Cosimo Martino

Dual-targeted therapy with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (HERACLES): a proof-of-concept, multicentre, open-label, phase 2 trial

BACKGROUND We previously found that dual HER2 blockade with trastuzumab and lapatinib led to inhibition of tumour growth in patient-derived xenografts of HER2-amplified metastatic colorectal cancer. In this study, we aimed to assess the antitumour activity of trastuzumab and lapatinib in patients with HER2-positive colorectal cancer. METHODS HERACLES was a proof-of-concept, multicentre, open-label, phase 2 trial done at four Italian academic cancer centres. We enrolled adult patients with KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive metastatic colorectal cancer refractory to standard of care (including cetuximab or panitumumab), an Eastern Cooperative Oncology Group performance status of 0 or 1, and at least one measurable lesion. We defined HER2 positivity in tumour samples by use of immunohistochemistry and fluorescence in-situ hybridisation in accordance with our previously validated colorectal cancer-specific diagnostic criteria. Eligible patients received intravenous trastuzumab at 4 mg/kg loading dose followed by 2 mg/kg once per week, and oral lapatinib at 1000 mg per day until evidence of disease progression. The primary endpoint was the proportion of patients achieving an objective response (defined as complete response or partial response), which was assessed by independent central review in the intention-to-treat population. This trial is registered with EudraCT, number 2012-002128-33. FINDINGS Between Aug 27, 2012, and May 15, 2015, we screened 914 patients with KRAS exon 2 (codons 12 and 13) wild-type metastatic colorectal cancer and identified 48 (5%) patients with HER2-positive tumours, although two died before enrolment. Of these patients, 27 were eligible for the trial. All were evaluable for response. At the time of data cutoff on Oct 15, 2015, with a median follow-up of 94 weeks (IQR 51-127), eight (30%, 95% CI 14-50) of 27 patients had achieved an objective response, with one patient (4%, 95% CI -3 to 11) achieving a complete response, and seven (26%, 95% CI 9-43) achieving partial responses; 12 (44%, 95% CI 25-63) patients had stable disease. Six (22%) of 27 patients had grade 3 adverse events, which consisted of fatigue in four patients, skin rash in one patient, and increased bilirubin concentration in one patient. No grade 4 or 5 adverse events were reported. We detected no drug-related serious adverse events. INTERPRETATION The combination of trastuzumab and lapatinib is active and well tolerated in treatment-refractory patients with HER2-positive metastatic colorectal cancer. FUNDING Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus, and Roche.

Assessment of a HER2 scoring system for colorectal cancer: Results from a validation study

We sought to develop criteria for ERBB2-positivity (HER2) in colorectal cancer to ensure accurate identification of ERBB2-amplified metastatic colorectal cancer patients suitable for enrolment in a phase II trial of ERBB2-targeted therapy (HERACLES trial). A two-step approach was used. In step 1, a consensus panel of pathologists adapted existing protocols for use in colorectal cancer to test ERBB2 expression and amplification. Collegial revision of an archival test cohort of colorectal cancer samples led to specific recommendations for adapting current breast and gastric cancer criteria for scoring ERBB2 in colorectal cancer. In step 2, from September 2012 to January 2015, colorectal-specific ERBB2 testing protocols and ERBB2 scoring criteria were used to centrally screen for ERBB2-positive KRAS wild-type colorectal cancer patients to be enrolled in the HERACLES trial (clinical validation cohort). In both archival test (N=256) and clinical validation (N=830) cohorts, a clinically sizeable 5% fraction of KRAS wild-type colorectal cancer patients was found to be ERBB2-positive according to the colorectal cancer-specific ERBB2 scoring criteria. ERBB2-positive tumors showed ERBB2 immunostaining consisting of intense membranous ERBB2 protein expression, corresponding to homogenous ERBB2 amplification, in >50% of cells. None of the immunohistochemistry 0 or 1+ cases was amplified. Concordance between SISH and FISH was 100%. In conclusion, we propose specific criteria for defining ERBB2-positivity in colorectal cancer (HERACLES Diagnostic Criteria). In a phase II trial of trastuzumab and lapatinib in a cetuximab-resistant population, HERACLES Diagnostic Criteria shaped the selection of patients and defined ERBB2 as a predictive marker for response to ERBB2-targeted therapy in metastatic colorectal cancer.

The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C

The cellular apoptosis susceptibility gene CAS/CSE1L is overexpressed in cancer, although it was originally identified as a gene that renders cells vulnerable to apoptotic stimuli. CAS/CSE1L has roles in the nucleocytoplasmic recycling of importin‐α and in the regulation of gene expression, cell migration, and secretion. We identified CAS/CSE1L as a survival factor for ovarian cancer cells in vitro and in vivo. In 3/3 ovarian cancer cell lines, CAS/CSE1L was down‐modulated by the unorthodox proapoptotic signaling of the MET receptor. CAS/CSE1L knockdown with RNA interference committed the ovarian cancer cells to death, but not immortalized normal cells and breast and colon cancer cells. In 70 and 95% of these latter cells, respectively, CAS/CSE1L was localized in the cytoplasm, while it accumulated in the nucleus in >90% of ovarian cancer cells. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells. In the nucleus, CAS/CSE1L regulated the expression of the proapoptotic Ras‐association domain family 1 gene products RASSF1C and RASSF1A, which mediated death signals evoked by depletion of CAS/CSE1L. Our data show that CAS/CSE1L protects ovarian cancer cells from death through transcriptional suppression of a proapoptotic gene and suggest that the localization of CAS/CSE1L dictates its function.—Lorenzato, A., Martino, C., Dani, N., Oligschläger, Y., Ferrero, A. M., Biglia, N., Calogero, R., Olivero, M., Di Renzo, M. F. The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C. FASEB J. 26, 2446‐2456 (2012). www.fasebj.org

Fumarase tumor suppressor gene and MET oncogene cooperate in upholding transformation and tumorigenesis

Loss of the fumarate hydratase (FH) tumor suppressor gene results in the development of benign tumors that rarely, but regrettably, progress to very aggressive cancers. Using mouse embryo fibroblasts (MEFs) to model transformation, we found that fh knockdown results in increased expression of the met oncogene‐encoded tyrosine kinase receptor through hypoxia‐inducible factor (hif) stabilization. MET‐in‐creased expression was alone able to stabilize hif, thus establishing a feed forward loop that might enforce tumor progression. The fh‐defective MEFs showed increased motility and protection from apoptosis. Motility, but not survival, relied on hif‐1α and was greatly enhanced by MET ligand hepatocyte growth factor. Met cooperated with a weakly oncogenic ras in making MEFs transformed and tumorigenic, as shown by in vitro and in vivo assays. Loss of fh was not equally effective by itself but enhanced the transformed and tumorigenic phenotype induced by ras and MET. Consistently, the rescue of fumarase expression abrogated the motogenic and transformed phenotype of fh‐defective MEFs. In conclusion, the data suggest that the progression of tumors where FH is lost might be boosted by activation of the MET oncogene, which is able to drive cell‐autonomous tumor progression and is a strong candidate for targeted therapy.—Costa, B., Dettori, D., Lorenzato, A., Bardella, C., Coltella, N., Martino, C., Cammarata, C., Carmeliet, P., Olivero, M., Di Renzo, M. F. Fumarase tumor suppressor gene and MET oncogene cooperate in upholding transformation and tumorigenesis. FASEB J. 24, 2680–2688 (2010). www.fasebj.org

Abstract CT005: Final results of the HERACLES trial in HER2-amplified colorectal cancer

Background: HER2 amplification is found in 5% of RAS wild type (RASWT) metastatic colorectal cancer (mCRC). Dual HER2 blockade with trastuzumab (T) and lapatinib (L), but not treatment with either drug alone, led to remarkable inhibition of tumor growth in patient-derived tumorgrafts of HER2-amplified mCRC. CRC-specific criteria for HER2 positivity by immunohistochemistry (IHC) and in situ hybridization (ISH) were defined retrospectively in 256 CRC paraffin embedded samples (HERACLES DGX criteria). The ensuing diagnostic algorithm was utilised to screen 1299 HER2-positive tumors for therapeutic targeting in patients in the HERACLES phase 2 trial. Methods: HERACLES was conducted at 4 Italian centres. Eligibility criteria were: RASWT exon 2, HER2 positivity, refractoriness to standard of care (including cetuximab or panitumumab), PS-ECOG 30% with a one-sided alpha level of 0.05 (EudraCT, number 2012-002128-33). Findings: Between August 27, 2012, and December 31, 2016, 69/1299 (5.3%) RASWT PTS were found HER2-positive. Of these, 33 were enrolled in HERACLES, and evaluable for response. At data cut-off (December 31, 2016), 10 (30.3%, 95% CI 17-47) of 33 heavily refractory PTS (median 5 prior regimens), achieved an objective response (2 complete and 8 a partial responses), 13 (39.3%, 95% CI 24-56) of 33 PTS had stable disease (SD) for a disease control rate of 70% (95% CI 52-82). Toxicity was mild with six (18%) of 33 PTS experiencing grade-3 side effects: fatigue (4), skin rash (1) elevated bilirubin (1). No drug-related SAEs were observed. Interpretation: HERACLES results prove that targeting HER2 is a successful therapeutic strategy in treatment-refractory HER2-positive mCRC patients, which can be easily implemented in clinical practice. Funding: Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus and Roche. Citation Format: Salvatore Siena, Andrea Sartore-Bianchi, Livio Trusolino, Cosimo Martino, Katia Bencardino, Sara Lonardi, Vittorina Zagonel, Francesco Leone, Erika Martinelli, Fortunato Ciardiello, Patrizia Racca, Alessio Amatu, Laura Palmeri, Emanuele Valtorta, Silvia Ghezzi, Angelo Vanzulli, Daniele Regge, Silvio Veronese, Alberto Bardelli, Silvia Marsoni. Final results of the HERACLES trial in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT005. doi:10.1158/1538-7445.AM2017-CT005

Abstract CT082: Pertuzumab and trastuzumab-emtansine in HER2-positive colorectal cancer: the HERACLES B trial

Rationale: The HERACLES A trial showed that a clinical sizeable subset of 3% of metastatic colorectal cancers (mCRC), identifiable by the amplification of the HER2 gene, can be targeted for effective, durable and safe anti-HER2 treatment with a combination of the small molecule kinase inhibitor lapatinib (L) and the monoclonal antibody trastuzumab (T) [Siena et al. J Clin Oncol 33, 2015.(suppl; abs 3508)]. As with any other targeted therapy, however, the L+T combination was not active in all patients, and even in responders its efficacy was limited in time by the engagement of growth-promoting cues that compensate for inhibition of the targeted kinase, eventually leading to disease progression. In both primary and secondary resistant HERACLES A patients the amplification of the HER2 gene was nevertheless present in more than 50% of the tumor cells. We reasoned that this finding could be exploited by adding to the anti HER2 strategy a ‘precision chemotherapy’ component, embodied by the HER2 antibody-drug conjugate trastuzumab-emtansine (T1). To this end, we conducted a preclinical trial in CRC0186, the only patient-derived xenograft (PDX) of our HER2+ mCRC PDX collection (N = 7), showing tumor inhibition rather than tumor shrinkage after L+T treatment. Six mice per arm were randomized to either control, pertuzumab (P), T1 or P+T1. Optimal tumor shrinkage, mirroring clinical complete response, was only observed in the T1 and the P+T1 arms, and persisted after treatment stop only in the latter arm (Bertotti, unpublished data). Notably, this post treatment response was not observed in comparable experiments testing the L+T or P+T combinations in more sensitive HER2+ PDXs (Bertotti et al., Cancer discovery 2011; Leto et al., Clin Cancer Res 2015). In light of the above results we designed the HERACLES B trial. Trial design: HERACLES B is an independent phase II trial of P+T (accrual starting March 2016). Response rate is the primary endpoint, progression free survival the secondary. Sample size was calculated according to the Fleming & A’Hern one stage design under the following assumptions: H0 = RR 10%, H1 = RR 30%. With a = 0.05 and β = 85%, we need to accrue 27 patient and observe ? 6 responses to declare the study positive. The clonal evolution of each patient tumor9s is monitored via fortnightly liquid biopsies. Eligibility criteria: mCRC patients HER2+ according to HERACLES criteria (Valtorta E. et al, Modern Pathology, 2015) and wild type for KRAS, NRAS and BRAF; prior treatment with fluoropirimidines, oxaliplatin, and irinotecan containing regimens, ± anti EGFR or antiangiogenic treatment; measurable disease (RECIST v1.1), ECOG PS ?1, and adequate organ function. Treatment: Pertuzumab (840 mg/kg iv load followed by 420mg/kg IV) in combination with TDM1 (3,6 mg/kg) both q3wks. EudraCT number: 2012-002128-33; Funded by AIRC Grant # 9970. Citation Format: Livio Trusolino, Andrea Bertotti, Sara Lonardi, Andrea Sartore-Bianchi, Cosimo Martino, Francesca Cottino, Valentina Vurchio, Emanuele Valtorta, Calogero Lauricella, Daniele Regge, Angelo Vanzulli, Vittorina Zagonel, Francesco Leone, Patrizia Racca, Fortunato Ciardiello, Andrea Ardizzoni, Silvia Marsoni, Salvatore Siena. Pertuzumab and trastuzumab-emtansine in HER2-positive colorectal cancer: the HERACLES B trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT082.

Daily administration of low molecular weight heparin increases Hepatocyte Growth Factor serum levels in gynaecological patients: pharmacokinetic parameters and clinical implications

BackgroundHepatocyte Growth Factor (HGF) enhances cytotoxicity of paclitaxel (PTX) and cisplatin (CDDP) in human ovarian cancer cells. Because of potential pitfalls of HGF exogenous administration, we investigated whether HGF serum concentration might be alternatively raised in vivo by administering low molecular weight heparin (LMWH).MethodsThe main HGF pharmacokinetic parameters were evaluated following acute and chronic LMWH treatment. First, women, operated on for gynaecological tumors, were treated with a single dose of calcium nadroparin and studied for 12 hours. Next, women operated on for benign or malignant gynaecological tumors were treated daily with calcic nadroparin for one month. Subsequently, the biological activity of the measured HGF serum levels was tested in assays of ovarian cancer cell sensitization to drugs.ResultsIn the short-term treated group, median HGF AUCss, Cmax and Caverage were about four-fold that of the control group, whereas Cmin was three-fold. In the patients treated chronically median HGF serum levels rose about six-fold in the first week, and decreased but remained significantly higher after one month. The pharmacokinetic of nadroparin-dependent HGF increase were similar in the two groups. The HGF concentrations measured after both acute and chronic treatment were found to be effective in sensitising ovarian cancer cells to chemotherapeutics.ConclusionsThis study raises the possibility of using LMWH to increase HGF serum concentration and to take advantage of its biological activities. In particular, nadroparin might be used as a chemo-potentiating agent in epithelial cell ovarian carcinoma through its action on HGF serum concentration.Trial registrationClinicalTrials.gov ID: NCT01523652

Abstract 2848: Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer

Targeting HER2 with trastuzumab and lapatinib is effective in ERBB2 amplified metastatic colorectal cancer (mCRC). Although at least 30% of the patients initially respond, secondary resistance occurs in most of the cases. Since the drivers of secondary resistance to trastuzumab and lapatinib in ERBB2 amplified mCRC are unknown, we exploited longitudinal plasma collections and patient-derived cell models to define the molecular bases of resistance to HER2 blockade. Levels of ERBB2 amplification in plasma circulating tumor DNA (ctDNA) paralleled response and relapse. The emergence of EGFR, ERBB2, RAS, BRAF and PIK3CA variants in ctDNA was associated with resistance. Radiographic measurements of individual metastases coupled with longitudinal liquid biopsies unveiled lesion-specific patterns of heterogeneous response in several patients. Phylogenetic tracking and functional analyses on tissue samples and patient-derived cell models established from eight metastases of a single case revealed new druggable oncogenic dependencies and genomic evolution associated with resistance. These data highlight the relevance of coupling imaging and liquid biopsies analyses in precision oncology and provide the rationale for additional lines of therapies in HER2 positive mCRC relapsing upon HER2 blockade. Citation Format: Giulia Siravegna, Luca Lazzari, Andrea Sartore-Bianchi, Giovanni Crisafulli, Benedetta Mussolin, Andrea Cassingena, Cosimo Martino, Richard Lanman, Rebecca Nagy, Giorgio Corti, Alice Bartolini, Pamela Arcella, Monica Montone, Francesca Lodi, Alice Vanzati, Emanuele Valtorta, Giovanni Cappello, Andrea Bertotti, Sara Lonardi, Vittorina Zagonel, Francesco Leone, Mariangela Russo, Antonella Balsamo, Mauro Truini, Federica Di Nicolantonio, Alessio Amatu, Erica Bonazzina, Silvia Ghezzi, Daniele Regge, Angelo Vanzulli, Livio Trusolino, Salvatore Siena, Silvia Marsoni, Alberto Bardelli. Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2848.

Radiologic and Genomic Evolution of Individual Metastases during HER2 Blockade in Colorectal Cancer

Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.

Abstract 215: CSE1L as a potential target to sensitize ovarian cancer cells to cisplatin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Human ovarian cancer is frequently (70% of cases) at advanced stage at presentation and requires multimodality treatment, consisting of cytoreductive surgery and chemotherapy based on platinum-drugs and taxanes. Patients are initially responsive to this treatment, but, in most cases, experience disease relapse because tumour cells acquire drug resistance. We demonstrated that the Hepatocyte Growth Factor (HGF) sensitizes to cisplatin (CDDP) and taxol an ovarian carcinoma cell line known to be resistant to platinum drugs: SK-OV-3, leading to an increased cancer cell death at very low drug doses both in vitro and in an animal model (Bardella et al., 2007; Rasola et al., 2004). This was an unexpected finding as HGF and its tyrosine kinase receptor, encoded by the MET oncogene, drives cell proliferation, survival and invasiveness during development and tumorigenesis. We also demonstrated that this effect is mediated by p38MAPK, which overwhelms the concomitant HGF activation of survival pathways, as the expression of a dominant negative form of p38MAPK abrogates the sensitization effect of HGF (Coltella et al., 2006). Using oligonucleotide microarrays, we studied the transcriptional responses of cell treated with CDDP in the presence or in the absence of HGF, and found that HGF pre-treatment modifies the transcriptional response to CDDP not only in SK-OV-3, but also in NIH-OVCAR-3, and TOV-21G ovarian cancer cells. The up- or down-modulation of the most differentially expressed genes found in common within these cell lines was reverted when the cells expressed a dominant negative form of p38MAPK, upholding once again the involvement of p38MAPK in this phenomenon. Among the top-ranked differentially expressed genes, which we identified, we focused functional studies on CSE1L/CAS. This gene is a nuclear transporter involved in both proliferation and apoptosis found to be amplified and over-expressed in ovarian cancer. Knocking down of CSE1L/CAS made ovarian cancer cells sensitized to low doses of cisplatin and triggered apoptosis while had no effect on other cancer and normal cell lines. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or to revert chemotherapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 215.