous carcinomas, P<0.0001). Aberrant p53 expression was found in 86/190 (45%) cancers, more commonly in serous carcinomas (77 vs 18% in high-grade endometrioid carcinomas, P<0.0001). BAF250a loss was associated with mismatch repair loss (P<0.0001) and normal p53 expression (P<0.0001). These associations were maintained in the subset analysis within the high-grade endometrioid (P=0.026 and P=0.0083, respectively) and serous carcinoma cases (P=0.0031 and P<0.0001, respectively). Survival analysis revealed a superior progression-free survival (P=0.017) for patients with BAF250a loss within the entire cohort but not within the high-grade endometrioid and serous subtypes. Additionally, data from The Cancer Genome Atlas were extracted to correlate mutations in ARID1A, TP53, and MMR genes; we found that ARID1A mutations were negatively associated with TP53 mutations but were unrelated to mismatch repair gene mutations. In conclusion, BAF250a loss is more common in high-grade endometrioid carcinomas than in other high-grade endometrial cancers and is associated with mismatch repair deficiency and normal p53 expression.

Frequent somatic mutations of the telomerase reverse transcriptase promoter in ovarian clear cell carcinoma but not in other major types of gynaecological malignancy.

Up‐regulated expression of telomerase reverse transcriptase (TERT) and subsequent maintenance of telomere length are essential in tumour development. Recent studies have implicated somatic gain‐of‐function mutations at the TERT promoter as one of the mechanisms that promote transcriptional activation of TERT; however, it remains unclear whether this genetic abnormality is prevalent in gynaecological neoplasms. We performed mutational analysis in a total of 525 gynaecological cancers, and correlated TERT promoter mutations with clinicopathological features. With the exception of ovarian clear cell carcinomas, in which mutations were found in 37 (15.9%) of 233 cases, the majority of gynaecological malignancies were wild‐type. TERT promoter mutation does not appear to be an early event during oncogenesis, as it was not detected in the contiguous endometriosis associated with ovarian clear cell carcinoma. Ovarian clear cell carcinoma cell lines with TERT promoter mutations exhibited higher TERT mRNA expression than those with wild‐type sequences (p = 0.0238). TERT promoter mutation tended to be mutually exclusive with loss of ARID1A protein expression (p = 4.4 × 10–9) and PIK3CA mutation (p = 0.0019) in ovarian clear cell carcinomas. No associations with disease‐specific survival were observed for ovarian clear cell carcinoma. The above results, in conjunction with our previous report showing longer telomeres in ovarian clear cell carcinomas relative to other types of ovarian cancer, suggests that aberrations in telomere biology may play an important role in the pathogenesis of ovarian clear cell carcinoma. Copyright

Combined hepatocellular cholangiocarcinoma : prognostic factors investigated by computed tomography/magnetic resonance imaging

This study was designed to assess the clinical usefulness of imaging for predicting the prognosis of patients with combined hepatocellular cholangiocarcinoma (cHCC‐CC). Between 1999 and 2004, 30 patients with histopathologically proven cHCC‐CC underwent computed tomography (CT) or magnetic resonance imaging (MRI). The imaging data and survival were analysed. Univariate log‐rank analysis of imaging findings revealed that tumour necrosis, bile duct invasion, major vascular branch invasion, multiplicity, bilobar distribution, regional lymph node involvement, regional organ invasion, distant metastasis and ascites had adverse influences on overall survival. Multivariate Cox proportional hazard analysis demonstrated that major vascular branch invasion, regional organ invasion, nodal and distant metastases were independent prognostic factors that adversely affected overall survival rates. Overall cumulative survival rates at 1, 3 and 5 years were 53%, 26% and 12%, respectively. Analysing the survival of our patients by using clinical stages of the newly updated American Joint Committee on Cancer (AJCC) classification for liver neoplasm based on the imaging findings, we found significant differences between stages I/II and III (p < 0.001) and between stages III and IV (p = 0.040). We conclude CT or MRI can be used to identify the prognostic factors and to estimate the outcomes of patients with cHCC‐CC.

Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Clinicopathologic features and outcomes following surgery for pancreatic adenosquamous carcinoma

BackgroundPancreatic adenosquamous carcinoma (ASC) is a rare pancreatic malignancy subtype. We investigated the clinicopathological features and outcome of pancreatic ASC patients after surgery.MethodsThe medical records of 12 patients with pancreatic ASC undergoing surgical treatment (1993 to 2006) were retrospectively reviewed. Survival data of patients with stage IIB pancreatic adenocarcinoma and ASC undergoing surgical resection were compared.ResultsSymptoms included abdominal pain (91.7%), body weight loss (83.3%), anorexia (41.7%) and jaundice (25.0%). Tumors were located at pancreatic head in 5 (41.7%) patients, tail in 5 (41.7%), and body in 4 (33.3%). Median tumor size was 6.3 cm. Surgical resection was performed on 7 patients, bypass surgery on 3, and exploratory laparotomy with biopsy on 2. No surgical mortality was identified. Seven (58.3%) and 11 (91.7%) patients died within 6 and 12 months of operation, respectively. Median survival of 12 patients was 4.41 months. Seven patients receiving surgical resection had median survival of 6.51 months. Patients with stage IIB pancreatic ASC had shorter median survival compared to those with adenocarcinoma.ConclusionAggressive surgical management does not appear effective in treating pancreatic ASC patients. Strategies involving non-surgical treatment such as chemotherapy, radiotherapy or target agents should be tested.

Clonality analysis of combined Brenner and mucinous tumours of the ovary reveals their monoclonal origin

The derivation of ovarian intestinal‐type mucinous tumours is not well established. Some are derived from teratomas but the origin of the majority is not clear. It has been recently proposed that the non‐germ cell group may be derived from Brenner tumours, as the association of a mucinous tumour with a Brenner tumour is frequently observed. In order to explore the histogenesis of these neoplasms, we undertook a clonality analysis of the two components of ten combined Brenner and mucinous tumours using a human androgen receptor gene (HUMARA) assay. All eight informative cases of ten showed a concordant X‐chromosome inactivation pattern between the two tumour components, indicative of a shared clonal origin (p = 0.0039). Microsatellite genotyping in five of the combined tumours displayed an identical heterozygous pattern with paired Fallopian tube tissue, indicative of a somatic cell origin. In addition, paired box protein 8, a highly sensitive Müllerian epithelial marker, was not detected by immunohistochemistry in either tumour component in any of the ten tumours, suggesting that this subset of mucinous tumours does not originate from Müllerian‐derived epithelium. In conclusion, this study demonstrates that in combined mucinous and Brenner tumours, there is a shared clonal relationship between the two different tumour components and suggests that some pure mucinous tumours may develop from a Brenner tumour in which the Brenner tumour component becomes compressed and obliterated by an expanding mucinous neoplasm. Copyright