Samuli Eldfors

Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase–signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.

Somatic mutations in clonally expanded cytotoxic T lymphocytes in patients with newly diagnosed rheumatoid arthritis

Somatic mutations contribute to tumorigenesis. Although these mutations occur in all proliferating cells, their accumulation under non-malignant conditions, such as in autoimmune disorders, has not been investigated. Here, we show that patients with newly diagnosed rheumatoid arthritis have expanded CD8+ T-cell clones; in 20% (5/25) of patients CD8+ T cells, but not CD4+ T cells, harbour somatic mutations. In healthy controls (n=20), only one mutation is identified in the CD8+ T-cell pool. Mutations exist exclusively in the expanded CD8+ effector-memory subset, persist during follow-up, and are predicted to change protein functions. Some of the mutated genes (SLAMF6, IRF1) have previously been associated with autoimmunity. RNA sequencing of mutation-harbouring cells shows signatures corresponding to cell proliferation. Our data provide evidence of accumulation of somatic mutations in expanded CD8+ T cells, which may have pathogenic significance for RA and other autoimmune diseases.

Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.Aggressive natural killer-cell leukemia (ANKL) has few targeted therapies. Here ANKL patients are reported to harbor STAT3, RAS-MAPK pathway, DDX3X and epigenetic modifier mutations; and drug sensitivity profiling uncovers the importance of the JAK-STAT pathway, revealing potential ANKL therapeutic targets.

Sequencing of Lynch syndrome tumors reveals the importance of epigenetic alterations

Genomic instability and epigenetic aberrations are important classifiers of human tumors, yet, their interrelations are poorly understood. We used Lynch syndrome (LS) to address such relationships. Forty-five tumors (11 colorectal adenomas, 18 colorectal carcinomas, and 16 ovarian carcinomas) were profiled for CpG Island Methylator Phenotype (CIMP) and somatic mutations. All tumors showed high-degree microsatellite instability. Panel sequencing of 578 cancer-relevant genes revealed the average number of 1433, 1124, and 657 non-synonymous somatic mutations per colorectal adenoma, colorectal carcinoma, and ovarian carcinoma, respectively. Genes harboring mutations with allele frequency 25 % or higher in at least 31 % of tumors were regarded to be possible drivers. Among 72 and 10 such genes identified in colorectal and ovarian tumors, respectively, the most frequently mutated genes BRD4 and MLL2 (62 % of colorectal tumors) and ARID1A (50 % of ovarian carcinomas) are involved in epigenetic regulation. The total number of somatic mutations or mutant genes per tumor were significantly associated with CIMP. Our results suggest that even in an inherited disease, tumor type-specific epigenetic changes are significant and may result from regulatory changes (CIMP) or structural events (mutations of epigenetic regulatory genes). The findings are clinically relevant since many of the affected pathways can be therapeutically targeted.

Clonal heterogeneity influences drug responsiveness in renal cancer assessed by ex vivo drug testing of multiple patient-derived cancer cells: Subclone-specific therapeutic approach for renal cancer

Renal cell cancer (RCC) has become a prototype example of the extensive intratumor heterogeneity and clonal evolution of human cancers. However, there is little direct evidence on how the genetic heterogeneity impacts on drug response profiles of the cancer cells. Our goal was to determine how genomic clonal evolution impacts drug responses. Finding from our study could help to define the challenge that clonal evolution poses on cancer therapy. We established multiple patient‐derived cells (PDCs) from different tumor regions of four RCC patients, verified their clonal relationship to each other and to the uncultured tumor tissue by genome sequencing. Furthermore, comprehensive drug‐sensitivity testing with 460 oncological drugs was performed on all PDC clones. The PDCs retained many cancer‐specific copy number alterations and mutations in driver genes such as VHL, PBRM1, PIK3C2A, KMD5C and TSC2 genes. The drug testing highlighted vulnerability in the PDCs toward approved RCC drugs, such as the mTOR‐inhibitor temsirolimus, but also novel sensitivities were uncovered. The individual PDC clones from different tumor regions in a patient showed distinct drug–response profiles, suggesting that genomic heterogeneity contributes to the variability in drug responses. Studies of multiple PDCs from a patient with cancer are informative for elucidating cancer heterogeneity and for the determination on how the genomic evolution is manifested in cancer drug responsiveness. This approach could facilitate tailoring of drugs and drug combinations to individual patients.

Converging endometrial and ovarian tumorigenesis in Lynch syndrome: Shared origin of synchronous carcinomas

OBJECTIVEnThe diagnosis of carcinoma in both the uterus and the ovary simultaneously is not uncommon and raises the question of synchronous primaries vs. metastatic disease. Targeted sequencing of sporadic synchronous endometrial and ovarian carcinomas has shown that such tumors are clonally related and thus represent metastatic disease from one site to the other. Our purpose was to investigate whether or not the same applies to Lynch syndrome (LS), in which synchronous cancers of the gynecological tract are twice as frequent as in sporadic cases, reflecting inherited defects in DNA mismatch repair (MMR).nnnMETHODSnMMR gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nationwide registry. Endometrial (nu202f=u202f35) and ovarian carcinomas (nu202f=u202f23), including 13 synchronous carcinoma pairs, were collected as well as endometrial hyperplasias (nu202f=u202f56) and normal endometria (nu202f=u202f99) from a surveillance program over two decades. All samples were studied for MMR status, ARID1A and L1CAM protein expression and tumor suppressor gene promoter methylation, and synchronous carcinomas additionally for somatic mutation profiles of 578 cancer-relevant genes.nnnRESULTSnSynchronous carcinomas were molecularly concordant in all cases. Prior or concurrent complex (but not simple) endometrial hyperplasias showed a high degree of concordance with endometrial or ovarian carcinoma as the endpoint lesion.nnnCONCLUSIONSnOur investigation suggests shared origins for synchronous endometrial and ovarian carcinomas in LS, in analogy to sporadic cases. The similar degrees of concordance between complex hyperplasias and endometrial vs. ovarian carcinoma highlight converging pathways for endometrial and ovarian tumorigenesis overall.

A6.02 Somatic mutations in clonally expanded CD8+ T cells in patients with newly diagnosed rheumatoid arthritis

Background and objectives Clonally expanded CD8+ lymphocytes are known to exist in patients with rheumatoid arthritis (RA). Interestingly, in large granular lymphocyte (LGL) leukaemia (a haematological malignancy with indolent disease course), CD8+ lymphocytes expand and the expanded clones harbour somatic mutations. LGL leukaemia patients with STAT3 mutated CD8+ lymphocytes have significantly increased risk of developing autoimmune manifestations, RA being a common autoimmune co-morbidity. As somatic mutations in CD8+ lymphocytes associate with autoimmunity in LGL leukaemia, we wanted to examine if similar phenomenon exists in newly diagnosed RA patients without known LGL lymphoproliferation. Materials and methods Lymphocyte clonality was analysed by flow cytometry using vbβ -staining kit (Beckman Coulter) and the major clones were confirmed by TCR deep sequencing. Deep sequencing of 986 selected immune-related genes was performed from CD8+ T cells using CD4+ lymphocytes as germline control. The coverage was 200–6000x enabling reliable calling of low frequency mutations. Candidate mutations were validated using targeted deep amplicon sequencing. Results 43% (n = 31) of the newly diagnosed RA patients in our patient cohort (n = 68) had CD8+ lymphocyte expansions exceeding 10% of the total CD8+ T cells. The largest clones were subjected to targeted deep sequencing. We identified somatic gene variants in five (n = 5) patients and in one healthy control. Most of these variants were non-synonymous coding single nucleotide changes in genes important for immune functions (C5, PADI4, IRF1, SLAMF6, BST1 and CLEC10A), or proliferation (PTPRO, PADI4, PROM1, CDK12 and CLIP2). Sequencing of FACS sorted CD8+ lymphocytes confirmed that the expanded clone harboured the identified mutations. Conclusions In RA patients, somatic mutations accumulate in the CD8+ T cells during clonal expansion. Related findings have recently been reported in the myeloid lineage, where somatic variants are found to accumulate even in healthy subjects in a process called clonal haematopoiesis. Importantly, we focused on CD8+ lymphocytes that have undergone thymic education and thus we report acquired somatic variants in mature cytotoxic lymphocytes in a non-malignant set-up. Although more research is needed to address the possible pathogenicity of the identified mutations, these findings provide an interesting link between autoimmunity and cancer.