Novel cell enrichment technique for robust genetic analysis of archival classical Hodgkin lymphoma tissues.

Approximately 15% of patients with classical Hodgkin lymphoma (cHL) die after relapse or progressive disease. Comprehensive genetic characterization is required to better understand its molecular pathology and improve management. However, genetic information on cHL is hard to obtain mainly due to rare malignant Hodgkin- and Reed-Sternberg cells (HRSC), whose overall frequencies in the affected tissues ranges from 0.1 to 10%. Therefore, enrichment of neoplastic cells is necessary for the majority of genetic investigations. We have developed a new high-throughput method for marker-based enrichment of archival formalin-fixed and paraffin-embedded (FFPE) tissue-derived HRSC nuclei by fluorescence-assisted flow sorting (FACS) and successfully applied it on ten cHL cases. Genomic DNA extracted from sorted nuclei was used for targeted high-throughput sequencing (HTS) of 68 genes that are frequently affected in lymphomas. Chromosomal copy number aberrations were investigated by the Agilent SurePrint 180k microarray. Our method enabled HRSC nuclei enrichment to 40-90% in sorted populations. This level of enrichment was sufficient for reliable identification of tumor-specific mutations and copy number aberrations. Genetic analysis revealed that components of JAK-STAT signaling pathway were affected in all investigated tumors by frequent mutations of SOCS1 and STAT6 as well as copy number gains of JAK2. Involvement of nuclear factor-κB (NF-κB) pathway compounds was evident from recurrent gains of the locus containing the REL gene and mutations in TNFAIP3 and CARD11. Finally, genetic alterations of PD-L1 and B2M suggested immune evasion as mechanisms of oncogenesis in some patients. In this work, we present a new method for HRSC enrichment from FFPE tissue blocks by FACS and demonstrate the feasibility of a wide-scale genetic analysis by cutting-edge molecular methods. Our work opens the door to a large resource of archived clinical cHL samples and lays foundation to more complex studies aimed to answer important biological and clinical questions that are critical to improve cHL management.

Mutations of CREBBP and SOCS1 are independent prognostic factors in diffuse large B cell lymphoma: mutational analysis of the SAKK 38/07 prospective clinical trial cohort.

BACKGROUND/PURPOSE: Recently, the mutational background of diffuse large B cell lymphoma (DLBCL) has been revealed, identifying specific genetic events that drive lymphomagenesis. However, the prognostic value of these mutations remains to be determined. Prognostic biomarkers in DLBCL are urgently needed, since the current clinical parameter-based factors (e.g., International Prognostic Index (IPI)) are insufficient, particularly in identifying patients with poor prognosis who might benefit from alternative treatments. METHODS: We investigated the prognostic value of somatic mutations in DLBCL in a clinical trial (NCT00544219) patient cohort homogenously treated with six cycles of rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP), followed by two cycles of R (R-CHOP-14). The primary endpoint was event-free survival (EFS) at 2 years. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). Targeted high-throughput sequencing (HTS) of tumor genomic DNA was performed on all exons or hotspots of 68 genes frequently mutated in B cell lymphomas. Mutational data was correlated with the endpoints to identify prognostic associations. RESULTS: Targeted HTS detected somatic mutations in 71/76 (93%) of investigated cases. The most frequently mutated genes were KMT2D, SOCS1, GNA13, and B2M. Survival analysis revealed that CREBBP- and EP300-mutated cases had significantly worse OS, PFS, and EFS. In addition, ATM mutations predicted worse outcomes for all three clinical endpoints in germinal center B cell-like DLBCL. In contrast, SOCS1 mutations were associated with better PFS. On multivariable analysis taken into account IPI and failure to achieve complete remission, CREBBP and EP300 mutations remained significant to predict worse OS, PFS, and EFS. CONCLUSION: Targeted mutation analysis of a uniformly treated prospective clinical trial DLBCL cohort identifies tumor-based genetic prognostic markers that could be useful in the clinical management of such patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00544219.