Jacqueline A. Brosnan

Clinical significance of the genetic landscape of pancreatic cancer and implications for identification of potential long-term survivors.

Purpose: Genetic alterations of KRAS, CDKN2A, TP53, and SMAD4 are the most frequent events in pancreatic cancer. We determined the extent to which these 4 alterations are coexistent in the same carcinoma, and their impact on patient outcome. Experimental Design: Pancreatic cancer patients who underwent an autopsy were studied (n = 79). Matched primary and metastasis tissues were evaluated for intragenic mutations in KRAS, CDKN2A, and TP53 and immunolabeled for CDKN2A, TP53, and SMAD4 protein products. The number of altered driver genes in each carcinoma was correlated to clinicopathologic features. Kaplan–Meier estimates were used to determine median disease free and overall survival, and a Cox proportional hazards model used to compare risk factors. Results: The number of genetically altered driver genes in a carcinoma was variable, with only 29 patients (37%) having an alteration in all 4 genes analyzed. The number of altered driver genes was significantly correlated with disease free survival (P = 0.008), overall survival (P = 0.041), and metastatic burden at autopsy (P = 0.002). On multivariate analysis, the number of driver gene alterations in a pancreatic carcinoma remained independently associated with overall survival (P = 0.046). Carcinomas with only 1 to 2 driver alterations were enriched for those patients with the longest survival (median 23 months, range 1 to 53). Conclusions: Determinations of the status of the 4 major driver genes in pancreatic cancer, and specifically the extent to which they are coexistent in an individual patients cancer, provides distinct information regarding disease progression and survival that is independent of clinical stage and treatment status. Clin Cancer Res; 18(22); 6339–47. ©2012 AACR.

Genetically defined subsets of human pancreatic cancer show unique in vitro chemosensitivity

Purpose: Pancreatic cancer is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for pancreatic cancer might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs. Experimental Design: Using a panel of genetically defined human pancreatic cancer cell lines, we tested gemcitabine (antimetabolite), docetaxel (antimicrotubule), mitomycin C (MMC; alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug), and artemisinin (control). Results: All pancreatic cancer cell lines were sensitive to triptolide and docetaxel. Most pancreatic cancer cells were also sensitive to gemcitabine and MMC. The vast majority of pancreatic cancer cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized pancreatic cancer cells to cisplatin and irinotecan by 2- to 4-fold, but they were modestly less sensitive to gemcitabine. Pancreatic cancer cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A-inactivated pancreatic cancer cells were 3- to 4-fold less sensitive to gemcitabine and MMC. Conclusions: Chemosensitivity of pancreatic cancer cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of pancreatic cancer in the future. Further work will need to confirm these responses and determine their magnitude in vivo. Clin Cancer Res; 18(23); 6519–30. ©2012 AACR.

A New Branch on the Tree: Next-Generation Sequencing in the Study of Cancer Evolution

Cancer is a disease caused by the accumulation of genetic alterations in association with successive waves of clonal expansion. Mapping of the human genome sequence, in conjunction with technical advances in the ability to sequence entire genomes, have provided new insight into the mutational spectra and genetic events associated with clonal evolution of cancer. Moving forward, a clearer understanding of those alterations that undergo positive and negative selection throughout carcinogenesis and leading to metastatic dissemination would provide a boon not only to our understanding of cancer evolution, but to the development of potential targets for therapeutic intervention as well.

Origin of metastases: subspecies of cancers generated by intrinsic karyotypic variations.

Conventional mutation theories do not explain (1) why the karyotypes of metastases are related to those of parental cancers but not to those of metastases of other cancers and (2) why cancers metastasize at rates that often far exceed those of conventional mutations. To answer these questions, we advance here the theory that metastases are autonomous subspecies of cancers, rather than mutations. Since cancers are species with intrinsically flexible karyotypes, they can generate new subspecies by spontaneous karyotypic rearrangements. This phylogenetic theory predicts that metastases are karyotypically related to parental cancers but not to others. Testing these predictions on metastases from two pancreatic cancers, we found: (1) Metastases had individual karyotypes and phenotypes. The karyotypes of metastases were related to, but different from, those of parental cancers in 11 out of 37 and 26 out of 49 parental chromosomal units. Chromosomal units are defined as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. (2) Metastases from the two different cancers did not share chromosomal units. Testing the view that multi-chromosomal rearrangements occur simultaneously in cancers, as opposed to sequentially, we found spontaneous non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are individual autonomous species differing from each other and parental cancers in species-specific karyotypes and phenotypes. They are generated from parental cancers by multiple simultaneous karyotypic rearrangements, much like new species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.

Pancreatic cancer genomics: insights and opportunities for clinical translation

Pancreatic cancer is a highly lethal tumor type for which there are few viable therapeutic options. It is also caused by the accumulation of mutations in a variety of genes. These genetic alterations can be grouped into those that accumulate during pancreatic intraepithelial neoplasia (precursor lesions) and thus are present in all cells of the infiltrating carcinoma, and those that accumulate specifically within the infiltrating carcinoma during subclonal evolution, resulting in genetic heterogeneity. Despite this heterogeneity there are nonetheless commonly altered cellular functions, such as pathways controlling the cell cycle, DNA damage repair, intracellular signaling and development, which could provide for a variety of drug targets. This review aims to summarize current knowledge of the genetics and genomics of pancreatic cancer from its inception to metastatic colonization, and to provide examples of how this information can be translated into the clinical setting for therapeutic benefit and personalized medicine.

Erratum: The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma (Nature (2012) 486 (266-270) DOI: 10.1038/nature11114)

This corrects the article DOI: 10.1038/nature11114

Erratum: Corrigendum: The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

This corrects the article DOI: 10.1038/nature11114

Corrigendum: The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

This corrects the article DOI: 10.1038/nature11114

Abstract 4006: Smad6 upregulation provides an alternative mechanism for BMP inactivation in SMAD4 wild type pancreatic cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Genetic inactivation of SMAD4, a central mediator of TGF-β superfamily signaling, is significantly correlated with metastatic behavior in pancreatic cancer patients. However, because some patients with pancreatic cancer have genetically intact TGF-β and BMP pathway components, we investigated the role of alternative mechanisms of inactivation in promoting pancreatic cancer metastasis. Methods: Eighteen cell lines for which the genetic status of all members of the TGF-β pathway were known were used in this study. TGF-β signaling levels were analyzed in each cell line using a luciferase reporter system under the control of a Smad Binding Element (SBE). Immunoblotting was performed on protein lysates from pancreatic cancer cell lines of known genotype. Cell lines that express high levels of Smad6 were transiently transfected with shRNA constructs targeting Smad6; cell lines that express low levels of Smad6 were transiently transfected with a construct to constitutively express Smad6. The effects of Smad6 modulation were assessed by proliferation assay, migration assay, and invasion assay. Immunohistochemistry was performed on primary and metastatic pancreatic cancer tissues and staining intensity correlated to clinical data. Results: Functional TGF-β and BMP signaling were eliminated in cell lines with known SMAD4 inactivation, as well as in several cell lines in which these pathways remain intact. Immunoblotting for known TGF-β superfamily antagonists in these cell lines revealed differential expression of Smad6, an inhibitory Smad. Modulation of Smad6 levels in vitro suggests that Smad6 overexpression contributes to increased levels of proliferation, migration, and invasion in SMAD4-intact cell lines. Overexpression of Smad6 did not restore TGF-β signaling, but did increase BMP response. Immunohistochemistry for Smad6 in patient samples revealed a nuclear localization pattern, suggesting that the pro-oncogenic roles of Smad6 are mediated by its ability to act as a transcription factor. High Smad6 levels correlated with worse prognosis and metastatic behavior among SMAD4-intact pancreatic cancers. Conclusions: Smad6, an inhibitory Smad, is differentially expressed in pancreatic cancer, both in cell lines and patient samples. High levels of Smad6 in patients at autopsy associate with widespread metastasis, irrespective of SMAD4 status. Preliminary studies in vitro support a metastasis-promoting function of Smad6: Smad6 overexpression is associated with increased levels of proliferation, migration, and invasion in SMAD4-intact pancreatic cancer cell lines. Experiments are ongoing to determine the mechanism through which Smad6 is acting in pancreatic cancer. Citation Format: Jacqueline A. Brosnan, Richard Morgan, Catherine M. White, Seung-Mo Hong, Shinichi Yachida, Michael Goggins, Barish Edil, Christine A. Iacobuzio-Donahue. Smad6 upregulation provides an alternative mechanism for BMP inactivation in SMAD4 wild type pancreatic cancers. [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 4006. doi:10.1158/1538-7445.AM2013-4006

Abstract 5318: DPC4 loss results in the activation of alternative oncogenic pathways in pancreatic cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Pancreatic cancer remains one of the most lethal forms of cancer, with a five-year survival rate of less than five percent. Many of these patients succumb to complications from metastasis. We have shown that genetic inactivation of DPC4, a major factor in TGF-β signaling, is significantly correlated with widespread metastasis in pancreatic cancer patients at autopsy. We hypothesize that DPC4 loss may be driving pancreatic cancer metastasis by forcing the TGF-β signal down an alternative pathway. Methods: DPC4 was stably restored into three cell lines that harbor a homozygous deletion of the locus (A2.1, A6L, and BxPC3), forming isogenic pairs of DPC4−/− and DPC4+ cells. Matched DPC4−/− and DPC4+ cells were treated with TGF-β1 for 24 hours prior to protein extraction. Immunoblotting was performed for total and phospho Akt (Pi3K pathway), p44-42 (ERK pathway), and Stat3 (JAK-Stat pathway), as well as nuclear and cytoplasmic p65 (NFκB pathway), as these networks have previously been identified as being activated by TGF-β. Immunohistochemistry was performed on 50 surgically resected pancreatic cancers for pAkt. Results: DPC4 was successfully introduced into DPC4−/− cells, as evidenced by semi-quantitative PCR for DPC4, as well as a TGF-β luciferase assay. DPC4+ cells displayed characteristic differences in cell behavior, such as decreased proliferation, migration, and invasion, compared to DPC4−/− cells. Western blot analysis of Smad-independent TGF-β pathways in three sets of DPC4-complemented pancreatic cancer cell lines revealed patterns of preferential activation of the Pi3K and ERK signaling pathways in cells lacking DPC4. In surgically-resected pancreatic cancers, activation of Pi3K was associated with the presence of lymph node metastases (p=0.03), while concomitant DPC4 loss and Pi3K pathway activity was associated with poor survival in pancreatic cancer patients (p=0.02). Conclusions: Restoration of DPC4 into DPC4−/− cells results in the abrogation of Pi3K and ERK signaling, and simultaneous DPC4 loss and Pi3K activation correlates to a poor prognosis of pancreatic cancer patients at surgery. Further studies will confirm whether genotype-specific activation of the ERK and Pi3K pathways plays a role in pancreatic cancer cell behavior, particularly those processes that directly impact metastasis (i.e. migration and invasion). Nevertheless, it is intriguing to consider the Pi3K and ERK pathways as therapeutic targets in pancreatic cancer patients with loss of DPC4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5318. doi:1538-7445.AM2012-5318