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Purpose: Using reverse phase protein array, we measured protein expression associated with response to primary chemotherapy in patients with advanced-stage, high-grade serous ovarian cancer.Experimental Design: Tumor samples were obtained from 45 patients with advanced high-grade serous cancers from the Gynecology Tumor Bank at the British Columbia Cancer Agency. Treatment consisted of platinum-based chemotherapy following debulking surgery. Protein lysates were prepared from fresh frozen tumor samples, and 80 validated proteins from signaling pathways implicated in ovarian carcinogenesis were measured by reverse phase protein array. Normalization of Ca-125 by the 3rd cycle of chemotherapy was chosen as the primary outcome measure of chemotherapy response. Logistic regression was used for multivariate analysis to identify protein predictors of Ca-125 normalization and Cox regression to test for the association between protein expression and progression-free survival. A significance level of P ≤ 0.05 was used.Results: The mean age at diagnosis was 56.8 years. epidermal growth factor receptor, YKL-40, and several transforming growth factor β (TGF-β) pathway proteins [c-jun–NH2–kinase (JNK), JNK phosphorylated at residues 183 and 185, plasminogen activator inhibitor 1, Smad3, TAZ] showed significant associations with Ca-125 normalization on univariate testing. On multivariate analysis, epidermal growth factor receptor (P < 0.02), JNK (P < 0.01), and Smad3 (P < 0.04) were significantly associated with normalization of Ca-125. Contingency table analysis of pathway-classified proteins revealed that the selection of TGF-β pathway proteins was unlikely because of false discovery (P < 0.007; Bonferroni adjusted).Conclusion: TGF-β pathway signaling likely plays an important role as a marker or mediator of chemoresistance in advanced serous ovarian cancer. On this basis, future studies to develop and validate a useful predictor of treatment failure are warranted. Clin Cancer Res; 16(10); 2852–60. ©2010 AACR.

Supplementary Table S1 from Functional Proteomic Analysis of Advanced Serous Ovarian Cancer Using Reverse Phase Protein Array: TGF-β Pathway Signaling Indicates Response to Primary Chemotherapy

Supplementary Figure 1 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Table 1 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Methods from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Figure Legends 1-2 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Table 2 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Figure 2 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Introduction Oncogenic KRAS mutations are absent in 10% of patients with pancreatic ductal adenocarcinoma (PDAC) and may represent a subgroup of PDAC with therapy options beyond standard-of-care chemotherapy. While distinct gene fusions have been implicated in KRAS wildtype (wt) PDAC, other traits unique to KRAS wt PDAC remain unexplored. Methods Patients with treatment-naive metastatic PDAC (n=63) received matched tumor/normal whole-genome and RNA sequencing and clinical follow-up as part of the PanGen trial (NCT02869802). Clinical data and mutation, gene fusion and copy alteration frequency was compared between KRAS wt (n=9) and mutant (n=54) groups. Metastatic colorectal adenocarcinoma and cholangiocarcinoma samples from the POG trial (NCT02155621) were incorporated for sequencing-based comparison. Results KRAS wt tumors showed lower frequency of TP53 SNV/indels (p=0.01) and distinct amplifications affecting chr1q genes NR5A2 (p=9.0e-4), MPC2 (p=0.002) and MCL1 (p=0.02) and chr8q gene SQLE (p=0.02). Oncogenic fusions involving NRG1, FGFR2, NTRK2 or BRAF were unique to KRAS wt tumors (67% of samples; p=1e-6). KRAS wt tumors showed expression patterns indicative of cholangiocytes and, when clustered alongside other tumor types, formed a distinct cluster with cholangiocarcinoma samples. Conclusion These data provide the first identification of distinct chr1q/chr8q amplification events unique to KRAS wt PDAC while highlighting novel and striking molecular similarities to cholangiocarcinoma. Taken together, results of the PanGen trial generate new hypotheses relevant to therapeutic targeting of KRAS wt PDAC and rationale towards incorporating KRAS mutation status as part of routine clinical testing in PDAC. Citation Format: James Topham, Erica Tsang, Joanna Karasinska, Andrew Metcalfe, Hassan Ali, Steve Kalloger, Veronika Csizmok, Laura Williamson, Emma Titmuss, Hui-Li Wong, Richard Moore, Andrew Mungall, Jonathan Loree, Oliver Bathe, Patricia Tang, Rachel Goodwin, Janessa Laskin, Marco Marra, Steven Jones, David Schaeffer, Daniel Renouf. Analysis of KRAS wildtype pancreatic ductal adenocarcinoma reveals mutation and expression-based similarities to cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB129.

587 Background: Oncogenic driver mutations in KRAS represent a hallmark genomic event in approximately 90% of pancreatic adenocarcinoma (PDAC). For the remaining 10% of patients with KRAS wildtype (wt) PDAC, distinct driver mutations have been described, but their transcriptional landscape has not been reported. Here, we leverage sequencing data from the PanGen trial to provide a comprehensive characterization of advanced KRASwt PDAC. Methods: 63 patients with advanced PDAC received whole genome and transcriptome sequencing prior to treatment for metastatic disease as part of the PanGen trial (NCT02869802). Clinical features, somatic mutation data and gene expression patterns were compared between KRASwt and mutant groups. PDAC samples were contrasted with 77 other metastatic carcinoma (colorectal and cholangiocarcinoma) samples from the Personalized OncoGenomics trial (NCT0215562). KRAS wt-associated genes were further investigated using 3 additional PDAC cohorts (COMPASS NCT02750657, TCGA, and ICGC). Results: 9 of 63 (14%) samples were KRASwt, with an earlier median age at diagnosis (51.4 vs. 60.9 years; p=0.03). Clinical features, including diabetes, family history of malignancy, and location of primary tumor, were comparable. CA 19-9 at baseline was lower in the KRASwt group, with median 58 vs. 4900 U/mL in the KRAS-mutant group ( p=0.03). Patients with KRASwt PDAC showed increased overall survival in univariable ( p=0.0024) and multivariable ( p=0.0089) analyses. 6 of 9 (67%) KRASwt tumors had fusions involving NRG1 (n = 3), FGFR2 (n = 1), BRAF (n = 1) or NTRK2 (n = 1), while known actionable fusions were not observed in KRAS mutant patients. KRASwt tumors showed increased expression of genes associated with cholangiocytes and grouped with cholangiocarcinoma samples in unsupervised clustering analysis. Validation using three independent PDAC cohorts revealed a core set of 70 KRAS wt-associated genes that converge on keratinization, ion transport, and hormone metabolism pathways. Conclusions: Patients with KRASwt PDAC show potentially targetable molecular traits with actionable fusions. We also highlight novel mutation and expression-based similarities between KRASwt PDAC and cholangiocarcinoma samples. Recurrent dysregulation of genes involved in cellular structure and metastasis provide impetus for further investigation into the developmental trajectory and potential therapeutic vulnerabilities of KRASwt PDAC.

Purpose: As we have published expertise in breast cancer xenograft models and clonal dynamics, our aim was to explore rates of engraftment based on type of tissue for attempted xenografting (primary vs relapsed/metastatic disease) and clinical breast biomarker subtype. Methods: Tissue from patients (pts) enrolled in a locally advanced/metastatic study and a breast tumour tissue repository (ie. resectable primaries) between Sept. 2008 and July 2015 underwent xenografting using NodScid/IL2rgKO (NSG) mice. Xenografts were passaged when tumour volume reached 1 cm3. Mice with no engraftment after 12 months (mos) were sacrificed. Pt charts were reviewed to determine biomarker status (hormone receptor [HR], HER2), date and type of tissue collection for xenografting. Prediction of successful engraftment based on tissue type and biomarker status was performed using nominal logistic regression. Results: A total of 70 tissue samples with known engraftment status were included in the analysis: 51 from primary breast tumour, 10 from relapsed disease (dz) with ≤ 1 line of therapy in the advanced setting and 9 from relapsed dz with > 1 line of therapy in the advanced setting. Tumours from pts treated with > 1 line of therapy were more likely to engraft compared to primary or recurrent dz with ≤ 1 line of therapy (89%, 35%, and 40% respectively; p=.008). HR- primary tumours were more likely to engraft compared to HR+ primary tumours: 71% of HR-/HER2- (triple negative) and 67% of HR-/HER2+ tumours versus 4% of HR+/HER2- and 38% of HR+/HER2+ tumours; p Conclusion: This preliminary study highlights potential differences in successful xenoengraftment based on biomarker status at diagnosis and type of tissue, primary vs relapsed tumour, the latter suggesting that the underlying biology of primary or first relapsed recurrent disease is distinct from more refractory disease, and warrants further exploration. This work is ongoing. (Funded by CBCRA, BCCF) Engraftment of primary tumour vs relapsed disease Primary tumour (N=52) N, (%)Recurrent disease and ≤ 1 line of Rx in advanced setting (N=10) N, (%)Recurrent disease and > 1 line of Rx in advanced setting (N=9) N, (%)Engraftment Yes18 (35)4 (40)8 (89)HR-/HER2-10 (55)1 (25)2 (25)HR-/HER2+4 (22)1 (25)1 (13)HR+/HER2+3 (17)00HR+/HER2-1 (6)2 (50)5 (62)Engraftment No33 (65)6 (60)1 (11)HR-/HER2-4 (12)00HR-/HER2+2 (6)1 (17)0HR+/HER2+5 (15)00HR+/HER2-22 (67)5 (83)1 (100) Citation Format: den Brok W-l, Chia S, Kalloger S, Bates C, Aparicio S, Mar C, Gelmon K, Eirew P. Rates of successful engraftment in breast cancer xenograft models based on tissue type: Primary vs relapsed disease [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-10.

Purpose: We have expertise in breast cancer xenograft models and have previously published data on clonal dynamics. Our aim was to explore clinical characteristics of those patients (pts) whose breast cancer tumours engrafted versus those that did not. Methods: Tissue from pts enrolled in a locally advanced/metastatic (MBC) study and a breast tumour tissue repository between Sept. 2008 and July 2014 underwent xenografting using NodScid/IL2rgKO (NSG) mice. Xenografts were passaged when tumour volume reached 1 cm3. Mice were sacrificed if no engraftment by 12 months (mos). Pt charts were reviewed to determine biomarker status (hormone receptor [HR], HER2), grade, LVI, time free from disease/progression, and pathologic complete response (pCR) for pts receiving neoadjuvant therapy. Results: A total of 64 pts had known xenograft status: 32 engrafters, 32 non-engrafters. Biomarker status did not predict likelihood of engraftment (p = .0695) and is shown in Table 1 along with site/timing of biopsy for tissue engraftment within biomarker groups. When HER2+ cases are excluded from analysis, the HR-/HER2- phenotype yields a 72% probability of engraftment compared to 39% for the HR+/HER2- group (p = .0418). For engrafters, 22/32 (68%) of pts had or went on to have relapsed or de novo MBC. For non-engrafters, 7/32 (22%) had or went on to have relapsed disease (p = .0004). Grade and LVI did not predict engraftment (p = .1806 and p = .8657 respectively). No grade 1 tumours engrafted (n = 3). There were 25 pts who received neoadjuvant chemotherapy: 14 engrafters, 11 non-engrafters. None of the engrafters achieved a pCR; 3 non-engrafters achieved a pCR. Median time to engraftment was 5 mos for pts with relapsed/advanced disease vs 9.8 mos for pts who did not relapse however, treatment was variable. Conclusion: This preliminary study highlights potential differences in clinical characteristics of engrafters vs non-engrafters in breast cancer xenograft models and warrants further exploration. (Funded by CBCRA, BCCF) TABLE 1.Biomarker status, tissue site/type in attempted xenografts.Engrafter (N = 32) N, (%)Non-engrafter (N = 32) N, (%)HR+/HER2-14 (44)22 (69)Tissue from:Primary tumour419Recurrence23Advanced dz on therapy80HR-/HER2-13 (40)5 (16)Tissue from:Primary tumour114Recurrence21Advanced dz on therapy00HER2+/HR+0 (0)2 (9)Tissue from:Primary tumour02Recurrence00Advanced dz on therapy00HER2+/HR-5 (16)3 (9)Tissue from:Primary tumour42Recurrence01Advanced dz on therapy10 Citation Format: Wendie D. den Brok, Stephen Chia, Cherie Bates, Steve Kalloger, Samuel Aparicio, Mar Mar, Karen Gelmon, Peter Eirew. Clinical characteristics of breast cancer xenograft models. [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 638.

Clear cell ovarian cancer histotypes exhibit metabolic features associated with resistance to hypoxia and glucose deprivation-induced cell death. This metabolic characteristic suggests that clear cell ovarian cancers activate survival mechanisms not typical of other epithelial ovarian cancers. Here we demonstrate that microtubule-associated protein 1 light chain 3A (LC3A), a marker of autophagy, is related to hypoxia and poor prognosis in clear cell ovarian cancer. In 485 ovarian tumours, we found that LC3A was significantly associated with poor progression-free (p = 0.0232), disease-specific (p = 0.0011) and overall patient survival (p = 0.0013) in clear cell ovarian cancer patients, but not in other subtypes examined. LC3A was an independent prognostic marker of reduced disease-specific [hazard ratio (HR): 2.55 (95% CI 1.21-5.37); p = 0.014] and overall survival [HR: 1.95 (95% CI 1.00-3.77); p = 0.049] in patients with clear cell ovarian carcinoma. We also found a strong link between autophagy and hypoxia as LC3A staining revealed a significant positive association with the hypoxia-related proteins carbonic anhydrase-IX and HIF-1α. The functional link between hypoxia and autophagy was demonstrated using clear cell and high-grade serous cell lines that were subjected to hypoxia or hypoxia + glucose deprivation. Clear cell carcinoma lines displayed greater autophagy induction and were subsequently more sensitive to inhibition of autophagy under hypoxia compared to the high-grade serous lines. Together, our findings indicate that hypoxia-induced autophagy may be crucial to the clinical pathology of clear cell ovarian cancer and is a potential explanation for histological subtype differences in patient disease progression and outcomes.

Serous carcinoma is the prototype of type 2 uterine carcinoma. In many cases, establishing a diagnosis is straightforward, but problems can arise in that papillary variants of endometrioid carcinoma may be mistaken for serous carcinoma, and glandular variants of serous carcinoma may be misdiagnosed as endometrioid carcinoma. Markers such as p53, oestrogen receptor and p16 may be of use in problematic cases, but there is overlap and these may not therefore be of value in an individual case. It has been shown recently that high-mobility group AT-hook 2 (HMGA2) is expressed by most ovarian serous carcinomas, and our aim was to ascertain whether it is also expressed in uterine serous carcinoma and of value in its distinction from endometrioid carcinoma.Whole tissue sections of uterine serous (n = 33) and endometrioid (n = 38) carcinoma were immunostained using HMGA2 antibody. As many of the diagnostic problems relate to the distinction between serous carcinoma and grade 3 endometrioid carcinoma, tissue microarrays (TMAs) containing uterine serous (n = 71) and uterine grade 3 endometrioid (n = 68) carcinomas were also stained. Staining was classified as negative (totally negative or occasional nuclei positive), 1+ (10% of nuclei positive), 2+ (10-49% of nuclei positive), 3+ (50-74% of nuclei positive), or 4+ (≥75% of nuclei positive). On the whole tissue sections, positive staining was also classified as weak, moderate, or strong, and an immunohistochemical composite score, taking into account both extent and intensity of staining, was calculated. On whole tissue sections, there was a statistically significant difference between HMGA2 staining in serous and endometrioid carcinomas with regard to both extent and composite score, with higher expression in serous carcinomas (P 0.0001). Thirty of 33 (91%) serous carcinomas were positive, usually with diffuse (3+ or 4+) staining. All five cases of serous endometrial intraepithelial carcinoma (EIC) (the postulated precursor of uterine serous carcinoma) were positive, as were 14 of 38 (37%) endometrioid carcinomas, usually with 1+ or 2+ staining. There was a statistically significant difference in HMGA2 staining in the TMAs between the serous and grade 3 endometrioid carcinomas, with higher expression in the former (P 0.0001).Immunoreactivity for HMGA2 is diffusely positive in whole tissue sections in most uterine serous carcinomas and negative in most endometrioid carcinomas, although, as with other markers, there is overlap in individual cases. In conjunction with other markers, HMGA2 may be of value in problematic uterine carcinomas where the differential diagnosis includes serous and endometrioid carcinoma. As HMGA2 is expressed in serous EIC, this suggests that it may be implicated in the early development of uterine serous carcinoma.