Francesca Sarno

Integrated Next Generation Sequencing and Avatar Mouse Models for Personalized Cancer Treatment

Background: Current technology permits an unbiased massive analysis of somatic genetic alterations from tumor DNA as well as the generation of individualized mouse xenografts (Avatar models). This work aimed to evaluate our experience integrating these two strategies to personalize the treatment of patients with cancer. Methods: We performed whole-exome sequencing analysis of 25 patients with advanced solid tumors to identify putatively actionable tumor-specific genomic alterations. Avatar models were used as an in vivo platform to test proposed treatment strategies. Results: Successful exome sequencing analyses have been obtained for 23 patients. Tumor-specific mutations and copy-number variations were identified. All samples profiled contained relevant genomic alterations. Tumor was implanted to create an Avatar model from 14 patients and 10 succeeded. Occasionally, actionable alterations such as mutations in NF1, PI3KA, and DDR2 failed to provide any benefit when a targeted drug was tested in the Avatar and, accordingly, treatment of the patients with these drugs was not effective. To date, 13 patients have received a personalized treatment and 6 achieved durable partial remissions. Prior testing of candidate treatments in Avatar models correlated with clinical response and helped to select empirical treatments in some patients with no actionable mutations. Conclusion: The use of full genomic analysis for cancer care is encouraging but presents important challenges that will need to be solved for broad clinical application. Avatar models are a promising investigational platform for therapeutic decision making. While limitations still exist, this strategy should be further tested. Clin Cancer Res; 20(9); 2476–84. ©2014 AACR.

Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2-/-IL2Rγnull immunodeficient mice

A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.

Clinical validation of prospective liquid biopsy monitoring in patients with wild-type RAS metastatic colorectal cancer treated with FOLFIRI-cetuximab

Cancer genomics and translational medicine rely on the molecular profiling of patients tumor obtained during surgery or biopsy. Alternatively, blood is a less invasive source of tumor DNA shed, amongst other ways, as cell-free DNA (cfDNA). Highly-sensitive assays capable to detect cancer genetic events from patients blood plasma became popularly known as liquid biopsy (LqB). Importantly, retrospective studies including small number of selected patients with metastatic colorectal cancer (mCRC) patients treated with anti-EGFR therapy have shown LqB capable to detect the acquired clonal mutations in RAS genes leading to therapy resistance. However, the usefulness of LqB in the real-life clinical monitoring of these patients still lack additional validation on controlled studies. In this context, we designed a prospective LqB clinical trial to monitor newly diagnosed KRAS wild-type (wt) mCRC patients who received a standard FOLFIRI-cetuximab regimen. We used BEAMing technique for evaluate cfDNA mutations in KRAS, NRAS, BRAF, and PIK3CA in twenty-five patients during a 2-y period. A total of 2,178 cfDNA mutation analyses were performed and we observed that: a) continued wt circulating status was correlated with a prolonged response; b) smoldering increases in mutant cfDNA were correlated with acquired resistance; while c) mutation upsurge/explosion anticipated a remarkable clinical deterioration. The current study provides evidences, obtained for the first time in an unbiased and prospective manner, that reinforces the utility of LqB for monitoring mCRC patients.

Lurbinectedin induces depletion of tumor-associated macrophages, an essential component of its in vivo synergism with gemcitabine, in pancreatic adenocarcinoma mouse models

ABSTRACT We explored whether the combination of lurbinectedin (PM01183) with the antimetabolite gemcitabine could result in a synergistic antitumor effect in pancreatic ductal adenocarcinoma (PDA) mouse models. We also studied the contribution of lurbinectedin to this synergism. This drug presents a dual pharmacological effect that contributes to its in vivo antitumor activity: (i) specific binding to DNA minor grooves, inhibiting active transcription and DNA repair; and (ii) specific depletion of tumor-associated macrophages (TAMs). We evaluated the in vivo antitumor activity of lurbinectedin and gemcitabine as single agents and in combination in SW-1990 and MIA PaCa-2 cell-line xenografts and in patient-derived PDA models (AVATAR). Lurbinectedin-gemcitabine combination induced a synergistic effect on both MIA PaCa-2 [combination index (CI)=0.66] and SW-1990 (CI=0.80) tumor xenografts. It also induced complete tumor remissions in four out of six patient-derived PDA xenografts. This synergism was associated with enhanced DNA damage (anti-γ-H2AX), cell cycle blockage, caspase-3 activation and apoptosis. In addition to the enhanced DNA damage, which is a consequence of the interaction of the two drugs with the DNA, lurbinectedin induced TAM depletion leading to cytidine deaminase (CDA) downregulation in PDA tumors. This effect could, in turn, induce an increase of gemcitabine-mediated DNA damage that was especially relevant in high-density TAM tumors. These results show that lurbinectedin can be used to develop ‘molecularly targeted’ combination strategies. Summary: Lurbinectedin-gemcitabine synergism in PDA models involves lurbinectedin-induced tumor macrophage depletion, triggering CDA downregulation and enhanced DNA damage, supporting the use of this combination to treat macrophage-infiltrated pancreatic tumors.

Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown. Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts. Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice. Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550–9. ©2018 AACR.

Whole-exome sequencing of plasma cell-free DNA portrays the somatic mutation landscape of refractory metastatic colorectal cancer and enables the discovery of mutated KDR/VEGFR2 receptors as modulators of anti-angiogenic therapies.

The non-invasive detection of cancer mutations is a breakthrough in oncology. Here, we applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens including VEGFR inhibitors. Using WES-cfDNA, we could detect 73% (54/74) of the somatic mutations uncovered by WES-tumor including a variety of mutation types: frameshift (indels), missense, noncoding (splicing), and nonsense mutations. Additionally, WES-cfDNA discovered 14 high-confidence somatic mutations not identified by WES-tumor. Importantly, in the absence of the tumor specimen, WES-cfDNA could identify 68 of the 88 (77.3%) total mutations that could be identified by both techniques. Of tumor biology relevance, we identified the novel KDR/VEGFR2 L840F somatic mutation, which we showed was a clonal mutation event in this tumor. Comprehensive in vitro and in vivo functional assays confirmed that L840F causes strong resistance to anti-angiogenic drugs, whereas the KDR/VEGFR2 hot-spot mutant R1032Q confers sensitivity to cabozantinib. Moreover, we found a 1-3% of recurrent KDR somatic mutations across large and non-overlapping cancer sequencing projects, and the majority of these mutations were located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK, suggesting a functional role. In summary, the current study highlights the capability of exomic sequencing of cfDNA from plasma of cancer patients as a powerful platform for somatic landscape analysis and discovery of resistance-associated cancer mutations. Because of its advantage to generate results highly concordant to those of tumor sequencing without the hurdle of conventional tumor biopsies, we anticipate that WES-cfDNA will become frequently used in oncology. Moreover, our study identified for the first-time KDR/VEGFR2 somatic mutations as potential genetic biomarkers of response to anti-angiogenic cancer therapies and will serve as reference for further studies on the topic.

Abstract B44: When murine tumors are thought to be human: A drawback of patient derived xenografts.

The use of preclinical models is essential in every aspect of translational cancer research, from the biologic understanding of the disease to the development of new treatments. Patient Derived Xenograft (PDX) models are increasingly used in translational cancer research due to their advantages over other preclinical models. However, although it has been widely demonstrate that these kind of models are predictive of clinical outcome, there are also some drawbacks when working with them. Analyzing our PDX model collection, we have observed that there is a small percentage of cases in which a murine tumor is developed instead of the original human one by a process that has been called horizontal oncogenesis. This process was already observed and described in the early 80´s, however, it is still not well understood and the molecular mechanisms by which it happens remain unknown. In order to determine the origin of the different murine tumors observed in our mice, we performed immunohistochemistry characterization of our altered models and found two different types of murine tumors: murine fibrosarcomas, which were observed in Colorectal and Pancreatic Ductal Adenocarcinoma PDX models; and murine lymphomas, that were observed mainly in Lung PDX models when they were implanted in Nod-scid mice. We also found a case in which a human lymphoproliferation grew in the animals instead of the sample from the small cell lung cancer obtained from the patient. As these murine tumors grow in the same place where the human donor tumor was implanted, it is important to have a quick and effective method to identify them before performing any study with these altered models. In this regard, we have implemented a simple technique based on PCR analysis with primers that recognize K-Ras. We have designed an estrategy in which we can obtain amplicons of different sizes depending on their human or murine origin. Although we still have to analize some of our PDX models, using this methodology we have confirmed that murine tumors appear in a very low percentage of cases (less than 5% to date). However, even with this low incidence we consider that is essential to identify those murine tumors as soon as possible in order to avoid wasting time, money and resources. Citation Format: Camino Menendez, Beatriz Salvador, Manuel Munoz, Natalia Banos, Francesca Sarno, Pedro P Lopez-Casas, Manuel Hidalgo. When murine tumors are thought to be human: A drawback of patient derived xenografts.. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B44.

Abstract B42: Patient-derived metastatic models of pancreatic cancer: An in-vivo system for modeling metastasis and preclinical drug screening

So far, there are few preclinical models that can recapitulate the full metastatic process of pancreatic cancer, therefore modeling of tumor progression to metastasis is urgently needed. In our laboratory, we established patient-derived cancer xenograft (PDX) metastatic models of pancreatic ductal adenocarcinoma (PDA) by orthotopic transplantation of human pancreatic tumor specimens into immunodeficient NOD/SCID/IL2λ-receptor null (NSG) mouse. Importantly, these preclinical orthotopic models preserve the histological and genetic characteristics of donor tumor and provide successful tumor growth with subsequent distal spread to the mouse liver and lung, which are the common metastatic sites of PDA patients. Furthermore, we found that in one of the PDX metastatic models, Gemcitabine and albumin-bound paclitaxel (Nab-paclitaxel) showed significant increase in survival of treated mice, compared to control group, as well as Nab-paclitaxel resulted in reducing the number of mice affected with metastasis. Currently, we are focused on identification and characterization of circulating tumor cells (CTCs) derived from PDX metastatic models, hypothesizing that CTCs population contain cells with clonal capacity to initiate distant metastases. To identify and isolate CTCs from PDX metastatic models of pancreatic cancer, we developed a magnetic separation assay using anti-human antibodies specific to the MHC-class1-surface antigens HLA-ABC, which allowed us to enumerate human tumor cells in mouse peripheral tissues. The molecular characterization of the human CTCs population at single cell level is performed via microfluidics-based platform (Fluidigm) for multiplex gene expression analysis in individual cells. This system allows us to study the simultaneous expression of groups of genes by RNAseq and resolve cellular diversity during pancreatic cancer metastasis by comparing gene expression signatures of the human tumor cells derived from orthotopic tumor, mouse metastatic organs and peripheral blood. We expect that this single cell gene expression analysis will help us to characterize the metastasis-initiating cell sub-clones within the whole heterogeneous tumor population and subsequently identify potential therapeutic targets to trigger the metastatic spread. In summary, our preclinical data provides a rationale for further use of these PDX metastatic models of pancreatic cancer for studying the molecular mechanisms involved in metastatic process, preclinical drug screening and personalized medicine strategies. Citation Format: Spas Dimitrov, Manuel Munoz, Natalia Banos, Camino Menendez, Victoria Bonilla, Yolanda Duran, Rodrigo Toledo, Francesca Sarno, Javier Perales-Paton, Fatima Al-Shahrour, Pedro P Lopez-Casas, Manuel Hidalgo. Patient-derived metastatic models of pancreatic cancer: An in-vivo system for modeling metastasis and preclinical drug screening. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B42.

Abstract A36: Assessment of effectiveness and molecular markers of CDK4/6 inhibitor Palbociclib in Pancreatic Ductal Adenocarcinomas

Pancreatic Ductal Adenocarcinoma (PDAC) continues to be the deadliest human cancer with a 5-year survival rate of 7%. One of the current standards of care for advanced PDAC is gemcitabine plus nab-paclitaxel, a regimen our group helped develop. Finding new agents to combine with this regimen remains necessary, particularly using drug targeting strategies. At the molecular level, genetic and genomic profiling identified CDKN2A as a very frequently disrupted gene in PDAC ( In the current study, we aimed to evaluate the effectiveness of palbociclib monotherapy and in combination with nab-paclitaxel and gemcitabine/nab-paclitaxel in a cohort of PDACs patient derived xenograft (PDX) models. PDX models obtained from late stage PDAC patients were treated with palbociclib alone and in combination with gemcitabine/nab-paclitaxel. The majority of models displayed >50% tumor growth inhibition (TGI) following treatment with single agent palbociclib. Treatment with palbociclib plus nab-paclitaxel or palbociclib plus gemcitabine/nab-paclitaxel increased TGI to a greater degree than the gemcitabine/nab-paclitaxel combination and also increased the duration of response as compared to the standard of care therapy. Moving forward, the palbociclib/nab-paclitaxel combination will be evaluated in a clinical trial for PDAC patients. We are currently expanding our analyses to a larger cohort of PDX models and performing molecular characterization of these models in order to gain insights on biomarkers and mechanism of action of the drug combinations in PDAC. Citation Format: Beatriz Salvador, Pedro P. Lopez-Casas, Camino Menendez, Natalia Banos, Francesca Sarno, Yin Min-Jean, Peter Olson, Todd VanArsdale, David J. Shields, Manuel Hidalgo. Assessment of effectiveness and molecular markers of CDK4/6 inhibitor Palbociclib in Pancreatic Ductal Adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A36.

Abstract 4534: In vivo antitumor activity of PM060184 in patient-derived xenografted tumors (avatar)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: PM060184 is a synthetic marine-derived compound originally isolated from the marine sponge Lithoplocamia lithistoides. PM060184 induces disorganization and disruption of the microtubule network as well as aberrant mitotic spindle multipolarization and chromosome missegregation. These effects give rise to prometaphase arrest and formation of multinucleated cells. Then, cells enter to caspase-driven apoptosis or are arrested in a pseudo-senescent state. PM060184 is currently under evaluation in Phase I clinical studies in patients with advanced cancer diseases. Material and Methods: Athymic female nu/nu mice were subcutaneously implanted with different patient-derived tumors: NSCLC (N=4)), pancreas (N=5), colon (N=4) and gastric (N=2). Tumor (ca. 300 mm3) bearing animals (N=6-10/group) were randomly allocated to receive PM060184 (16 mg/kg) or placebo. Treatments were administered weekly for 5 consecutive weeks. Antitumor effect was calculated using ΔT/ΔC (%), defined as a percentage of the change in tumor size for treated (T) and placebo (C) groups during the placebo-treated survival time (D). Complete tumor regression (CR) was defined when tumor volume < 63 mm3 for 2 or more consecutive measurements. Results: The treatment with PM060184 produced lowest ΔT/ΔC values summarized as follows: View this table: During the treatment, complete remissions (CR) of tumors were also seen in animals bearing the following xenografts: Pulm-005 (7 CR/7 mice), Pulm-016 (10 CR/10 mice) and Gastric-010 (9 CR/9 mice). Conclusion: The treatment with PM060184 demonstrated significant in vivo antitumor activity in patient-derived xenografts of human NSCLC, pancreas, colon and gastric tumors. Citation Format: Manuel Hidalgo, M. Jose Guillen, Pedro Pablo Lopez-Casas, Francesca Sarno, Oscar Cataluna, Mandy Palomares, Carmen Cuevas, Pablo M. Aviles. In vivo antitumor activity of PM060184 in patient-derived xenografted tumors (avatar). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4534. doi:10.1158/1538-7445.AM2014-4534