Wood Hudson Cancer Research Laboratory

Abstract Transforming growth factor β (TGFβ) signals through the TGFβ Receptor 1 (TGFβR1) and is implicated in many aspects of malignancy. TGFβR1 gene is frequently mutated in ovarian carcinomas (OvCa) (Chen, T., et al, Cancer Research 61, 4679-4682, 2001). Susceptibility to numerous cancers is linked to two germline variants of TGFβR1, a G to A single nucleotide polymorphism in intron 7 (Int 7G24A) and a nine base pair deletion in exon 1 (TGFβR1*6A) although the mechanism(s) for this association is still unclear. Since the canonical pathway for TGFβR1 signaling is via phosphorylation of SMAD, the goal was to determine the association of either or both variants with development of subtypes of OvCa and to measure phosphorylation of pSMAD in the epithelium and stroma of OvCas from women with either or both Int 7G24A and TGFβR1*6A. FFPE tissues from 122 women without a history of cancer, and 59 women with OvCa were obtained from St. Elizabeth Healthcare (N. KY) and from 63 women with OvCa through the Cooperative Human Tissue Network (Birmingham Ala). OvCa patients and non-cancer controls were age matched. St. Elizabeth Healthcare IRB gave permission for this study. Tumors were diagnosed in H&E stained histologic sections by a Board Certified Pathologist (LED) and were classified as either Low Malignant Potential (LMP), Type 1 (clear cell, mucinous and low grade serous and endometrioid) or Type 2 (high grade serous, high grade endometrioid, or carcinosarcoma). Variants were identified in extracted DNA from FFPE tissues using PCR, capillary electrophoresis (CE), RFLP, and SSCP. Histologic sections were stained by IHC using the DAKO LSAB2 kit (Agilent) and anti pSMAD 2 (Millipore AB3849-1). Stain was evaluated as cytoplasmic and nuclear Histoscores (stain Area X Intensity) by two observers (LED, JHC). Data were evaluated by “Gene Code” with Gene 1 wild type for both variants, Gene 2 homozygous or heterozygous for Int 7G24A, Gene 3 homozygous or heterozygous for TGFβR1*6A, and Gene 4 having both variants. Usual chi-tests were used to determine significance of contingency tables, ANOVA with multiple comparison adjusted were used for markers. KM plots and proportional hazards were used in survival tests. The frequency in Gene 1, 2, 3, and 4 was significantly different in controls vs. OvCa patients (p = 0.0010); 57.7% of OvCa patients had a TGFβR1 variant vs. 36.6% of controls. Frequency of TGFβR1 variants in 62 patients with Type 2 OvCa was 62.9%. Patient survival differed significantly between patients with LMP, Type 1, and Type 2 OvCa (p <0.0001). Differences in nuclear expression of pSMAD in both tumor epithelial and stromal cells between OvCa types were highly significant (p < 0.0001, p< 0.0092). pSMAD expression decreased significantly in the nuclei of both epithelial and stromal cells in Type 2 tumors. These data indicate that germline variations in the TGFβR1 gene are associated with high grade ovarian cancers and altered SMAD phosphorylation. Citation Format: Julia H. Carter, James P. Schaeper, Taiping Chen, Diane W. Fritz, Leah Focke, Adrian Guy, James A. Deddens, Larry E. Douglass. Alterations in TGFβ signaling in ovarian cancer patients with TGFβ receptor 1 variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2267.

Abstract Stomach cancer is the third most common cause of cancer mortality worldwide. Most are adenocarcinomas. Adenocarcinomas of the stomach fall into two obvious groups based on site of origin; those arising in the area of the gastroesophageal junction (GEJ) and those arising from a more distal location, which we term gastric in this study. Adenocarcinomas of the stomach fall into four obvious groups based on a combination of genomic, molecular biologic, clinical and histopathologic characteristics (Nagara A, Kikuchi O, Bass AJ, Cancer Discovery 2019; DOI: 10.1158/2159-8290.CD-19-0487). We are reporting herein a study of 99 patients with adenocarcinoma of the stomach. This study evaluates the location of the tumor, the histopathologic features of the tumor, the immunohistochemically determined nuclear and cytoplasmic expression of P21 activated kinase (Pak 1), Transforming Growth Factor α (TGFα), and Ki67, a marker of cell proliferation. This study correlates these findings with patient survival. The study reveals: 1. significantly lower survival in patients with diffuse type adenocarcinoma compared to intestinal type, in both GEJ and gastric locations. 2. both GEJ and gastric adenocarcinomas had a poor prognosis. 3. increased expression of both TGFα and Pak1 in GEJ adenocarcinomas. 4. Ki67 expression is significantly less in diffuse type than the intestinal type (p=0.01 for GEJ and 0.03 for gastric). 5. cytoplasmic Pak1 and cytoplasmic TGFα were highly positively correlated (p=0.0001). 6. Ki67 expression was significantly inversely correlated with both nuclear Pak1 (p=0.04) and nuclear TGFα (p=0.02). 7. significantly increased expression of nuclear Pak1 in gastric diffuse type compared with gastric intestinal (p=0.0002) and GEJ intestinal (p=0.0001). These data suggest that Pak1 and TGFα might serve as diagnostic biomarkers of subtypes of adenocarcinomas of the stomach and gastroesophogeal junction. Citation Format: Larry E. Douglass, Michael Parritt, Adam Neltner, Mariah Dooley, Michelle Robillard, Molly Frydl, Leah Focke, Kyle Damen, James A. Deddens, Julia H. Carter. Pak1, TGFα, Ki 67 and survival in human gastric and gastroesophageal junction adenocarcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 747.

Abstract Cholangiocarcinoma (CCA) originates from biliary tract epithelium and can be classified anatomically into intrahepatic or extrahepatic CCA. Although a relatively rare disease in the United States and Europe, the incidence of intrahepatic CCA is on the rise with unknown causes. CCA incidence is higher in Asia and the etiology is associated with infections such as liver fluke and hepatitis B/C. Prognosis at diagnosis is poor with median survival time of < 1 year, and only 10-20% of patients are eligible for tumor resection at diagnosis. This study examined the prevalence of MET over-expression, activating single point mutations of KRAS and IDH1/2, and ROS1 gene fusions in ∼100 intrahepatic and extrahepatic CCA FFPE tumor tissues obtained from non-Asian (n=40) and Asian (n=60) patients. Immunohistochemistry performed with an anti-MET-specific antibody (A2) demonstrated that MET is expressed in the majority of all intrahepatic CCA samples analyzed, with ∼50% of samples reported with membranocytoplasmic scores of 2+ (moderate intensity) or 3+ (strong intensity) on a 0-3+ scale. Interestingly, most non-malignant bile ducts and much vascular endothelia also stained lightly positive for MET. To determine mutation frequencies of KRAS and IDH1/2, competitive allele-specific Taqman® PCR (castPCR) was performed on DNA extracted from FFPE CCA tissue (limit of mutation detection 0.1%). Fourteen KRAS activating mutations (G12S, G12R, G12A, G13C, G13S, G13R, G12D, G13D, G12V, G12C, Q61R, Q61L, Q61H (c.183A>C and c.183A>T for Q61H), 5 IDH1 mutations (R132G, R132S, R132H, R132C, R132L), and 5 IDH2 mutations (R172G, R172M, R172K, R172W, R172S) were analyzed. Overall, 25% of analyzed samples were positive for KRAS mutation, and G12D was the predominant mutation (∼60%). One-third of Asian samples were positive for KRAS mutation, whereas less than one-fifth of non-Asian samples contained KRAS mutations. For IDH1, the frequency of mutation was less than 10% overall, and the majority of patients with IDH1 mutations were non-Asian. The R132C mutation was the predominant IDH1 mutation, and all tissues that were positive for IDH1 mutations were of intrahepatic origin. Interestingly, 2 out of the 7 samples positive for IDH1 mutations (R132C) were also positive for G12D KRAS mutation. There is no trend of MET expression correlating with either KRAS or IDH1 mutations. IDH2 analyses by castPCR and FISH studies examining ROS1 gene fusion are ongoing. Based on these data, inhibitors of receptor tyrosine kinases and their signaling pathways such as MET and ROS1 may merit clinical evaluation in CCA patients. LY2801653, a MET inhibitor which also has inhibitory activity against ROS1 and MKNK1/2 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). Citation Format: Megan N. Thobe, S. Betty Yan, Kelly M. Credille, Aejaz Nasir, Jessica A. Roseberry Baker, Mary Lajiness, Nathan A. Brooks, Darryl W. Ballard, Donna M. Farley, Victoria L. Peek, Suzane L. Um, G. Jason Jin, Raymond Gilmour, Christoph Reinhard, Jeremy R. Graff, Andrew E. Schade, Aaron M. Gruver, Bruce Colligan, Larry Douglass, Julia Carter, Richard A. Walgren. Prevalence of MET expression, activating mutations of KRAS and IDH1/2, and ROS1 fusions in cholangiocarcinoma patient tumor samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2013-2339

Abstract The carcinogenic potential of chlorinated organics is of direct importance in human risk assessment. Most drinking water chlorinated organics are disinfection by products (DBPs) of water chlorination and many test positive in rodent bioassays. Trihalomethanes (THMs) are the most prevalent DBPs generated in chlorinated drinking water and chloroform (TCM) is the THM in highest concentration in finished drinking water. Human exposure to TCM occurs through ingestion of drinking water, inhalation and dermal exposure. TCM is carcinogenic to the liver and kidney of rodents including male and female B6C3F1 mice and male F-344/N and Osborne-Mendel rats. The carcinogenic mechanism of TCM in the rodent is not completely understood. Four mechanisms have been proposed: 1) mutagenicity; 2) reparative hyperplasia; 3) altered gene expression; and 4) secondary genotoxicity. Here, histopathology, immunohistochemistry, and quantitative image analysis were used to examine reparative cell proliferation and altered gene expression as potential mechanisms of TCM-induced hepatocellular carcinogenesis in male F-344/N rats. Animals were exposed to concentrations of 803 + 5 or 1592 + 21 mg/L in the drinking water for 78 or 100 weeks. Distilled water was the vehicle control. The high TCM dose increased the prevalence (% of animals with a lesion) of hepatocellular neoplasia (carcinoma and adenoma) 17.5% vs. 5.1% (p<0.05) and marginally enhanced the prevalence of combined preneoplastic and neoplastic tumors (20.5% vs 7.7%; 0.05<p<0.1). Cell proliferation (proliferating cell nuclear antigen), apoptosis (terminal transferase assay) and expression of tumor associated markers, transforming growth factor alpha (TGF-α) and the p21 Ras oncogene were evaluated in normal liver, altered foci (AF), large foci of cellular alteration (LFCA) and adenomas (AD). PCNA, TGF-α, and p21 Ras increased significantly during TCM-induced neoplastic progression (p = 0.0021, 0.0105, and 0.0167, respectively). A significant change in apoptosis was not found during neoplastic progression, however, a progressive imbalance between cell proliferation and death occurred (p = 0.0022). The phenotype of hepatocellular lesions in rats receiving TCM in their drinking water differed from that of the “spontaneous” lesions arising in rats given distilled water, suggesting a different mechanistic origin. Taken together the data are consistent with a carcinogenic mechanism for TCM that includes adaptation to a hepatotoxic chemical and selection of resistant cells. (This abstract does not reflect the views of the EPA). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3451.

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHP's stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.

Huggins and Morii (J. Exp. Med., 114: 741, 1961) reported that massive adrenal necrosis occurs in 79 and 100% of female Sprague-Dawley rats receiving 20 and 30 mg, respectively, of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Here, adrenal necrosis and regeneration were studied in 158 rats for up to 21 days post-DMBA by radioautography of the adrenals of animals given 50 microCi [3H]thymidine 30 min before sacrifice. Adrenal cell proliferation was markedly inhibited 21 days post-DMBA. Regenerated adrenals were more susceptible to this adrenocorticolytic effect. To investigate if alterations in adrenal function modify tumorigenesis, animals underwent adrenalectomies (ADX), hypophysectomies, ovariectomies, and pituitary transplants alone or in combination 6 days after receiving DMBA (20 mg/100 g intragastrically) at 50 days of age. To prevent adrenal necrosis, 24 animals were pretreated with metyrapone. Methylprednisolone acetate, 1 mg i.m., was given to 40 animals every 5 days beginning 6 days post-DMBA. There were 50 non-DMBA-treated intact and surgical controls. DMBA was necessary but not sufficient to induce mammary tumors. No tumors developed in controls or in 46 animals hypophysectomized 6 days after DMBA. Metyrapone reduced tumor incidence and yield. ADX after DMBA treatment increased the tumorigenic response and eliminated resistance to tumorigenesis in older rats. Only three tumors developed in DMBA-treated rats receiving methylprednisolone acetate. Mammary tumorigenesis was increased by pituitary transplant 6 days after DMBA to intact and ADX animals. Ovariectomy 6 days after DMBA was as effective as methylprednisolone acetate in preventing tumorigenesis; ADX did not overcome either inhibition. We conclude that adrenal hormones inhibit proliferation of initiated mammary cells.