GATA6 Expression Distinguishes Classical and Basal-Like Subtypes in Advanced Pancreatic Cancer Grainne M

Published OnlineFirst March 10, 2020; DOI: 10.1158/1078-0432.CCR-19-3724

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

GATA6 Expression Distinguishes Classical and Basal-like Subtypes in Advanced Pancreatic Cancer Grainne M. O’Kane1,2, Barbara T. Grunwald€ 1,8, Gun-Ho Jang1, Mehdi Masoomian3, Sarah Picardo2, Robert C. Grant1,2, Robert E. Denroche1, Amy Zhang1, Yifan Wang4,5, Bernard Lam1, Paul M. Krzyzanowski1, Illinca M. Lungu1, John M.S. Bartlett1, Melanie Peralta2,8, Foram Vyas2,8, Rama Khokha3, James Biagi6, Dianne Chadwick7, Stephanie Ramotar2, Shawn Hutchinson2, Anna Dodd2, Julie M. Wilson1, Faiyaz Notta1,8, George Zogopoulos4,5, Steven Gallinger1,2,9,10, Jennifer J. Knox2, and Sandra E. Fischer3

ABSTRACT ◥ Purpose: To determine the impact of basal-like and classical ORR was 10% versus 33%, respectively (P ¼ 0.02). In patients with subtypes in advanced pancreatic ductal adenocarcinoma basal-like tumors treated with modified FOLFIRINOX (n ¼ 22), (PDAC) and to explore GATA6 expression as a surrogate the progression rate was 60% compared with 15% in classical biomarker. PDAC (P ¼ 0.0002). Median OS in the intention-to-treat popu- Experimental Design: Within the COMPASS trial, patients lation (n ¼ 195) was 9.3 months for classical versus 5.9 months for proceeding to chemotherapy for advanced PDAC undergo tumor basal-like PDAC (HR, 0.47; 95% confidence interval, 0.32–0.69; biopsy for RNA-sequencing (RNA-seq). Overall response rate P ¼ 0.0001). GATA6 expression by RNA-seq highly correlated (ORR) and overall survival (OS) were stratified by subtypes and with the classifier (P < 0.001)andISHpredictedthesubtypeswith according to chemotherapy received. Correlation of GATA6 with sensitivity of 89% and specificity of 83%. In a multivariate analysis, the subtypes using gene expression profiling, in situ hybridization GATA6 expression was prognostic (P ¼ 0.02). In exploratory (ISH) was explored. analyses, basal-like tumors, could be identified by keratin 5, were Results: Between December 2015 and May 2019, 195 patients more hypoxic and enriched for a T-cell–inflamed gene expression (95%) had enough tissue for RNA-seq; 39 (20%) were classified as signature. basal-like and 156 (80%) as classical. RECIST response data were Conclusions: The basal-like subtype is chemoresistant and can available for 157 patients; 29 basal-like and 128 classical where the be distinguished from classical PDAC by GATA6 expression.

Introduction paclitaxel (GnP) are the most commonly used regimens resulting in median survival less than 1 year (2, 3). While the need to discover novel By 2030, pancreatic ductal adenocarcinoma (PDAC) will become approaches is obvious, it is equally important to understand how to the second leading cause of cancer-related mortality in North Amer- select the aforementioned regimens for current patients. There are no ica (1). The majority of patients with PDAC present with advanced randomized data to show superiority of either combination and disease where the mainstay of treatment remains combination che- patient inclusion differences are evident in the two pivotal phase III motherapy. Modified FOLFIRINOX (mFFX) and gemcitabine-nab- trials (2, 3). The only molecular predictor of response is prior knowledge of a pathogenic germline variant in a homologous recombination fl 1Ontario Institute for Cancer Research, Toronto, Ontario, Canada. 2Wallace repair gene, which may in uence the regimen of choice (4). Other McCain Centre for Pancreatic Cancer, Princess Margaret Cancer Centre, Univer- currently targetable genomic variants are uncommon in PDAC. sity Health Network, Toronto, Ontario, Canada. 3Division of Pathology, Uni- Gene expression profiling, primarily in resected pancreatic tumors, versity Health Network, Toronto, Ontario, Canada. 4The Research Institute of the describes a number of subtypes with considerable overlap, yet pres- 5 McGill University Health Centre, Montreal, Quebec, Canada. The Goodman ently these do not inform clinical practice (5–7). Collisson and 6 Cancer Research Centre of McGill University, Montreal, Quebec, Canada. King- colleagues documented three subtypes (classical, quasimesenchymal, ston General Hospital, Kingston, Ontario, Canada. 7Department of Laboratory Medicine and Pathobiology, University of Toronto, University Health Network, and exocrine-like; ref. 6), Bailey and colleagues four subtypes (immu- fi Toronto, Ontario, Canada. 8Division of Research, Princess Margaret Cancer nogenic, progenitor, ADEX, and squamous; ref. 5), and Mof tt and Centre, University Health Network, Toronto, Ontario, Canada. 9Lunenfeld Tanen- colleagues two subtypes (classical and basal-like; ref. 7). The squamous baum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. (Bailey), quasimesenchymal (Collisson), and basal-like (Moffitt) 10 Hepatobiliary/Pancreatic Surgical Oncology Program, University Health Net- cohorts align well across the classifiers and all three are associated work, Toronto, Ontario, Canada. with a poor prognosis in these studies. Despite this, varying tumor Note: Supplementary data for this article are available at Clinical Cancer cellularity and heterogeneity in clustering methodologies leaves uncer- Research Online (http://clincancerres.aacrjournals.org/). tainty as to the most appropriate classifier and furthermore, the clinical Corresponding Authors: Grainne M. O’Kane, Princess Margaret Cancer Centre, application of these subtypes to advanced stage disease is unclear. 610 University Ave, Toronto, Ontario M5G 2M9, Canada. Phone: 416-946-2000, In an effort to reconcile and apply existing knowledge, we ext. 4853; Fax: 416-946-6546; E-mail: grainne.O’[email protected]; and Sandra E. established the COMPASS trial [Comprehensive Molecular Char- Fischer, Laboratory Medicine Program, University Health Network, 200 acterization of Advanced Pancreatic Ductal Adenocarcinomas Elizabeth Street, 11E207, Toronto, Ontario M5G 2C4, Canada. Phone: 416- 340-3723; Fax: 416-340-5517; E-mail: [email protected] (PDAC) for Better Treatment Selection: A Prospective Study, NCT NCT02750657]. Unique to this prospective study is the acquisition Clin Cancer Res 2020;XX:XX–XX of tissue prior to chemotherapy in the advanced setting, which then doi: 10.1158/1078-0432.CCR-19-3724 undergoes laser capture microdissection (LCM) to ensure high 2020 American Association for Cancer Research. tumor cellularity. The primary endpoint of feasibility in obtaining

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(hg38) and transcriptome (Ensembl v84) using STAR v.2.5.2a (11). Translational Relevance Duplicated reads were marked using Picard v. 1.121 (https://github. The transcriptomic basal-like subtype is highly chemoresistant com/broadinstitute/picard). Gene expression was calculated in frag- and patients have a shorter median overall survival compared with ments per kilobase of exon per million reads mapped using the classical pancreatic ductal adenocarcinoma (PDAC). In this study, cufflinks package v. 2.2.1 (12). A modified Moffitt classification survival was lowest in basal-like PDAC treated with modified (classical vs basal-like) was applied to each sample with sufficient FOLFIRINOX. GATA6 expression by both RNA-sequencing and RNA for analysis (Supplementary Fig. S1). Cut-off threshold levels for in situ hybridization is highly associated with the classifier where GATA6 expression were determined using the maximal x2 method on low or absent GATA6 is seen in the basal-like subtype. RECIST response and dichotomized GATA6 expression.

GATA6 RNA in situ hybridization Given our early results, the COMPASS trial was amended (February a high-quality genome report within 8 weeks in the first 50 patients 1, 2017) to include GATA6 staining using an RNAscope In Situ has been published (8). In this earlier analysis, we determined that a Hybridization (ISH) Assay (Advanced Cell Diagnostics Inc.). A semi- modified Moffitt RNA signature, optimized for use in advanced quantitative score was used by the study pathologist (S.E. Fischer; stage PDAC (classical vs basal-like; Supplementary Fig. S1) may Supplementary Fig. S2A) as reported previously (8). Scoring was have prognostic impact (8). Furthermore, we found that GATA6,a applied blinded to results of the modified Moffitt classifier. transcription factor required for normal pancreas development (9), which has been shown to align with the classical subtype, could IHC analysis of GATA6 and keratins represent a surrogate marker for classical PDAC (8). Here, we To provide more widely applicable diagnostic tests for PDAC evaluated the modified Moffitt basal-like and classical subtypes subtypes, we optimized a protocol for GATA6 IHC (Supplemen- together with GATA6 expression on outcomes in patients receiving tary Materials and Methods) using a polyclonal anti-GATA6 mFFX or GnP regimens on the expanded COMPASS trial. We antibody from R&D (catalog number AF1700), and secondary further explored specific clinical and pathologic characteristics of antibody from Vector (catalog number VECTABA5000). DABþ the subtypes and evaluated GATA6 as a surrogate biomarker and (3,3-diaminobenzidine tetrahydrochloride plus, DAKO, catalog num- clinical tool. Post hoc exploratory analyses were performed to seek ber K3468) was used as chromogen and nuclei were counterstained additional positive biomarkers for the basal-like subtype. with Mayer hematoxylin (Supplementary Fig. S2B). To assess the pattern of GATA6 staining across larger tumor regions, we used whole sections (n ¼ 30) from a previously described resection cohort with Materials and Methods matched RNA-seq data (10) together with biopsies (n ¼ 41) from the Patient population advanced cohort. The COMPASS trial is a prospective multi-institutional Canadian In an exploratory analysis, we sought additional clinical markers to cohort study. Patient eligibility for the study has been described aid subtype identification; cytokeratins associated with GATA6 expres- previously (8). Briefly, patients require a radiologic or histologic sion were identified from RNA-seq data and further explored by IHC diagnosis of locally advanced or metastatic PDAC, suitable for com- (Supplementary Materials and Methods). bination chemotherapy, and must consent to a fresh tumor biopsy prior to treatment start. Biopsies can be taken from the primary lesion Image analysis or any metastatic sites. Patients must not have had prior treatment for To control for potential bias of manual scorings of both ISH or IHC, advanced disease. Treatment decisions are at the discretion of their we performed image analysis on preannotated tumor sections using medical oncologist. Response to therapy is assessed using CT and image analysis software QuPath v0.1.3 (13). Detection parameters measured using RECIST 1.1. Demographics and treatment details, were established on unequivocal GATA6-high versus -low versus including subsequent treatments, are prospectively collected using an -absent tumors and confirmed by the study pathologist. Semiquan- electronic MEDIDATA database. This report includes all patients titative scores were also predicted from image analysis data using the enrolled from December 2015 until May 2019 and follow-up censored maximal x2 method. on August 30, 2019. Patients on this study were enrolled at the Princess Margaret Cancer Centre (Toronto, Ontario, Canada), McGill Univer- Statistical analysis sity Health Centre (MUHC, Montreal, Quebec, Canada), and Kingston Qualitative variables were compared by Fisher exact test, and General Hospital (Kingston, Ontario, Canada) and the study was quantitative variables by Wilcoxon rank-sum test for pairwise com- conducted in accordance with the Declaration of Helsinki. The parison and the Kruskal–Wallis test for multiple group comparison. COMPASS trial has been approved by participating site Institutional All patients receiving at least one cycle of chemotherapy were included Review Board (University Health Network, Toronto, Ontario, Canada; in the analysis of overall response rate (ORR). Survival curves were MUHC Centre for Applied Ethics, Montreal, Quebec, Canada; and plotted using the Kaplan–Meier method and HRs were calculated Queen's University Health Sciences and Affiliated Teaching Hospitals using Cox proportional hazard regressions with P values calculated Research Ethics Board, Kingston, Ontario, Canada); each patient using the Wald statistic. All tests were two-sided. Multiple tests provided written informed consent prior to study entry. P values were adjusted using Benjamini and Hochberg method (14) for independent tests or Benjamini and Yekutieli method (15) for RNA-sequencing and GATA6 expression dependent tests, respectively. Statistical significance was set at P ¼ 0.05. Frozen biospecimens underwent LCM for tumor enrichment. All analyses were conducted in R version 3.2 (16). Spearman corre- RNA-sequencing (RNA-seq) analysis was performed at the Ontario lation coefficients were ascertained for evaluating gene expression. Institute of Cancer Research (Ontario, Canada) as described previ- Sensitivity, specificity, and accuracy scores were computed to assess ously (10). Briefly, reads were aligned to the human reference genome prediction quality.

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Consort diagram

Enrolled May 30, 2019 (N = 250) No biopsy (n = 18) • Withdrawal of consent (n = 14) • Unsafe biopsy (n = 3) • Did not meet eligibility criteria (n = 1) Biopsies (N = 232) • Pancreas (n = 56) • Liver (n = 151) • Omentum/peritoneum (n = 21) • Other (n = 4)

Nonparcipants (n = 26) • Other histology/subtype (n = 10) • Insuﬃcient tumor (n = 7) • Withdrawal of consent (n = 1) • Declined chemotherapy (n = 7) • Did not meet eligibility (n = 1)

Eligible (N = 206) RNA-seq available: (N = 195)

Data cutoff August 30, 2019 • Alive on first line: n = 29 • Alive off study: n = 23 • Dead: n = 143

Figure 1. Consort diagram of patients enrolled and included on the COMPASS trial. A total of 250 patients were enrolled and 232 patients underwent biopsies. Biopsy sites included liver, pancreas, and peritoneum/omentum. A total of 195 patients were eligible with RNA-seq data representing the study population.

Results 19 received only one cycle of chemotherapy; five of these 23 had basal- Patient characteristics at baseline like PDAC. One patient receiving mFFX did not have measurable Between December 30, 2015 and May 30,2019, 250 patients were disease at enrollment. Accordingly, RECIST response data were enrolled and 206 were eligible (Fig. 1, consort). Of these, 195 available for 157 patients (81%) including 29 patients with basal- patients (95%) had enough tissue for RNA analysis and are included like tumors and 128 with classical tumors (Fig. 2A). The ORR in in this report. Table 1 shows baseline clinical and pathologic classical PDAC was 33% versus 10% in basal-like PDAC (P ¼ 0.02). characteristics of those patients. Using the modified Moffitt clas- The rates of progression by RECIST criteria at first CT image were sifier, 39 (20%) baseline tumor samples were basal-like and 156 much higher in basal-like versus classical PDAC (52% vs 16%; P < (80%) classical. Locally advanced disease at diagnosis was present in 0.0001). Figure 2A shows the percentage change in target lesions, 24 (12%) and these cases, in this small subset, were all identified as demonstrating chemoresistance of the basal-like subtype. In patients classical (P ¼ 0.005). Liver metastases were present in 97% of basal- treated with mFFX and evaluable for response (n ¼ 91), progression like tumors compared with 69% of classical tumors (which includes was evident in 60% of basal-like versus 15% of classical PDAC (P ¼ the locally advanced cases; P < 0.0001). Although basal-like tumors 0.0002; Fig. 2B). The ORR was 29.6% versus 10% in classical versus were more frequent in male patients (P ¼ 0.02) the overall sample basal-like PDAC (P ¼ 0.09). One patient in the latter group with a size was small. Other characteristics were similar between the partial response had a germline BRCA2 pathogenic variant and groups (Table 1). displayed genomic characteristics of homologous recombination deficiency. The numbers treated with GnP regimens and available for Response to chemotherapy according to modified Moffitt response were small, progression of disease was seen in three of nine classification. (33%) patients with basal-like versus eight of 54 (15%) with classical Of the 195 patients, 14 (7%) did not receive any chemotherapy and tumors (P ¼ 0.18; Fig. 2C). The ORR was 39% versus 11% in classical were considered nonevaluable (NE). A further 23 patients (12%) died versus basal-like PDAC, respectively (P ¼ 0.14). Of note, 20 of 63 as a result of rapid functional decline prior to their first scan, of which (32%) received additional experimental agents in this group.

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Table 1. Baseline characteristics of patients included (n ¼ 195). GATA6 expression for the Moffit subtype also demonstrated the prognostic impact of GATA6 in the model, again supporting its use Classical Basal-like as a biomarker of the subtypes. Of note, stage (locally advanced vs N ¼ N ¼ ( 156) ( 39) metastatic) or chemotherapy type had no impact in this observational N (%) N (%) P cohort study (Supplementary Fig. S3). To further explore whether Median age (years) 64.0 (29–84) 65.0 (44–83) 0.75 there was a significant interaction between mFFX or GnP and the Sex subtypes, we performed an interaction analysis. There was no statis- Male 83 (53) 29 (74) tically significant difference to suggest one chemotherapy regimen for Female 73 (47) 10 (26) 0.02 one particular subtype, although basal-like tumors trended toward Stage improved survival with GnP (P ¼ 0.08). Of note, the modified Moffitt Metastatic 132 (85) 39 (100) classifier used in this study outperforms the previously published Locally advanced 24 (15) 0 (0) 0.005 Moffitt classifier in identifying the poor prognostic basal-like subtype Race White 119 (79) 27 (77) (Supplementary Fig. S1B). Asian 28 (19) 6 (17) African/other 4 (3) 2 (6) GATA6 expression by RNA-seq and RNA ISH is associated with Unknown 5 4 0.23 modified Moffitt subtypes. Prior resection GATA6 expression remained strongly associated with the modified Yes 13 (8) 3 (8) Moffitt transcriptomic classifier (P < 0.001) in this expanded cohort No 143 (92) 36 (92) 0.99 ( Fig. 4A, left). In addition, the proposed RNA ISH semiquantitative – – CA19.9 (median, range) 1832 (1 371847) 1124 (1 71956) 0.24 score was highly associated with GATA6 gene expression (RNA-seq; Ever smoker P < 0.001; Fig. 4A, right). Matched RNA-seq and ISH results were Yes 80 (51) 23 (59) available in 106 patients (23 with basal-like and 83 with classical No 76 (49) 16 (41) 0.47 GATA6 fi Type II DM ≥18 months subtypes). Semiquantitative scoring of ISH con rmed higher GATA6 – Yes 32 (21) 8 (21) expression (74/83 score 2 4) in classical versus basal-like No 120 (79) 30 (79) 0.99 PDAC (19/23 score 0–1). Furthermore, GATA6 ISH correlated with Unknown 4 1 modified Moffitt with a sensitivity of 89%, specificity of 83%, and Liver metastases accuracy of 88%. Both manual scores and modified Moffitt calls could Yes 108(69) 38 (97) be predicted from image analysis data with concordance of 92% and No 48(31) 1 (3) <0.0001 fi a 81%, respectively, con rming reproducibility of semiquantitative HRD genotype assessment (n ¼ 106). The modified Moffitt signature remained Yes 14 (9) 2 (5) prognostic in this staining subcohort (Fig. 4B); both GATA6 ISH SQ No 142 (91) 37 (95) 0.74 First chemotherapy scoring (Fig. 4C) and subtyping inferred from image analysis of mFFX 81 (52) 22 (59) GATA6 ISH (Fig. 4D) predicted outcome in a similar manner. GnP regimens 61 (39) 10 (26) GnP alone 43 8 GATA6 IHC may discriminate basal-like from classical PDAC GnP þ experimental 18 2 Matched IHC and ISH results were available in only 78 advanced Cisplatin/gem or gem 5 (3) 2 (5) 0.25 PDAC cases. GATA6 levels by IHC and ISH were well correlated using alone quantitative assessment (Supplementary Fig. S4A) and semiquantita- None 9 (6) 5 (13) tive scoring (concordance 88%), indicating that GATA6 protein levels mirror RNA expression and could aid subtype identification when Note: Baseline characteristics of cases enrolled according to modified Moffitt fi RNA detection is not feasible. Indeed, IHC-based semiquantitative classi cation (classical vs basal-like). fi Abbreviations: DM, diabetes mellitus; gem, gemcitabine; HRD, homologous scoring identi ed most patients with classical subtype tumors by recombination deficiency. strong and moderate GATA6 staining (52/63 with scores 2–4), while aIncludes both somatic and germline HRD cases. basal-like subtype patients mostly exhibited no or weak GATA6 staining (9/15 with scores 0–1), so that GATA6 protein detection by IHC was associated with modified Moffitt subtypes in advanced PDAC Overall survival according to the modified Moffitt classification with a sensitivity of 83%, specificity of 60%, and accuracy of 78%. Once Overall survival (OS) in the intention-to-treat population (n ¼ 195) more, this was confirmed by quantitative assessment (Supplementary is shown in Fig. 3A. Median follow-up was 7.17 months. Median OS Fig. S4B) and the concordance between prediction of GATA6 scoring according to receipt of chemotherapy is shown in Fig. 3B. In patients from image analysis to manual scoring of GATA6 was 90%. receiving mFFX (n ¼ 103), where performance status was less likely to confound results, median OS was 6.5 months in basal-like versus Tissue distribution of GATA6 by IHC in a subset or resectable 10.6 months in classical subgroups [HR, 0.33; 95% confidence interval and metastatic PDAC (CI), 0.19–0.60; P ¼ 0.0001). These observations suggest favorable Recent data are emerging that basal and classical subtypes can impact of mFFX in classical PDAC but little impact of mFFX in the coexist in PDAC (17, 18). We therefore explored potential variation in basal-like population (Fig. 3C). In contrast, there was no difference GATA6 expression patterns. We used whole sections from selected between subgroups when treated with GnP regimens, where median resected cases (n ¼ 30) in addition to needle biopsies (n ¼ 41). OS, was 8.12 months in basal-like versus 8.19 months in classical Although early stage tumors may not necessarily reflect the biology groups, respectively (HR, 0.80; 95% CI, 0.40–1.60; P ¼ 0.53; Fig. 3D). of advanced disease, adequate tumor content is available for more In a multivariate Cox proportional hazard regression analysis, the complete evaluation. GATA6 staining (IHC) in resected specimens Moffitt subtype remained highly prognostic (P ¼ 0.018). Substituting that were basal-like (n ¼ 14) and classical (n ¼ 16) also associated with

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Mod Moffitt

Tumor size change Fisher exact test P : 0.0008 n = 37 Nonevaluable

X XX XX X X XXX X XX X Tumor size change (percent)

B C Mod Moffitt

Mod Moffitt Tumor size change Tumor size Fisher exact test P : 0.2619 change n = 8 Fisher exact test P : 0.0007 n = 11 Noneval Noneval

X X

XXXX XXX X X Tumor size change (percent) Tumor size change (percent)

Figure 2. Waterfall plots demonstrating tumor size change according to modified Moffitt classifier. A, Tumor size change in all patients included (n ¼ 194): This includes anychemotherapyreceived.Thenonevaluablepatientsdidnothaveimagingtodetermineresponse.B, Tumor size change in patients receiving first-line mFFX (n ¼ 102). C, Tumor size change in patients receiving GnP regimens (n ¼ 71). , one patient with nonmeasurable disease is not included; ,newlesions. the Moffitt subtypes: extensive immunopositivity for GATA6 (>50% of marker in PDAC (ref. 21; Supplementary Fig. S7B). Among these, tumor cells with score 2 or higher) was found in nine of 16 (56%) keratin 5 (CK5) demonstrated the strongest expression differences classical tumors versus one of 14 (7%) basal-like tumors (Supplemen- between basal-like and classical tumors and was found to be comple- tary Table S1). Interestingly, variable GATA6 immunopositivity mentary to GATA6 expression in our cohort (Supplementary 8). (<50% of tumor cells with score 2 or higher) was present in four of Furthermore, GATA6 and keratin 5 often demonstrated complemen- 16 (25%) and four of 14 (28%) of classical and basal-like tumors, tary staining pattern by IHC in PDAC tissues, including in 41 respectively, documenting a group where these subtypes may coexist. COMPASS biopsies and 30 resected PDAC whole sections (Fig. 5; This was furthermore observed in a number of advanced PDAC Supplementary Table S1). From these specimens, we observed the biopsies, which also exhibited variable GATA6 expression by ISH presence of both GATA6 and CK5 staining in a subset of cases (Fig. 5, and IHC (Supplementary Fig. S5), demonstrating that regional bottom). Indeed, the intratumoral staining pattern of the two markers GATA6 heterogeneity can exist in resectable and advanced-stage was predominantly inversely correlated in 149 individual tumor tumors. These differences were also observed at the cellular level by regions from the 30 resected cases (Fig. 6A). Of note, this analysis image analysis (Supplementary Fig. S6). In sum, GATA6 staining revealed a small number of regions that contained considerable þ þ patterns were widely comparable across whole sections of 22 of 30 number of both CK5 and GATA6 cells (Fig. 6A). Double immu- þ þ (73%) resection cases. Variable GATA6 immunopositivity was present nostaining confirmed distinct GATA6 /CK5 and GATA6 /CK5 in a subset of both, classical and basal-like subtypes, in resectable and regions within the same tumor (Fig. 6B) and in individual ducts advanced disease, which may point at the presence of classical and (Fig. 6C), which further support the notion that basal-like and classical basal regions in the same tumor. programs can coexist in the same tumor. Overall, many basal-like cases of advanced PDAC showed CK5 positivity (10/19, 53%), whereas most Keratin 5 may positively identify the basal-like subtype classical tumors (22/23, 96%) exhibited scant (<10%) or negative CK5 GATA6 positively identifies classical PDAC, but markers for the staining. Keratin 5 was thus highly specific and also showed remark- basal subtype are lacking. able intratumoral complementarity to GATA6 staining suggesting a In an exploratory analysis, we evaluated keratin markers associated clinically relevant biomarker of the basal-like subtype. with GATA6 expression. In-line with their use as basal markers in other tumor types (19, 20), keratins 15, 5/6, 23, and 14 were inversely Additional molecular characteristics of the basal-like correlated with GATA6 expression and thus the classical subtype phenotype (Supplementary Fig. S7A). In this post hoc analysis, none of the Among the 195 eligible COMPASS patients, all eight (4%) with identified cytokeratins were superior to GATA6 in their association adenosquamous histology were basal-like and stained positive for with modified Moffitt subgroups, including keratin 17, a prognostic keratin 5 by IHC, with negligible GATA6 expression by RNA ISH.

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Figure 3. Kaplan–Meier OS curves according to modified Moffitt subtype and chemotherapy received. A, OS in the intention-to-treat population (n ¼ 195) which includes patients who did not receive chemotherapy or who were NE. B, OS in patients receiving first-line mFFX or GnP regimens (at least one cycle) and is presented according to modified Moffitt subtype (n ¼ 174). This graph integrates curves in C and D. C, OS in patients receiving ≥ 1 cycle mFFX (n ¼ 103) according to modified Moffitt subtype. D, OS in patients receiving ≥ 1 cycle GnP regimens (n ¼ 71) according to modified Moffitt subtype.

We have previously shown that the basal-like subgroup is enriched in a the aforementioned trials, histologic groups were not documented in hypoxia-associated gene signature by gene set enrichment analysis (22) either study, which is not unusual because many patients have a and this observation is retained in this expanded dataset (P ¼ 0.0003). diagnosis made from very small samples or brushings. In contrast, In addition, we found higher PD-L1 expression in the basal-like cohort the histologic classification in resected specimens can be more easily (P < 0.001), higher PD-1 expression (P < 0.001), and enrichment of a reported and the PRODIGE24/CCTG PA6 trial of mFFX in the T-cell–inflamed signature reported previously (refs. 23, 24; P ¼ 0.007; adjuvant setting documented the prognostic impact of tumor grade Supplementary Fig. S9). Tumor mutational burden was not different in multivariate analysis (26) In patients receiving mFFX, those with between groups (2.02 mutations/Mb vs 1.96 mutations/Mb) and was well-differentiated tumors benefited the most (HR, 0.52; 95% CI, 0.34– consistent with that seen in an unselected PDAC cohort (25). 0.81), whereas the impact in poorly differentiated tumors was not significant. Although limitations to the three-tiered histologic classification (poor, moderate, and well-differentiated) in PDAC have been Discussion noted (27), well-differentiated tumors highly express the classical Combination chemotherapy is used in the treatment of most program and GATA6 (28). patients with advanced PDAC, yet the field is lacking robust biomar- The resistance of the basal-like subtype to mFFX is supported by a kers of outcome to guide regimen selection. Here, we show that recent collaborative study by Tiriac and colleagues (29) demonstrating patients with tumors of a modified “basal-like” phenotype, or those that patient-derived organoid chemotherapy signatures may predict with low GATA6 RNA expression, have inferior outcomes compared treatment response. The signatures indicative of individual cytotoxic with those with the “classical” phenotype. The latter are accurately agents were applied to our COMPASS cohort suggesting that the basal- identified by high GATA6 expression and positive GATA6 staining by like cohort subgroup was most likely to have a nonoxaliplatin-sensitive ISH. Our data also suggests that basal-like tumors are particularly signature (29). We furthermore hypothesize that basal-like tumors resistant to mFFX, warranting further investigation. may have limited sensitivity to 5-fluorouracil (5-FU). Martinelli Both the PRODIGE4/ACCORD 11 and the MPACT PDAC trials of and colleagues demonstrated GATA6 loss in resected PDAC with a metastatic disease demonstrated an improvement in survival with basal-like phenotype in the ESPAC-3 trial, and shorter survival in FOLFIRINOX and GnP across all subcohorts compared with gemci- these patients when treated with adjuvant 5-FU. This study also tabine alone (2, 3), yet they provide little insight into which subgroups showed that GATA6-low cell lines derived from patient-derived might benefit the most. Notably, our study shows no superiority in xenografts were particularly resistant to 5-FU but not gemcitabine (30). either regimen in an unselected population with regard to survival. In Notably oxaliplatin was not evaluated. In search of treatment

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Figure 4. GATA6 expression is associated with modified Moffitt subtypes in advanced PDAC. A, Gata6 expression by RNA-seq versus modified Moffitt subtypes (left), and GATA6 expression by RNA-seq versus GATA6 ISH (right). Scores of 0–1reflect the basal-like subtype and 2–5 the classical subtype. B, Kaplan–Meier curve of OS by modified Moffitt in patients with matched tissue for RNA-seq and GATA6 ISH analysis (n ¼ 106). C, Kaplan–Meier curves of OS by GATA6 ISH semiquantitative analysis in patients with matched tissue for RNA-seq and GATA6 ISH analysis (n ¼ 106). D, Kaplan–Meier curve of GATA6 by QuPath image analysis in those patients with matched tissue for RNA-seq and GATA6 ISH (n ¼ 106).

alternatives, we report here that basal-like tumors had higher hypoxia receptor–positive, HER2 tumors. The impact of immune populations scores, and higher PD-1 and PD-L1 expression with enrichment of a within subtypes in PDAC will require further investigation (31). T-cell–inflamed signature (24), which may be predictive of immuno- Clinical applicability of RNA-seq and tumor enrichment by LCM therapy 6 in this chemoresistant group. Similarly, triple-negative is currently limited given tissue acquisition, cost, and time to breast cancers, although associated with worse outcomes, have higher reporting. GATA6 detection from formalin-fixed, paraffin- levels of tumor-infiltrating lymphocytes compared with hormone embedded needle biopsies at diagnosis is therefore an attractive

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Figure 5. Pathology images comparing GATA6 staining by ISH and IHC, together with CK5 IHC staining. A, Advanced PDAC cases: COMP-022: classic with glandular architecture [hematoxylin and eosin (H&E)], GATA6 ISH score 3, GATA6 IHC score 2, and CK5-negative (rare positive cells); magnification, 100. COMPA-0234: basal-like with squamous features (H&E), GATA6 ISH score 1, GATA6 IHC score 1 (weak/focal), and CK5 positive; magnification, 100. COMP-0135: basal-like with poor differentiation (H&E), GATA6 ISH score 2 (variable distribution), GATA6 IHC score 2 (variable distribution), and CK5 positive; magnification, 100. B, Resected PDAC cases: expression pattern of GATA6 and CK5 in resected PDAC. PCSI_639: classic with glandular architecture (H&E), GATA6 ISH score 3, GATA6 IHC score 2, and CK5 negative; magnification, 100. PCSI_588: basal-like with squamous features (H&E), GATA6 IHC score 1 (weak/focal), and CK5 positive; magnification, 100. PCSI_645: classic with dual phenotype (glandular and squamous) on HE, GATA6 IHC score 2 (variable distribution), and CK5 positive; magnification, 25.

surrogate for transcriptomic classifiers. We demonstrate concor- much lower for IHC compared with ISH, a prospective study with dance of GATA6 ISH with the subtypes with sensitivity and adequate tissue for matched analysis is needed. Recognizing that the specificity of over 80% in our tumor-enriched samples. Of note, identification of the basal-like subtype is critical and that GATA6 is the GATA6 geneisnotpartoftheoriginalMoffitt subtype signa- a negative marker, we sought additional positive keratin biomarkers tures but rather the Bailey squamous classifier, which largely over- thatmaybemorefeasibleforthepracticingclinician.Ofthese, laps with Moffitt calls in high purity samples (5). The number of keratin 5 predicted outcomes best after GATA6 expression and was tissue specimens available for matched ISH, IHC, and RNA-seq was found to exhibit high complementarity to GATA6 staining pattern low in our study (n ¼ 78, 40%). Therefore, although specificity was and RNA expression levels. Moreover, combined keratin 5 and

AB Matched regions on PDAC serial secons Quanﬁcaon Double immunostaining Keran 5 IHC GATA6 IHC Keran 5 GATA6 GATA6 Keran 5 Percent posive cells

C Resectable PDAC Advanced PDAC

Figure 6. Tissue pattern of GATA6 and keratin 5 expression. A, IHC staining of GATA6 and keratin 5 on serial sections from resected PDAC specimen (n ¼ 30). Representative images, magnification, 25 (left). Quantification of the percentage of GATA6þ or CK5þ cells, respectively, in 149 matched regions on adjacent sections (right). B, Dual immunostaining of GATA6 (brown) or CK5 (magenta) in resected PDAC revealing distinct regions of GATA6 or CK5 immunoreactivity, magnification, 25n. C, Dual immunostaining of GATA6 (brown) or CK5 (magenta) revealing GATA6 and CK5 immunoreactivity in the same tumor ducts. Resected PDAC (left); advanced PDAC (right); magnification, 400.

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GATA6 stainings on serial sections and by double immune-staining Disclosure of Potential Conflicts of Interest have consistently suggested that basal-like and classical elements G.M. O’Kane reports receiving speakers bureau honoraria from Roche and Eisai. J. can coexist in a subset of PDAC cases, which strongly reinforces the M.S. Bartlett reports receiving commercial research grants from Thermo Fisher fi needforapositivebasal-likebiomarkerandhasmajorimplications Scienti c, Genoptix, Agendia, NanoString Technologies, Inc., Stratifyer GmbH, and Biotheranostics, Inc., speakers bureau honoraria from NanoString Technologies, Inc., for rationalizing subtype-specific treatments. We are currently Oncology Education, and Biotheranostics, Inc., and is an advisory board member/ evaluating combined staining of GATA6 and keratin 5 on the unpaid consultant for Insight Genetics, Inc., BioNTech AG, Biotheranostics, Inc., COMPASS trial. Pfizer, Rna Diagnostics, and oncoXchange. J.J. Knox is an employee/paid consultant Notably microdissected tissue, although impractical in laboratory for Merck, and reports receiving commercial research grants from Astra Zeneca, medicine practice, most accurately detects tumor gene expression, with Ibsen, Roche, and Merck. No potential conflicts of interest were disclosed by the other comparatively less exocrine and immune compartments compared authors. with TCGA datasets, as recently described (32). This therefore implies Authors’ Contributions that more reliable biomarkers can be determined from highly cellular specimens. CA-19.9 is the only approved biomarker for monitoring Conception and design: G.M. O'Kane, M. Masoomian, J.J. Knox, S.E. Fischer Development of methodology: G.M. O'Kane, B.T. Grunwald,€ J.J. Knox, S.E. Fischer disease in the advanced setting (33) and the POLO trial has now Acquisition of data (provided animals, acquired and managed patients, provided documented a benefit for maintaining PARPi in patients with germline facilities, etc.): G.M. O'Kane, B.T. Grunwald,€ G.-H. Jang, M. Masoomian, S. Picardo, BRCA mutations (4). Robust subtyping of pancreatic cancer will be Y. Wang, B. Lam, P.M. Krzyzanowski, I.M. Lungu, J.M.S. Bartlett, F. Vyas, R. Khokha, critical to advancing the field, GATA6 as a single biomarker and highly J. Biagi, D. Chadwick, S. Ramotar, S. Hutchinson, A. Dodd, J.M. Wilson, correlated with the Moffitt classifier will now be evaluated in a G. Zogopoulos, S. Gallinger, J.J. Knox, S.E. Fischer prospective trial. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G.M. O'Kane, B.T. Grunwald,€ G.-H. Jang, R.C. Grant, This study is limited by few progression biopsies to understand the R.E. Denroche, Y. Wang, F. Vyas, F. Notta, S. Gallinger, J.J. Knox stability of the subtypes under selective pressure during chemotherapy. Writing, review, and/or revision of the manuscript: G.M. O'Kane, B.T. Grunwald,€ This is especially interesting in light of the coexistence of basal-like and G.-H. Jang, R.C. Grant, Y. Wang, J. Biagi, G. Zogopoulos, S. Gallinger, J.J. Knox, classical elements, documented here and elsewhere (17, 18). In addi- S.E. Fischer tion, the numbers of basal-like tumors treated with GnP regimens is Administrative, technical, or material support (i.e., reporting or organizing data, low and the GnP group is potentially confounded by performance constructing databases): G.M. O'Kane, S. Picardo, R.E. Denroche, A. Zhang, I.M. Lungu, J.M.S. Bartlett, M. Peralta, R. Khokha, S. Ramotar, S. Hutchinson, status. The interaction term for chemotherapy type and subtype was A. Dodd, J.M. Wilson, G. Zogopoulos, S. Gallinger, J.J. Knox, S.E. Fischer not significant in this study although numbers were low. We therefore Study supervision: G.M. O'Kane, P.M. Krzyzanowski, J.J. Knox, S.E. Fischer cannot conclude whether GnP is a better strategy in the basal-like Other (collection of data): M. Masoomian cohort, rather our data suggests alternative therapies are urgently Other (primary study coordinator): S. Ramotar needed, and clinical trials to evaluate this particular group are war- ranted. With mFFX as current treatment of choice in the adjuvant Acknowledgments setting, understanding chemotherapy response to subtypes has This study was conducted with support of the Ontario Institute for Cancer Research (PanCuRx Translational Research Initiative) through funding provided by increasing importance. It should also be noted that the response rates the Government of Ontario, the Wallace McCain Centre for Pancreatic Cancer and survival between those receiving mFFX and GnP were not supported by the Princess Margaret Cancer Foundation, the Terry Fox Research statistically different in this analysis. This is supported by the recent Institute, the Canadian Cancer Society Research Institute, and the Pancreatic Cancer HALO trial 109–321 study where response rates and OS are compa- Canada Foundation. The study was also supported by charitable donations from the rable with historic outcomes with mFFX (2, 34). This furthermore Canadian Friends of the Hebrew University (Alex U. Soyka). J.J. Knox is the recipient supports the need to understand which populations can benefit most of the Lewitt Chair in Pancreatic Cancer. G.M. O'Kane is supported by the Lewitt fellowship. S. Gallinger is the recipient of an Investigator Award from OICR. G. from these regimens and a prospective trial has now been planned. Zogopoulos is a clinical research scholar of the Fonds de recherche du Quebec–Sante. In the major tumor types of lung and colorectal cancer, factors such B.T. Grunwald€ was supported by the Princess Margaret Cancer Foundation, EMBO as histologic subtype, molecular profile, and PD-L1 status can influ- (ALTF 116-2018), and the Alexander-von-Humboldt Foundation (DEU 1199182 ence the choice of upfront systemic treatment in advanced disease and FLF-P). We acknowledge the contributions of team members at OICR within the have resulted in survival gains (35–37). Because PDAC will soon Diagnostic Development platform and the Genomics & Bioinformatics platform become the second leading cause of cancer-related mortality, it (genomics.oicr.on.ca). behooves the oncology community to invest in biomarkers helpful fi The costs of publication of this article were defrayed in part by the payment of page for selecting standard chemotherapy. In this study, we con rm the advertisement fi fi charges. This article must therefore be hereby marked in accordance prognostic impact of the modi ed Mof tt subtypes and demonstrate with 18 U.S.C. Section 1734 solely to indicate this fact. that basal-like PDAC responds poorly to mFFX. The basal-like cohort can be accurately identified by GATA6 RNA expression, providing a Received November 13, 2019; revised February 12, 2020; accepted March 6, 2020; putative single important biomarker in clinical trial design. published first March 10, 2020.

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OF10 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

GATA6 Expression Distinguishes Classical and Basal-like Subtypes in Advanced Pancreatic Cancer

Grainne M. O'Kane, Barbara T. Grünwald, Gun-Ho Jang, et al.

Clin Cancer Res Published OnlineFirst March 10, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-3724

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