Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR FMC63 as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.

Neuroblastoma is the most common extracranial solid tumour in children, representing about 7% of all malignancies under 15 years of age. Despite new treatment options, the median overall survival for high-risk patients is around 50%, and overall survival for patients with relapsed/refractory disease is dismal. Thus, new drugs and therapeutic strategies are urgently needed. Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy that has revolutionised the field of oncology, leading to dramatic improvement in the treatment of haematologic malignancies. Unfortunately, this success has not been replicated in solid tumours so far despite a huge research activity in this field. Recent data on CAR-T cells targeting GD2 in neuroblastoma patients demonstrated safety and remarkable tumour response.1 These results have created excitement for the use of CAR-T cells against neuroblastoma and the importance of identifying optimal cell-surface targets to increase CAR-T-cell efficacy. Targeting anaplastic lymphoma kinase (ALK) has emerged as one of the most promising and specific approaches for neuroblastoma therapy. Most neuroblastomas express the full-length ALK receptor,2, 3 and high expression of ALK correlates with poor prognosis.4 Activating mutations of full-length ALK are found in 8%−12% of neuroblastomas, including familial neuroblastoma, and ALK amplification is found in 2%−3% of neuroblastomas, increasing the risk of relapse.5 Several studies have demonstrated that activated ALK is an oncogenic driver in neuroblastoma, especially when associated with MYCN amplification.6 These findings provide a rationale to target ALK in neuroblastoma. Recently, promising clinical responses have been observed with the third-generation ALK inhibitor lorlatinib in patients with neuroblastoma carrying ALK genetic alterations.7 Also, antibody–drug conjugate directed to ALK demonstrated some efficacy against neuroblastoma in preclinical models.2 Initial approaches of targeting ALK with CAR-T cells using the ALK48 scFv-binding domain demonstrated limited preclinical efficacy owing to insufficient ALK target density.8 Recently, we demonstrated robust preclinical efficacy of an ALK-CAR created with a different scFv binder.3 ALK.CAR-Ts were as effective as GD2.CAR-Ts against a large panel of neuroblastoma lines. In vivo, ALK.CAR-T cells achieved 100% cure rate in a mouse model of human metastatic neuroblastoma with ALK amplification. In contrast, ALK.CAR-Ts and GD2.CAR-Ts were less potent against neuroblastoma tumours with lower ALK or GD2 expression, confirming the importance of antigen density for CAR-T-cell efficacy. These findings raise the challenge of targeting tumours with low antigen expression. We discovered a mechanism to increase the surface display of the ALK target on tumour cells. Treatment of neuroblastoma cells with ALK inhibitors enhanced total and surface expression of ALK, especially in ALK-mutated cells. The combination of lorlatinib and CAR-T cells selectively potentiated the efficacy of ALK.CAR-Ts but not of GD2.CAR-Ts, supporting the specificity and synergy of this combination (Figure 1). Mutated ALK receptors are mainly intracellular due to defective glycosylation, but regain cell-surface localisation upon kinase inactivation mediated by tyrosine kinase inhibitors (TKIs),9 enhancing susceptibility to ALK.CAR-Ts. Notably, while an increase in ALK expression was clearly detectable in neuroblastoma with ALK mutations, the enhanced killing by ALK.CAR-Ts was also observed in neuroblastoma with wild-type ALK. Processing and regulation of wild-type ALK remain unclear; however, TKIs can block the internalisation and degradation of the engaged receptor and block pathways involved in ALK transcription. Stratifying eligible neuroblastoma patients for ALK.CAR-T therapy could use the ALK genetic status and expression levels as a biomarker; nonetheless, the majority of neuroblastoma patients could benefit from this combination therapy irrespective of their ALK genetic status. Although the combination of lorlatinib with ALK.CAR-Ts enhanced anti-tumour activity, neuroblastoma tumours with low ALK expression were not fully eradicated. These residual tumoural cells may represent a clonal expansion or a subset of cells with very low ALK expression in which lorlatinib does not induce sufficient increase of surface expression. Reacquisition of ALK expression upon ex vivo cultivation suggested that strategies such as intermittent ALK TKI administration, higher doses, or enhanced cell surface stabilisation may further improve the efficacy of ALK.CAR-Ts. Importantly, limited ALK expression in healthy tissues is fundamental to avoid toxicities by ALK.CAR-T cell therapy. ALK expression in normal cells is restricted to developing organs in embryos and is believed to play a role in the regulation of neuronal differentiation during human embryogenesis. Analysis of mRNA expression profiles in an extended dataset of human tissues showed low levels of ALK transcripts in the hypothalamus, colon, pituitary gland, and testis.3, 10 However, comprehensive assessment of ALK expression in normal human tissues from healthy donors by immunohistochemistry showed low or undetectable ALK expression, apart from some staining in enteric neurons. The safety and specificity of ALK.CAR-Ts were testable in mouse models due to the cross-reactivity of ALK.CAR-Ts against both human and mouse ALK. Consistent with ALK limited expression in normal tissues, we did not observe detectable toxicities of ALK.CAR-Ts in mice. In keeping with this absence of toxicities, histology and immunohistochemistry for human CD3+ T cells in the hypothalamus and the enteric neurons in the intestinal wall did not show evidence of tissue damage or ALK.CAR-Ts accumulation. However, increased infiltration of T cells was observed in the small and large intestines of mice treated with lorlatinib together with ALK.CAR-Ts. These findings warrant for potential intestinal toxicity of lorlatinib-ALK.CAR-Ts co-administration, but they also underscore the likely on-target specificity of this approach. Importantly, the combination was well tolerated in mice. Similar intestinal infiltrates were observed in mice treated with GD2.CAR-Ts, and intestinal toxicity is manageable in patients treated with GD2.CAR-Ts. Therefore, there is suggestion that toxicity eventually arising with the combination of lorlatinib with ALK.CAR-Ts could be managed in patients. Notably, the rapid reversal of ALK expression within 12 h upon discontinuation of ALK TKI and its subsequent reoccurrence with lorlatinib treatment provides the option to suspend the drug to normalise ALK levels. This suggests that implementing treatment cycles with lorlatinib interspersed with wash-off periods could be a strategy to manage patients showing toxicity. Overall, these preclinical data support the implementation of a phase 1 clinical trial to test ALK.CAR-T cells in combination with lorlatinib in children with refractory/relapsed neuroblastoma. This trial will give us important information about the efficacy and safety of ALK.CAR-T cells and their combination with ALK inhibitors. Moreover, eventual positive results could be translated to other tumours expressing ALK, thus expanding the number of patients who could benefit from this treatment. Elisa Bergaggio and Roberto Chiarle conceived and wrote the manuscript. The figure was created with BioRender.com. E.B. and R.C. filed a patent covering the development of ALK.CAR-T cells.

Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have extended the survival of patients with ALK-rearranged cancers, including non-small cell lung cancer (NSCLC). Unfortunately, acquired resistance develops within 2-3 years, highlighting the urgent need for novel and effective therapeutic strategies for these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes against two human ALK immunogenic peptides we previously identified by mass spectrometry in biopsies from patients with ALK-rearranged NSCLC (PMID:37430060) and to develop TCR-T cell therapies for ALK-positive NSCLCs. Methods: BALB/c mice were vaccinated with the murine ALK peptide PGPGRVAKI and transgenic mice expressing human HLA-B*07:02 were vaccinated with human ALK peptides RPRPSQPSSL and IVRCIGVSL. All mice received two priming injections (days 1 and 14) followed by two boosters, and activated CD8+ T cells were sorted and subjected to single-cell sequencing. Results: As proof of concept, we first vaccinated BALB/c mice with the ALK peptide PGPGRVAKI and identified 381 unique ALK-specific clonotypes. Among these, we cloned the most expanded 5 clonotypes and confirmed that these clonotypes were functional as evidenced by the release of significant amounts of IFN-γ and IL2, and potent in vitro killing activity following co-culture with murine ALK-positive lung cancer cells. To identify TCR clonotypes for the development of TCR-T therapy in patients with ALK-positive cancers, we next vaccinated transgenic mice expressing human HLA-B*07:02 with human ALK peptides RPRPSQPSSL and IVRCIGVSL. We again identified multiple unique ALK-specific TCR clonotypes among CD8+/CD137+ cells from these transgenic mice vaccinated with RPRPSQPSSL peptide (N=353 TCR clonotypes) and IVRCIGVSL peptide (N=742 TCR clonotypes). Single-cell RNA sequencing of the expanded clonotypes (TCR clonotype frequency ≥4) versus non-expanded clonotypes (TCR clonotype frequency <4) revealed significant upregulation of Ccl3, Ccl4, Ccl1, Ifng, Xcl1, and Il13 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p <0.05). Gene set enrichment analysis (GSEA) confirmed significant upregulation of multiple pathways of adaptive immune response, including T cell activation, IFN-γ signaling and production, cytokine release, and T cell proliferation (adjusted p <0.05), suggesting functional activity of these TCR clonotypes against ALK. Conclusion: Here, we first demonstrated the feasibility of isolating ALK-specific TCR clonotypes by mice vaccination that exerted anti-tumor activity. Additionally, we identified ALK-specific TCR clonotypes from transgenic mice expressing human HLA-B*07:02 vaccinated with two human ALK peptides. This discovery lays the basis for the successful development of TCR-T cell therapies for ALK-positive NSCLCs, and possibly other ALK-positive cancers. Citation Format: Carmen Mecca, Ana Azambuja, Luca Alessandrí, Elisa Bergaggio, Simone Piane, Marcos Simoes-Costa, Ellis L. Reinherz, Rafael Blasco-Patiño, Roberto Chiarle. Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 21.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

Anaplastic lymphoma kinase ( ALK )-rearranged non-small cell lung cancer (NSCLC) is treated with ALK tyrosine kinase inhibitors (TKIs), but the lack of activity of immune checkpoint inhibitors (ICIs) is poorly understood. Here, we identified immunogenic ALK peptides to show that ICIs induced rejection of ALK + tumors in the flank but not in the lung. A single-peptide vaccination restored priming of ALK-specific CD8 + T cells, eradicated lung tumors in combination with ALK TKIs and prevented metastatic dissemination of tumors to the brain. The poor response of ALK + NSCLC to ICIs was due to ineffective CD8 + T cell priming against ALK antigens and is circumvented through specific vaccination. Finally, we identified human ALK peptides displayed by HLA-A*02:01 and HLA-B*07:02 molecules. These peptides were immunogenic in HLA -transgenic mice and were recognized by CD8 + T cells from individuals with NSCLC, paving the way for the development of a clinical vaccine to treat ALK + NSCLC.

Background Neuroblastoma is the most common extracranial solid tumor of childhood1 and accounts for 12-15% of cancer-related deaths in children.2 The survival of patients with refractory or relapsed neuroblastoma remains dismal.3 In neuroblastoma, chimeric antigen receptor (CAR) T cells against GD2 have shown encouraging clinical results, but relapses are associated with loss of antigen expression. The selection of the best target is critical for the therapeutic success of CAR-T cells in hematologic malignancies and solid tumors. The Anaplastic Lymphoma Kinase (ALK) receptor is expressed by most neuroblastoma while virtually absent in the majority of normal tissues. It is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. All these features render ALK a great candidate for CAR-T therapy. Methods We generated seven ALK.CAR constructs using the single-chain variable fragment derived from different anti-ALK monoclonal antibodies that recognize the ALK extracellular domain into a CAR construct that included the CD28 costimulatory endodomain. Their ability to target and kill was tested in vitro and in vivo against neuroblastoma cells expressing different intensities of ALK. The activity of ALK.CAR-T cells was compared to GD2.CAR-T cells. Results ALK CAR-T cells showed potent activity without on-target or off-target toxicity against neuroblastoma with high ALK expression. Combination with ALK inhibitors specifically potentiated the activity of ALK.CAR-T cells, but not GD2.CAR-T cell, against neuroblastoma with low ALK expression in cell lines and in a patient-derived xenograft (PDX), where the combination of ALK inhibitors with ALK.CAR-Ts significantly reduced tumor growth and extended mice survival. Mechanistically, ALK inhibitors impaired tumor growth and upregulated the expression of ALK, thereby improving the targeting of neuroblastoma tumors by ALK.CAR-T cells. Conclusions These data indicate that ALK. CAR-T cells are effective and safe as monotherapy against neuroblastoma with high ALK expression. Furthermore, treatment with ALK inhibitors increases the efficacy of ALK.CAR-T cells by enhancing ALK targeting. References Maris JM. Recent advances in neuroblastoma. The New England Journal of Medicine 2010; 362:2202-2011. Park JR, Eggert A, Caron H. Neuroblastoma: biology, prognosis, and treatment. Pediatr Clin North Am 2008;55:97-120. Irwin MS, Park JR. Neuroblastoma: paradigm for precision medicine. Pediatr Clin North Am 2015;62:225-256. Ethics Approval All mouse experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Boston Children's Hospital (Protocol 00001530).

The discovery of isocitrate dehydrogenases (IDHs) mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. More recently it has been suggested that the enzymatic inhibition of IDHs may have therapeutic potentials also for wild-type IDH cancers. Specifically, IDH2 inhibition can sensitize multiple myeloma cells to proteasome inhibitors. However, inhibitors directed against native IDHs are not present on the market. Here, we exploited an allosteric inhibitor of mutant IDH2 (AGI-6780), known to also decrease the activity of wild-type IDH2. Since AGI-6780 effectiveness in vivo is limited by its high hydrophobicity and very low bioavailability, the drug was loaded into mesoporous silica nanoparticles (MSNs) with the aim to enhance its efficacy. Furthermore, to enable high drug retention into the silica pores, improve biocompatibility, and reduce the off-target delivery of the drug, a Supported phosphoLipidic Bilayer (SLB) was self-assembled on the outer MSN surface. The silica nanoparticles were thus coated with three different lipid formulations and characterized in terms of structure, size, and morphology. We demonstrated that MSN@SLB nanoparticles have improved colloidal stability and hemocompatibility with respect to pristine MSN. We showed that MSN@SLB formulation displays an excellent loading and retention of the IDH2 inhibitor AGI-6780, with a limited drug leakage depending on the lipid formulation. Finally, we proved that AGI-6780-loaded MSN@SLB nanoparticles efficaciously inhibited the IDH2 enzymatic activity of multiple myeloma cells. Overall, this study provides a proof of concept of drug delivery to multiple myeloma cells by repurposing a neglected/dismissed drug (AGI-6780) with the use of smart nanoparticles and enabling the sensitization of multiple myeloma cells towards other possible treatments.

Abstract Neuroblastoma (NB) is the most deadly cancer in children with dismal survival in high-risk patients. The majority of NB express the full length anaplastic lymphoma kinase (ALK) receptor, that typically acts as driver oncogene together with MYCN. In contrast to ALK-driven lung cancer or lymphoma, targeted therapies with ALK tyrosine kinase inhibitors (TKIs), despite encouraging, induce only partial responses in NB. Therefore, additional tools to improve NB treatment are strongly needed. To specifically target NB cells, we developed a series of ALK chimeric antigen receptor (CAR) T constructs from antibodies that recognize both human and mouse ALK. Murine ALK CAR-T cells are able to control the growth of ALK+ leukemia and NB in syngeneic tumor models without detectable toxicity. From the leading candidate, we generated fully humanized ALK CAR-T cells that showed potent in vitro killing activity against a large panel of human NB lines, with activity comparable to GD2 CAR-T cells that are currently in clinical trials for NB patients. Remarkably, ALK CAR-T treatment synergized in vivo with the ALK inhibitor lorlatinib. Mechanistically, lorlatinib not only reduced tumor growth, but enhanced ALK expression on the surface of tumor cells, thereby facilitating ALK CAR-T targeting. Combination of ALK CAR-T cells with lorlatinib resulted in enhanced killing of NB cells and cure or markedly increased survival in mouse models of human metastatic NB even with low ALK expression. These findings support the clinical development of ALK CAR-T cells for NB therapy. Citation Format: Elisa Bergaggio, Wei-Tien Tai, Andrea Aroldi, Ineês Mota, Diego Alvarado, Elisa Landoni, Jasna Metovic, Manuel Nüesch, Rafael Blasco-Patiño, Mauro Papotti, Gianpietro Dotti, Roberto Chiarle. Generation of ALK CAR-T cells for neuroblastoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1544.

Isocitrate dehydrogenases (IDHs) are enzymes that catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (αKG) and CO2. The discovery of IDH1 and IDH2 mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. Here, we summarized findings addressing the impact of IDH mutants in rare pathologies and focused on the relevance of non-mutated IDH enzymes in tumors. Several pieces of evidence suggest that the enzymatic inhibition of IDHs may have therapeutic potentials also in wild-type IDH cancers. Moreover, IDHs inhibition could enhance the efficacy of canonical cancer therapies, such as chemotherapy, target therapy, and radiotherapy. However, further studies are required to elucidate whether IDH proteins are diagnostic/prognostic markers, instrumental for tumor initiation and maintenance, and could be exploited as targets for anticancer therapy. The development of wild-type IDH inhibitors is expected to improve our understanding of a potential non-oncogenic addition to IDH1/2 activities and to fully address their applicability in combination with other therapies.

Proteasome inhibitors (PI) are extensively used for the therapy of multiple myeloma (MM) and mantle cell lymphoma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. Here, to identify targets that synergize with PI, we carried out a functional screening in MM cell lines using a short hairpin RNA library against cancer driver genes. Isocitrate dehydrogenase 2 (IDH2) was identified as a top candidate, showing a synthetic lethal activity with the PI carfilzomib (CFZ). Combinations of US Food and Drug Administration-approved PI with a pharmacological IDH2 inhibitor (AGI-6780) triggered synergistic cytotoxicity in MM, mantle cell lymphoma, and Burkitt lymphoma cell lines. CFZ/AGI-6780 treatment increased death of primary CD138+ cells from MM patients and exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone marrow-derived stromal cells. Mechanistically, the CFZ/AGI-6780 combination significantly decreased tricarboxylic acid cycle activity and adenosine triphosphate levels as a consequence of enhanced IDH2 enzymatic inhibition. Specifically, CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), thus limiting IDH2 activation through the NAD+-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT or SIRT3 inhibitors impaired IDH2 activity and increased MM cell death. Finally, inducible IDH2 knockdown enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. Taken together, these findings indicate that NAMPT/SIRT3/IDH2 pathway inhibition enhances the therapeutic efficacy of PI, thus providing compelling evidence for treatments with lower and less toxic doses and broadening the application of PI to other malignancies.

The introduction of proteasome inhibitors (PIs) into the clinic has transformed the treatment of patients affected by multiple myeloma (MM) and mantle-cell lymphoma (MCL) establishing new standards of care. Despite these improvements, patients continuously relapse or are intrinsically resistant to PIs. Here, to identify druggable targets that synergize with PIs, we carried out a functional screening in MM cell lines using a short hairpin RNA library targeting 152 cancer driver genes, highly representative of all signaling pathways. The Isocitrate Dehydrogenase 2 (IDH2) gene was identified as a top candidate, showing a synthetic lethal activity with the PI Carfilzomib (CFZ). IDH2 is a NADP(+) dependent mitochondrial enzyme which catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate in the tricarboxylic acid (TCA) cycle. We demonstrated that combinations of the pharmacological IDH2 inhibitor AGI-6780 with FDA approved PIs significantly increased apoptotic cell death in ten MM cell lines, both sensitive and resistant to PIs. Combined treatments triggered synergistic cytotoxicity also in four MCL and in two Burkitt9s lymphoma cell lines. Importantly, CFZ/AGI-6780 treatment increased death of primary CD138-positive cells from nine MM patients and exhibited a favorable cytotoxicity profile towards peripheral blood mononucleated cells and bone marrow-derived stromal cells. Mechanistically, CFZ/AGI-6780 combination significantly decreased TCA cycle activity and ATP levels, as a consequence of enhanced IDH2 enzymatic inhibition. In contrast, only a slight increase of mitochondrial reactive oxygen species (ROS) was observed. CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme required for IDH2 activation through the NAD(+)-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT (FK866) or SIRT3 (AGK7) inhibitors impaired IDH2 activity and increased MM cell death, thus phenocopying CFZ/AGI-6780 effects and putting the proteasome in a direct link with IDH2 inhibition. Finally, inducible IDH2 knock-down enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. In conclusion, our data demonstrate that IDH2 inhibition increases the therapeutic efficacy of PIs, thus providing compelling evidence for treatments with lower and less toxic doses, and broadening the application of PIs to other malignancies. Citation Format: Elisa Bergaggio, Chiara Riganti, Giulia Garaffo, Elisabetta Mereu, Nicoletta Vitale, Cecilia Bandini, Elisa Pellegrino, Paola Omede, Katia Todoerti, Valentina Audrito, Antonio Rossi, Francesco Bertoni, Silvia Deaglio, Antonino Neri, Antonio Palumbo, Roberto Piva. IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies [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 LB-269.

Recent studies reported the expression of anaplastic lymphoma kinase (ALK) in malignant melanomas. The aim of this study was to investigate whether ALK expression is associated with specific clinical and molecular characteristics of melanoma metastases, and to evaluate its correlation with survival outcomes. Seventy-one patients with metastatic melanoma were investigated. Clinical features and survival outcomes were analyzed and correlated to ALK expression, as detected by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction, and to the mutational status of BRAF, KRAS, NRAS, and PIK3CA. No translocations or ALK alternative isoforms were identified. ALK expression was mainly detected in NRAS mutated metastatic lesions. Interestingly, among NRAS-mutated patients, ALK positive samples displayed a significantly more favorable outcome in terms of disease specific survival, as compared to ALK negative ones. In conclusion, we suggest that ALK positive/NRAS mutated metastases represent a specific subset of metastatic melanomas, associated with a better prognosis. Validation of these observations in larger cohorts could contribute to understand the molecular events cooperating to melanoma progression, in addition to open new perspectives in the clinical and therapeutic management of this subgroup of patients.

Identification of a new subclass of ALK-negative ALCL expressing aberrant levels of ERBB4 transcripts Irene Scarfò,* Elisa Pellegrino,* Elisabetta Mereu,* Ivo Kwee, Luca Agnelli, Elisa Bergaggio, Giulia Garaffo, Nicoletta Vitale, Manuel Caputo, Rodolfo Machiorlatti, Paola Circosta, Francesco Abate, Antonella Barreca, Domenico Novero, Susan Mathew, Andrea Rinaldi, Enrico Tiacci, Sara Serra, Silvia Deaglio, Antonino Neri, Brunangelo Falini, Raul Rabadan, Francesco Bertoni, Giorgio Inghirami, Roberto Piva, and the European T-Cell Lymphoma Study Group

Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5' RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5' untranslated regions.

Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily diagnosed, no specific markers and molecular events leading to ALK- ALCL transformation have been identified so far.

Anaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK(+) and ALK(-) systemic forms. Whereas ALK(+) ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK(-) ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK(-) ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK(-) ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols.