Joan Seoane

TGF-β signalling-related markers in cancer patients with bone metastasis

Abstract We measured transforming growth factor (TGF)-β-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-β inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-β1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml−1) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-β inhibitor.

New approach to cancer therapy based on a molecularly defined cancer classification

Translational and clinical cancer research, as well as clinical trials and treatment of cancer, are essentially structured based on the organ in which tumors originate. However, the recent explosion of knowledge about the molecular characteristics of tumors is opening a new way to tackle cancer. This article proposes a different approach to the classification of cancer with important implications for treatment and for basic, translational, and clinical research. The authors postulate that cancers from diverse organs of origin with similar molecular traits should be managed together. The common molecular features observed in different tumors determine clinical actions in a better way than organ‐based classification. Thus, comparisons between tumors residing in different locations but with shared molecular characteristics will improve the therapeutic approach and the understanding of the biology of cancer. CA Cancer J Clin 2014;64:70–74.

The Taming of the TAMs

Tumor-associated macrophages and microglia (TAMs) are considered crucial elements in cancer progression. Recent work reveals the molecular mechanisms underlying acquired resistance to colony-stimulating factor-1 receptor (CSF-1R) inhibitors in glioblastoma (GBM) and shows that targeting TAMs with CSF-1R inhibitors leads to an antitumor response in GBM followed by the acquisition of resistance to treatment through the induction of insulin-like growth factor 1 (IGF-1) expression in TAMs.

Abstract 930: Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnBackground: Genetic characterization and monitoring of brain tumors is challenging given the restricted sampling of tumors and the limited abundance of brain tumor-derived circulating tumor DNA (ctDNA) in the plasma. Here, we sought to define whether cerebrospinal fluid (CSF) could serve as a ‘liquid biopsy’ for the genetic characterization of tumor DNA originated from the central nervous system (CNS) of patients (pts) with brain tumors.nnMethods: CSF, plasma and tumor tissue were obtained from pts with glioblastoma (GBM), brain metastases from breast (BMBC) and lung cancers (BMLC) and leptomeningeal carcinomatosis (LC). Whole exome sequencing was done in CSF and tumor (n = 3) and was coupled with digital PCR for monitoring CSF and plasma ctDNA (n = 6). Targeted capture massively parallel sequencing of 39 samples from 4 BMBC pts, including samples from CSF, plasma and matched metastatic sites obtained at autopsy was performed using two independent platforms (MSK-IMPACT (341 genes) and BC panel (254 genes most frequently mutated in BC)) comprising 488 unique genes. Sequencing was performed on an Illumina HiSeq2000. Single nucleotide variants were defined using MuTect, and indels using Varscan. Copy number alterations were assessed using Varscan2 and GISTIC.nnResults: Whole exome sequencing of tumor and CSF DNA revealed a high proportion of tumor-derived cell-free DNA in the CSF of 3 pts (1 GBM, 2 BMBC). We identified actionable somatic mutations (EGFR L858R, IDH1 R132H) and also IDH1 mutations associated with molecular diagnosis of proneural GBM. In CSF and plasma ctDNA of 6 pts with GBM, BMBC, BMLC, we observed that the mutations present in the CSF ctDNA, but not plasma ctDNA, and their mutant allele fractions (MAFs) correlated with brain tumor burden. In 3 pts with suspected LC, we compared the results of cytopathologic analysis and CSF ctDNA obtained from samples used for cytopathologic diagnosis, and observed that CSF ctDNA was more robust and sensitive for the diagnosis of LC. The analysis of synchronous CSF ctDNA, plasma ctDNA and intra- and extra-cranial metastases from 4 autopsied BMBC pts showed that CSF ctDNA recapitulated the somatic genetic alterations present in the intra-cranial lesions. We detected similar MAFs for the truncal mutations (RB1, KMT2D, AHNAK2) in both CSF and plasma DNA of BMBC3, a pt with Li-Fraumeni syndrome and a diagnosis of concurrent BMBC and esthesioneuroblastoma; however, mutations in PIK3CB, PAK7, MSH5 found only in the CNS implant of each disease were only detected in the CSF but not in the plasma DNA.nnConclusions: Brain tumor-derived ctDNA is abundantly present in the CSF of brain cancer pts and compared to plasma ctDNA, CSF ctDNA is more representative of the brain lesions. Our results demonstrate that massively parallel sequencing can be performed using CSF DNA, allowing for the non-invasive genomic characterization and monitoring of brain lesions.nnCitation Format: Leticia De Mattos-Arruda, Regina Mayor, Charlotte K. Y. Ng, Britta Weigelt, Francisco Martinez-Ricarte, Davis Torrejon, Mafalda Oliveira, Carolina Raventos, Alexandra Arias, Elena Guerini-Rocco, Elena Martinez-Saez, Sergio Lois, Oscar Marin, Xavier de la Cruz, Salvatore Piscuoglio, Russell Towers, Ana Vivancos, Vicente Peg, Santiago Ramon y Cajal, Jordi Rodon, Enriqueta Felip, Joan Sahuquillo, Josep Tabernero, Javier Cortes, Jorge S. Reis-Filho, Joan Seoane. Analysis of cell-free tumor DNA in cerebrospinal fluid to characterize and monitor the genetic alterations of brain tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 930. doi:10.1158/1538-7445.AM2015-930

Abstract IA17: Intratumoral heterogeneity in glioblastoma

Cells within tumours have diverse genomes, epigenomes, and interact differentially with their surrounding microenvironment generating intratumour heterogeneity, which has critical implications for treating cancer patients. I will present our current approach to understand the intratumour heterogeneity in glioblastoma through the analysis of patient-derived models. I will present examples of how we perform functional analysis to understand genomic alterations in the context of inter- and intra-tumoral heterogeneity taking special interest on their therapeutic implications. Linked to the concept of intratumor heterogeneity in the cellular state of differentiation, I will explain our studies related to cancer-initiating cells in glioblastoma, and I will focus on the TGF-beta pathway which has a critical role in glioblastoma. Citation Format: Joan Seoane. Intratumoral heterogeneity in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA17. doi:10.1158/1538-7445.CHTME14-IA17

Abstract 4727: Genomic characterization of brain metastases reveals divergent evolution and metastasis specific mutations

Background: Brain metastases represent an unmet need in current oncologic care. Approximately 8-10% of cancer patients will develop brain metastases, and more than half of these patients will pass away within a few months of their diagnosis. We have a limited understanding of how brain metastases genetically evolve from their primary tumors. Our objectives were to (1) elucidate the genomic evolutionary patterns leading to brain metastases (2) identify whether brain metastases harbor clinically significant genetic differences compared to their primary tumors and other extracranial metastatic sites, and (3) examine the extent of genetic heterogeneity across regionally separated and anatomically distinct sites of brain metastasis. Methods: We subjected 104 matched primary tumor biopsies, brain metastases, and normal tissue to whole exome sequencing, including 20 cases with regionally and anatomically separated brain metastasis sites, regional lymph nodes, and distal extracranial metastases. We performed an integrative analysis of somatic single nucleotide variants and copy-number alterations to reconstruct phylogenetic trees relating the subclones from each patient. We analyzed evolutionary relationships between related cancer samples and annotated phylogenetic trees with clinically significant genetic alterations. Results: Every brain metastasis displayed branched evolution: the brain metastasis and primary tumor shared a common ancestor yet both the primary tumor and brain metastasis continued to evolve independently. We found novel clinically actionable genetic alterations that were exclusive to brain metastases in 56% of cases. The brain metastases were also enriched for several pathways, some pathways specific to a particular histology. Distal extracranial metastases and regional lymph nodes were highly divergent from brain metastases, and in no cases, did we observe an extracranial site that closely resembled the brain metastasis. In contrast, regionally and anatomically separated brain metastasis sites were genetically homogenous and shared nearly all genetic alterations detected. Conclusions: Brain metastases are genetically divergent from primary tumors. Clinically, these observations demonstrate that biopsies of primary tumors fail to capture the heterogeneity with patients with brain metastases, potentially missing clinically actionable mutations in these life-threatening metastases. Notably, regional lymph nodes and distal extracranial metastases were not reliable genetic surrogates for brain metastases. When clinically feasible, characterization of even a single brain metastasis lesion is superior to that of a primary or lymph node biopsy for selection of a targeted therapeutic agent. Citation Format: Priscilla K. Brastianos, Scott L. Carter, Sandro Santagata, Amaro Taylor-Weiner, Robert T. Jones, Eli Van Allen, Keith L. Ligon, Josep Tabernero, Joan Seoane, Elena Martinez-Saez, Daniel Cahill, William T. Curry, Ian F. Dunn, Sun Ha Paek, Paul Van Hummelen, Aaron R. Thorner, Bruce E. Johnson, Nancy U. Lin, Toni K. Choueiri, Michael S. Rabin, Rameen Beroukhim, Anat Stemmer-Rachamimov, Matthew Meyerson, Levi Garraway, Tracy Batchelor, Jose Baselga, David N. Louis, William C. Hahn, Gad Getz. Genomic characterization of brain metastases reveals divergent evolution and metastasis specific mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4727. doi:10.1158/1538-7445.AM2015-4727

Abstract PD4-5: Longitudinal massively parallel sequencing analysis of circulating cell-free tumor DNA: A feasibility study

Background: Plasma-derived cell-free tumor DNA (ctDNA) has been shown to constitute a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that the genetic data obtained from massively parallel sequencing analysis of ctDNA may be more informative than analysis of single tumor tissue biopsies, and would, therefore, constitute a tool to identify the presence of potentially actionable driver somatic genomic alterations, and monitor changes in the genetic landscape during the course of therapy.nnMethods: Our index case is a patient with an estrogen receptor (ER)+/ HER2 -, highly proliferative, grade 2, mixed ductal/lobular breast cancer and synchronous bone and liver metastases at diagnosis. DNA extracted from archival tumor material and plasma, and from peripheral blood leukocytes was subjected to targeted massively parallel sequencing using a platform comprising 280 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor.nnResults: Average read depths of >100x were obtained from the archival primary tumor sample and between 200x and 900x from the ctDNA samples. Sixteen somatic non-synonymous mutations were detected at high frequencies in all plasma samples, of which 11 (CDKN2A, AKT1, TP53, CDH1, TSC1, NF1, JAK3, ESR1, MML3, EPHB1, PIK3C2G) were also detected in >5% of alleles obtained from the primary tumor sample. Importantly, allelic frequencies of approximately 50% for missense mutations of AKT1(p.E17K) and ESR1(p.E380Q) were detected in the plasma ctDNA, providing strong evidence to suggest that most of the cell-free plasma DNA obtained from this patient was tumor-derived. The allelic frequency of ESR1 in the tumor sample was <5%, whereas for AKT1 it was 80%. In ctDNA samples, there was enrichment for mutant ESR1 alleles as compared to the primary.nnThe mutant allelic frequencies identified in ctDNA samples were reduced following the targeted therapy administered, which mirrored the pharmacodynamic response as assessed by PET-CT. Subsequently, the mutant allelic frequencies were increased again at the time of progression. Single agent treatment with an AKT inhibitor provided benefit in terms of long-lasting biochemical and radiologic responses (stable disease: 8 months) as shown by CA15.3 levels and radiologic assessment (RECIST 1.1), respectively.nnConclusions: Targeted capture massively parallel sequencing of plasma-derived ctDNA represents a potential tool to uncover and monitor tumor somatic alterations during the course of targeted therapy. The clinical and therapeutic impact of actionable mutations in luminal/ER+ breast cancers warrants further investigation.nnCitation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-5.

TGF-β Signaling in Homeostasis and Cancer

TGF-β is a member of a large family of cytokines with a crucial role in embryonic development and tissue homeostasis. Disruption of the TGF-β signaling pathway has been implicated in many human diseases including cancer. In normal epithelial cells, TGF-β acts as a tumor suppressor by inhibiting cellular proliferation. During cancer progression, tumor cells escape from the TGF-β antiproliferative response either by acquiring mutations in components of the TGF-β pathway or by selectively inactivating the pathway that leads to cell cycle arrest. In the latter case, TGF-β becomes an oncogenic factor. Over the last years, some of the molecular mechanisms implicated in the TGF-β antiproliferative response have been elucidated and we are beginning to understand how TGF-β is transformed from an anti-tumorigenic factor into an oncogenic factor during cancer progression. This allows a better understanding of cancer biology and helps in the design of better therapeutic protocols against this deadly disease.