Integrated Omics to Dissect the Landscape in Chemosensitive and Refractory Extranodal Natural-Killer T-Cell Lymphoma (ENKTCL): A Global Study of the PETAL (PEripheral T-Cell Lymphoma) Consortium
Authors: Fernandez Turizo Maria J. J. , Nam Yoonhee , Lenart Alexandra W , McCabe Sean M , Rider Anna B , Sohani Aliyah R. , MacVicar Caroline , Chopra Kusha , Ford Josie G , Merrill Mwanasha H. , Jacobsen Eric D. , Inghirami Giorgio , Cabrera Maria Elena , Villegas Pablo , Eipe Thomas , Shet Tanuja , Jain Hasmukh , Sengar Manju , Kim Won-Seog , Cho Junhun , Yoon Sang Eun , Malpica Castillo Luis Enrique , Beltran Brady , Castro Denisse , Quinones Pilar , Vasquez Jule F , Barrionuevo Carlos , Duenas Daniela E. , Leautaud Veronica , Rabadan Raul , Jain Salvia 
Published: December 2024

Background: The molecular mechanisms underlying sensitivity or resistance to chemotherapy in ENKTCL remain elusive. To address this issue, we assembled a topographically diverse series of cases with varying responses to chemotherapy from various international sites including the United States (US, n=27), Chile (n=6), India (n=5), Peru (n=5), and South Korea (n=6). We included a total of 49 cases and 18 controls with a median age of 50 years (IQR, 48-61.5) with the nasal cavity (n=26) as the primary site of involvement. From the available clinical information, there are 19 female and 17 male cases. Among the cases with sufficient clinical information to calculate PINK scores (n=20), 14 were high risk (4 from Chile, 4 from Peru, and 6 from the US), 3 were intermediate risk (1 from Chile and 2 from the US), and 3 were low risk (1 from Chile and 2 from the US). Frontline chemotherapy regimens included CHOP/CHOEP (n=4), SMILE (n=8), DeVIC (n=9), palliative radiation (n=1), GELOX (n=1), P-GemOx (n=1), and Cisplatinum (n=1). Regarding response to chemotherapy, 18 patients were classified as chemosensitive, and 17 as refractory. The response status is being collected for the remaining patients. Following first-line therapy, 10 patients underwent radiation and 1 received consolidative allogeneic SCT. For second-line, regimens included Ifosfamide-Methotrexate-Etoposide-Prednisone (n=1), Brentuximab Vedotin (n=1), SMILE (n=2), DeVIC (n=2), Pembrolizumab (n=1), GemOX with ruxolitinib and asparaginase (n=1), and GemOx plus dexamethasone (n=1). One patient received auto-SCT after second-line treatment. Third-line treatments included romidepsin (n=1), cytotoxic T-lymphocytes on a trial (n=1), CHOEP (n=1), and palliation (n=1).

Methods: We conducted whole-genome sequencing (60x) (WGS), whole-exome sequencing (150x) (WES), and bulk-RNA sequencing on diagnostic FFPE-tumor tissues and used peripheral blood mononuclear cells from 18 geographically representative healthy donors as controls. WGS and WES data were analyzed using GATK (Genome Analysis Toolkit) and Mutect2 for variant calling, and Control-FREEC for the detection of genomic copy number alterations (gCNAs). Bulk-RNA seq data was analyzed using VIPER (Virtual Inference of Protein-activity by Enriched Regulon analysis) and DESeq2. In parallel, we have completed in situ spatial transcriptomic profiling using the 10x Genomics Xenium platform on 12 FFPE ENKTL tumor samples using a custom ENKTCL-specific 480 target genome panel. Correlations between genomic alterations and spatial transcriptomics for these cases are currently underway.

Results: We report on the preliminary results of our ongoing genomic analyses. Our initial findings from 16 WGS cases revealed distinct gCNAs between chemo-sensitive (n = 10) and refractory cases (n = 6). In particular, we observed a higher frequency of copy number amplifications in 17q chromosome regions in chemo-sensitive cases. Additionally, differential gene expression analysis using RNA-seq from these 16 samples identified 67 differentially expressed genes (BH adjusted p &lt;0.05) between chemo-sensitive and chemo-refractory cases, including MYC overexpression in the chemo-refractory cases (log2FoldChange = 1.18, BH adjusted p =0.024). Pathway enrichment analysis indicated that highly expressed genes in chemo-refractory cases were enriched in pathways associated with cell proliferation (FDR-adjusted P = 4.38e-04) and PI3K-AKT signaling pathway (FDR-adjusted P = 0.033).

Conclusion: Our findings suggest that unique molecular signatures, including specific genomic copy number alterations and pathway regulations, are intricately linked with chemotherapy sensitivity in patients with ENKTCL. This first of its kind, highly multiplexed approach integrates genomics and spatial transcriptomics across a unique and diverse global set of ENKTCL. This approach has the potential to develop biomarkers of response and resistance to chemotherapy in patients with ENKTCL and prioritize therapies.