Search

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient (MMRp) and unresponsive to immunotherapy, while MMR deficient (MMRd) tumors often respond to immune checkpoint blockade (ICB). We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB) and, sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR proficient and KRAS mutant mCRC patients receive priming therapy with TMZ. Analysis of solid tissue biopsies and circulating tumor DNA (ctDNA) obtained after TMZ treatment revealed the emergence of TMZ mutational signature, alterations in MMR genes and increased TMB in 14 out of 16 patients. Genetic mutations induced by TMZ were dose-dependent and multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes such as the MSH6 T1219I variant which was detected in ctDNA and tissue of 13/14 (93%) of the cases. A subset of the patients whose tumors after TMZ priming displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment. Overall, we provide proof-of-concept that treatment of MGMT deficient/MMR proficient KRAS mutant mCRCs with TMZ can be tracked by mutational signature analysis and lead to inactivation of the MMR pathway, emergence of the TMZ mutational signature, TMB increase, and, in some cases, to disease stabilization during ICB. Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Alessio Amatu, Marco Macagno, Ludovic Barault, Andrea Cassingena, Alice Bartolini, Paolo Luraghi, Gianluca Mauri, Paolo Battuello, Nicola Personemi, Valeria Pessei, Pietro Paolo Vitiello, Federica Tosi, Laura Idotta, Emanuele Valtorta, Emanuela Bonoldi, Giovanni Germano, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6262.

Introduction: Mismatch repair (MMR) protein expression in colorectal cancer (CRC) is usually homogeneously retained or lost. However, rare CRC lesions may show a heterogeneous pattern of MMR protein expression. We aimed to evaluate the molecular effect of a heterogeneous expression of the MMR proteins in a complex context of concomitant loss of one of the other effectors of the DNA repair mechanism. Experimental procedures: We evaluated MMR protein expression (by immunohistochemistry -IHC- for MLH1, MSH2, MSH6 and PMS2) in 200 CRCs, identifying 3 groups with proficient (MMRp), deficient (MMRd) and heterogeneous (MMRh) MMR. MMRh tumors were micro-dissected based on the expression of the heterogeneous marker, DNA was extracted and subjected to targeted sequencing (TSO500 panel, Illumina). RNA was purified from bulk tumors of all MMRh cases and in a control series of 10 MMRp and 10 MMRd CRCs and analyzed using the IO360 panel (NanoString). The degree of tumor infiltrating lymphocytes (TILs) was calculated on H&E slides and on IHC sections stained with antibodies raised against CD8. Results: Twenty-nine out of 200 cases were MMRd (14.5%). Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, resulting in a microsatellite instable (MSI) phenotype. Five out of the 9 cases were suitable for separate mutational analysis of IHC-positive and IHC-(double)negative components of the tumor. Both components of all the lesions exhibited a high tumor mutation burden (mean TMB: 59 mut/Mb) in line with the MSI status, nevertheless a significant increase in TMB in the double-negative components was observed (mean TMB: neg. 67 mut/Mb vs pos. 53 mut/Mb), due to a higher number of sub-clonal, non-synonymous variants compared to the positive component. Comparative gene expression analyses between MMRd, MMRp and MMRh CRCs highlighted differential gene expression patterns, with MMRh tumors displaying a strong activation of angiogenesis, MAPK and PI3K-AKT axes. Moreover, these tumors showed the highest number of TILs, characterized by a substantial population of exhausted CD8+ lymphocytes. Conclusions: We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins, in a background of concomitant loss of one of the other markers. This heterogeneity is associated with i) an increase of TMB and of unstable loci in the negative component, with additional markedly sub-clonal variants, ii) a high number of TILs, mostly exhausted CD8+ lymphocytes, and iii) a differential activation of signaling pathways. Taken together these data provide indirect evidence that heterogeneous loss of MMR proteins may impact on the biology of CRCs. Whether this pattern may influence response to immune checkpoint inhibition remains to be determined. Citation Format: Enrico Berrino, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, Anna Sapino, Silvia Marsoni, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Emanuela Bonoldi, Caterina Marchio. A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5419.

Background. Metastatic colorectal cancer (CRC) remains mostly incurable, with a survival of about two years only. It has been recently proved that CRCs with genetic defects in the mismatch-repair pathway (MMRd), occurring in 15% of early CRC but only in 5% of metastatic CRC, present with a high tumor mutational burden (TMB), which results in an increased number of neoantigens that can be recognized by the immune system. Indeed, treatment with the anti-programmed cell death protein 1 (PD-1) immune checkpoint inhibitor pembrolizumab or nivolumab is effective in inducing durable objective responses in metastatic CRC MMRd cases. These results are quite remarkable considering that the clinical efficacy was independent from RAS mutations, which constrain the use of targeted treatments and negatively affect prognosis. We recently showed in preclinical models that the pharmacological treatment with temozolomide (TMZ) can induce the inactivation of MMR genes and consequently trigger an increase in immunogenic neoantigens. This suggests that TMZ could be used to prime MMR proficient (MMRp) tumors for response to checkpoint inhibitors. Accordingly, mCRC patients recruited in previous clinical trials where TMZ was administered, acquired alterations of MMR genes upon treatment and showed remarkable increase in TMB at disease progression (PD). We thus designed the ARETHUSA clinical trial to test whether a priming course with TMZ in patients can sensitize mCRC to the anti-PD1 inhibitor pembrolizumab. Methods. Arethusa is a 2-cohorts, phase II trial consisting of three different phases (NCT03519412). During screening-phase, 344 mCRC patients with RAS-extended mutations who failed standard therapies will be tested for MMR status. MMRd CRC patients will proceed directly to trial-phase for immediate pembrolizumab treatment (expected N=14). MMR-proficient (MMRp) patients will be further tested for TMZ sensitivity via assessment of expression of O6-methylguanine-DNA methyltransferase (MGMT) by immunohistochemistry and by promoter methylation analysis. Expected 67 IHC-negative, promoter methylation-positive MMRp patients will thus be eligible for priming-phase and will receive TMZ until PD; TMB will then be assessed on tumor biopsies at resistance. Those patients that will have >20 mutations/megabase (expected N=20) will proceed to that trial-phase and will be treated with pembrolizumab. Overall response rate (primary outcome), Progression Free, and Overall Survival, and treatment related toxicities (secondary outcomes) in MMRp pembrolizumab-treated patients will be estimated. Treatment efficacy and toxicity within pembrolizumab-treated MMRd cohort will be used for comparison. Pre- and post-TMZ biopsies and longitudinal blood and stool collection during priming and trial phases will allow for discovery of predictive molecular markers and for the assessment of integrated tumor and (immune)environment evolution in response to therapy. Citation Format: Silvia Marsoni, Giovanni Germano, Andrea Sartore Bianchi, Filippo Pietrantonio, Nicola Personeni, Alessio Amatu, Emanuela Bonoldi, Emanuele Valtorta, Ludovic Barault, Federica Di Nicolantonio, Filippo de Braud, Lorenza Rimassa, Armando Santoro, Silvia Ghezzi, Andrea Cassingena, Giovanna Marrapese, Loredana Lupica, Giulia Siravegna, Giuseppe Rospo, Cosimo Martino, Luca Lazzari, Paolo Luraghi, Nabil Amirouchene-Angelozzi, Alberto Bardelli, Salvatore Siena. Pharmacological inactivation of DNA repair to improve response to immunotherapy: The Arethusa trial in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT215.

Background The overall survival of mCRC patients has been increased by the availability of new cytotoxic and targeted agents and today potentially by the advent of immunotherapies. However, the impact of these advances has been incremental rather than transformative, and a number of unmet medical needs still await rational solutions. In order to navigate the co-evolutionary pathways of CRC tumors during their natural history and under the Darwinian pressure of therapies, we need to feed the experimental laboratories with “the right sample, at the right time, for the right experiment”. This can be accomplished through the design of master protocols that represent a new subset of observational trials aimed to empower the bi-directional collaboration between pre-clinical and clinical research, an essential prerequisite to feed and implement precision oncology. Methods AlfaΩmega (protocol number IFOM-CPO003/2018/PO002) has been designed to streamline the study of the co-evolutionary landscape between tumor and host cells in a stage-mixed cohort of at least 500 CRC patients, with the aim of understanding how their outcomes can be significantly improved. This resource for integrative clinical data and sample collection will allow the molecular story-telling of CRC metastatic spread along time and space and the selection of appropriate patients for experimentally-driven trials. To achieve the required level of ‘experimental precision’, patients can enter AlfaΩmega at two different ‘therapeutic checkpoints’: i) prior to a surgical event or ii) prior to a systemic treatment. Moreover, to optimize the enrollment of patients, the longitudinal collection of data/samples and their logistic management, AlfaΩmega has been designed as a flexible infrastructure organized in TIERs, each of which represents a building block for the stepwise comprehension of the biological processes that drive tumor evolution and that will have an independent informed consensus process: § TIER1, Monitoring: the ability to follow CRC evolution under standard of care treatments and to define new evolution-linked biomarkers: access to clinical & imaging data, FFPE, plasma and PBMCs. TIER1 informed consent is mandatory for the enrolment. § TIER2, Modelling: the ability to develop pertinent experimental models to study evolutionary mechanisms and define evolution-targeting therapeutic strategies: access to fresh tissue, blood, stools, buccal swabs and other fluids. Sample collection in TIER2 is discretionary, i.e. may not be applied to all patients entering TIER1. § TIER3, Linking: the ability to access data and samples of patients enrolled in proof-of-concept trials to prove the efficacy and study/understand resistance mechanisms of evolution-targeting therapies: linking with AlfaΩmega logistic backbone and IT architecture. Citation Format: Luca Lazzari, Paolo Luraghi, Nabil Amirouchene-Angelozzi, Andrea Bertotti, Enzo Medico, Federica Di Nicolantonio, Fabrizio d'Adda di Fagagna, Sergio Abrignani, Daniele Regge, Anna Sapino, Emanuela Bonoldi, Angelo Vanzulli, Vittorina Zagonel, Filippo de Braud, Livio Trusolino, Alberto Bardelli, Salvatore Siena, Silvia Marsoni. AlfaOmega- a master protocol empowering precision research in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT214.

Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to Sézary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance. [Cancer Res 2008;68(17):7137–46]