Signifi cance of molecular diagnosti cs in therapy of chronic lymphocytic leukemia

Chronic lymphocyti c leukemia (CLL) is a malignancy of mature CD5+ B lymphocytes that is characterized by excepti onal clinical and biological heterogeneity. The Rai and Binet staging systems, developed in the late 1970s to early 1980s, are used in clinical practi ce to strati fy CLL pati ents into risk categories and to help guide clinical follow-up opti ons: to treat or to watch and wait. However, in early-stage disease, these systems are unable to predict what pati ents will face the progression to a more aggressive disease. That means, a number of molecular markers with prognosti c and/ or predicti ve impact exist and their assessment is strongly recommended in all pati ents prior to treatment initi ati on. One of the fi rst recognized prognosti c genomic aberrati ons in CLL include those detected by fl uorescence in situ hybridizati on (FISH): del(17p), del(11q), trisomy 12 and del(13q), and the immunoglobulin heavy variable (IGHV) gene somati c hypermutati on (SHM) status. Moreover, the rapid development of genomics techniques greatly expanded the understanding of CLL at the molecular level in the past decade. This resulted in the discovery of many newer prognosti c markers based on chromosomal aberrati ons or gene mutati ons. For instance, next-generati on sequencing (NGS) studies have led to the discovery of recurrently mutated genes in CLL, such as NOTCH1, SF3B1, BIRC3, XPO1, POT1, NFKBIE and EGR2, that are associated with poor clinical outcome. Among all of these biomarkers, the disti ncti on between markers of prognosti c and predicti ve values should be made. Prognosti c markers refer to biomarkers that can provide informati on regarding the pati ent's outcome regardless of treatment. They are o����� en assessed before treatment to help guide decisions on to treat or not. Markers associated with overall survival (OS) or ti me to fi rst treatment (TTFT) represent such examples. On the other hand, predicti ve markers are related to therapeuti c interventi ons with the ability to predict treatment response to a drug. These markers are normally assessed when pati ents receive the parti cular therapy. Some markers can be both prognosti c and predicti ve. The Nati onal Comprehensive Cancer Network guideline recommends testi ng of TP53 geneti c alterati ons, IGHV mutati on status, and several well-established cytogeneti c markers for CLL prognosti cati on. Of these, TP53 mutati ons, IGHV unmutated status, del(17p), and del(11q), as well as complex karyotype (the presence of three or more unrelated clonal chromosomal abnormaliti es in a sample), are associated with poor prognosis. Normal karyotype and trisomy 12 are considered as intermediate prognosti c factors, whereas del(13q) is associated with a favorable prognosis. The higher frequencies of the previously menti oned unfavorable markers (except for IGHV) found in the treated populati on usually imply the clonal evoluti on during disease progression or change in clonal dynamics induced by therapies, especially chemotherapies. Diff erent molecular and genomic techniques are employed for detecti ng molecular biomarkers in CLL. For IGHV mutati on status, the preferred method is Sanger sequencing to detect mutati ons in genomic DNA or cDNA following PCR, and align the resulti ng sequences to the germline IGHV using the IMGT/V-QUEST analyti c tool, where ≥ 98% homology to the germ line is interpreted as unmutated, >2% nonhomology as mutated, and 97.0% to 97.9% is interpreted as borderline. Prognosti cally signifi cant chromosomal abnormaliti es are frequently detected using fl uorescence in situ hybridizati on, array comparati ve genomic hybridizati on or conventi onal karyotyping. Fluorescence in situ hybridizati on, although off ers a high sensiti vity and specifi city, requires prior knowledge of chromosomal lesions for the probe designs and are limited to the chosen panel genes. The technique has limitati ons in detecti ng possible complex cytogeneti c abnormaliti es, as well. On the other hand, karyotyping and array comparati ve genomic hybridizati on provide genome-wide coverage. Despite the fact that array comparati ve genomic hybridizati on does not eff ecti vely detect balanced chromosomal rearrangements, it uncovers more genomic abnormaliti es than karyotyping as the probe-based technology examines the chromosomal structure at a much higher resoluti on. Development in NGS technology in the past two decades, made the technique, especially targeted sequencing of gene panels, much less costly and accessible. Currently, in Serbia, geneti c techniques such as FISH, conventi onal karyotyping, Sanger sequencing and NGS are available for detecti on of CLL biomarkers. Advances in the understanding of CLL pathogenesis have consequently led to the development of several highly eff ecti ve targeted therapies, including Bruton tyrosine kinase (BTK), phosphati dylinositol 3-kinase, and BCL2 apoptosis regulator (BCL2) directed inhibitors. B-cell survival and proliferati on is regulated by the BCR signaling pathway. In normal B cells, BCR is triggered by anti gen ligati on, leading to acti vati on of a cascade of tyrosine kinases, including BTK. BCR signaling is aberrantly acti vated in many B-cell malignancies, including CLL. Ibruti nib has demonstrated high clinical effi cacy acti ng as an irreversible potent inhibitor of Bruton's tyrosine kinase and targets several key components of the BCR pathway. However, despite having 80% to 90% response rate, 10% to 15% of CLL pati ents, who respond initi ally, develop ibruti nib resistance and disease relapse in 2 to 3 years on ibruti nib treatment, mainly because of the acquisiti on of a BTK C481S mutati on. The mutati on prevents the drug from forming a covalent bond with the C481 residue that weakened the drug-BTK binding by 500-fold. As a result, BCR signaling and cell proliferati on were restored in the tumor cells. BTK mutati ons may be found in approximately 70% of CLL pati ents who progressed on ibruti nib treatment. Another resistance mechanism is through acquired acti vati ng mutati ons in PLCG2, which is found in approximately 10% of the cases. Given these evidences, the current Nati onal Comprehensive Cancer Network guideline recommends testi ng for BTK and PLCG2 mutati ons for CLL pati ents receiving ibruti nib who are suspected of having disease progression. NGS has become the opti mal method for detecti ng BTK or PLCG2 mutati ons in the se����� ng of ibruti nib treatment, as multi ple mutati ons in both genes may occur in the same specimen. Currently, approximately 20% of CLL pati ents who progressed on ibruti nib do not have either BTK or PLCG2 mutati ons; thus, with NGS, it is possible to uncover other less common but yet undefi ned drug-resistance mutati ons. In additi on to BTK and PLCG2 mutati ons known to confer ibruti nib resistance, other molecular markers have been associated with an upfront high risk of relapse on ibruti nib treatment. It has been reported that complex karyotype, del(17p)/TP53 mutati on, and del(18p) at baseline before ibruti nib treatment are strongly associated with disease relapse. Other approved targeted agents for CLL treatment include the phosphati dylinositol 3-kinase inhibitors idelalisib and duvelisib and BCL2 inhibitor venetoclax. For venetoclax, a novel BCL2-G101V mutati on was identi fi ed to prevent drug acti vity through drug-protein interacti on. Each pati ent with CLL may have several clinical and molecular markers of confl icti ng prognosti c signifi cance simultaneously, making the precise prognosti cati on challenging. Today is of the greatest importance to apply ultrasensiti ve techniques to reveal molecular relapses a����� er therapy initi ati on and to detect minimal residual disease a����� er pati ents achieve complete responses

Systematic analysis of cell-intrinsic and extrinsic factors in chronic lymphocytic leukemia to understand functional consequences for drug response and clinical outcome

Chronic lymphocytic leukemia (CLL) is an indolent B-cell malignancy with a very heterogeneous clinical course. Even though many aspects of the biology of CLL have been thoroughly described, the underlying molecular cause for this heterogeneity has still not been completely understood. To fill this gap, this thesis presents a comprehensive analysis of cancer cell-intrinsic and extrinsic factors which modify drug response phenotypes and patient outcome in a cohort of 81 primary CLL patient samples. Some cancer cell-intrinsic factors, like the genome or transcriptome of CLL, have been comprehensively explored. However, proteomic profiling of a large CLL patient cohort and its integration with other molecular layers is currently lacking. Therefore, this study performed a thorough characterisation of multiple CLL cell-intrinsic factors, including the proteome, the transcriptome and the genome. These were additionally linked to ex-vivo drug response profiles (43 drugs). This revealed associations between the different layers and functional consequences for drug response and clinical outcome. nsupervised clustering of protein levels uncovered a previously unappreciated poor prognosis CLL subgroup, which was independent of established risk factors and characterised by a distinct protein and drug response profile. The existence of this subgroup could be validated in an external cohort. This comprehensive multi-omics analysis represents the first proteogenomic study of a large CLL patient cohort. CLL cells additionally depend on cell-extrinsic signals provided by the microenvironment, such as the bone marrow niche. Such signals can modify and reduce the activity of selected drugs. However, a systematic analysis of how the bone marrow microenvironment influences drug response and resistance is lacking, because appropriate bone marrow model systems for high-throughput drug screening do currently not exist. To this end, a high-throughput co-culture drug-sensitivity testing platform was established. During the careful evaluation of different stroma cells as CLL cell support for the system, an unexpected phenomenon was discovered. Some bone marrow stroma cells had the ability to phagocytose apoptotic cells in large amounts. Phagocytosis decreased the total amount of cells and, thus, artificially increased the percentage of alive cells. This has implications for co-culture studies in general, as phagocytosis can cause a systematic bias and the misinterpretation of results if left unconsidered. Consequently, nonphagocytic stroma cells were chosen for the final screening platform. Using this optimised system, responses to 43 different drugs were measured. A linear model was employed to distinguish between the effect of stroma cells on spontaneous and on druginduced apoptosis of CLL cells. In accordance with the literature, stroma cells protected CLL cells from spontaneous apoptosis ex-vivo. Interestingly, effect sizes varied between patients and especially samples with unmutated immunoglobulin heavy chain variable region and high degrees of spontaneous apoptosis profited from co-culturing. Moreover, the influence of stroma cells on drug responses was systematically assessed. While some drugs, like chemotherapeutics, were less active in co-cultures, other drugs had unchanged activity or were even more effcient in the context of stroma cells. Especially Janus kinase inhibitors could overcome the protective effect by stroma cells and kill CLL cells despite the presence of stroma. The systematic analysis of the impact of the bone marrow niche on drug response can help to understand and overcome microenvironment-induced resistances. In conclusions, this thesis provides a systematic overview of how leukemia cell-intrinsic layers of CLL and the microenvironment determine drug response and patient outcome

Future prospects for mitosis-targeted antitumor therapies

Dysregulation of cell cycle progression is a hallmark of cancer cells. In recent years, efforts have been devoted to the development of new therapies that target proteins involved in cell cycle regulation and mitosis. Novel targeted antimitotic drugs include inhibitors of aurora kinase family, polo-like kinase 1, Mps1, Eg5, CENP-5 and the APC/cyclosome complex. While certain new inhibitors reached the clinical trial stage, most were discontinued due to negative results. However, these therapies should not be readily dismissed. Based on recent advances concerning their mechanisms of action, new strategies could be devised to increase their efficacy and promote further clinical trials. Here we discuss three main lines of action to empower these therapeutic approaches: increasing cell death signals during mitotic arrest, targeting senescent cells and facilitating antitumor immune response through immunogenic cell death (ICD). © 2021 The Author(s

Chromosome banding analysis and genomic microarrays are both useful but not equivalent methods for genomic complexity risk stratification in chronic lymphocytic leukemia patients

Genome complexity has been associated with poor outcome in patients with chronic lymphocytic leukemia (CLL). Previous cooperative studies established five abnormalities as the cut-off that best predicts an adverse evolution by chromosome banding analysis (CBA) and genomic microarrays (GM). However, data comparing risk stratification by both methods are scarce. Herein, we assessed a cohort of 340 untreated CLL patients highly enriched in cases with complex karyotype (CK) (46.5%) with parallel CBA and GM studies. Abnormalities found by both techniques were compared. Prognostic stratification in three risk groups based on genomic complexity (0-2, 3- 4 and ¿5 abnormalities) was also analyzed. No significant differences in the percentage of patients in each group were detected, but only a moderate agreement was observed between methods when focusing on individual cases (kappa=0.507; P<0.001). Discordant classification was obtained in 100 patients (29.4%), including 3% classified in opposite risk groups. Most discrepancies were technique-dependent and no greater correlation in the number of abnormalities was achieved when different filtering strategies were applied for GM. Nonetheless, both methods showed a similar concordance index for prediction of time to first treatment (TTFT) (CBA: 0.67 vs. GM: 0.65) and overall survival (CBA: 0.55 vs. GM: 0.57)

Development and application of methodologies and infrastructures for cancer genome analysis within Personalized Medicine

[eng] Next-generation sequencing (NGS) has revolutionized biomedical sciences, especially in the area of cancer. It has nourished genomic research with extensive collections of sequenced genomes that are investigated to untangle the molecular bases of disease, as well as to identify potential targets for the design of new treatments. To exploit all this information, several initiatives have emerged worldwide, among which the Pan-Cancer project of the ICGC (International Cancer Genome Consortium) stands out. This project has jointly analyzed thousands of tumor genomes of different cancer types in order to elucidate the molecular bases of the origin and progression of cancer. To accomplish this task, new emerging technologies, including virtualization systems such as virtual machines or software containers, were used and had to be adapted to various computing centers. The portability of this system to the supercomputing infrastructure of the BSC (Barcelona Supercomputing Center) has been carried out during the first phase of the thesis. In parallel, other projects promote the application of genomics discoveries into the clinics. This is the case of MedPerCan, a national initiative to design a pilot project for the implementation of personalized medicine in oncology in Catalonia. In this context, we have centered our efforts on the methodological side, focusing on the detection and characterization of somatic variants in tumors. This step is a challenging action, due to the heterogeneity of the different methods, and an essential part, as it lays at the basis of all downstream analyses. On top of the methodological section of the thesis, we got into the biological interpretation of the results to study the evolution of chronic lymphocytic leukemia (CLL) in a close collaboration with the group of Dr. Elías Campo from the Hospital Clínic/IDIBAPS. In the first study, we have focused on the Richter transformation (RT), a transformation of CLL into a high-grade lymphoma that leads to a very poor prognosis and with unmet clinical needs. We found that RT has greater genomic, epigenomic and transcriptomic complexity than CLL. Its genome may reflect the imprint of therapies that the patients received prior to RT, indicating the presence of cells exposed to these mutagenic treatments which later expand giving rise to the clinical manifestation of the disease. Multiple NGS- based techniques, including whole-genome sequencing and single-cell DNA and RNA sequencing, among others, confirmed the pre-existence of cells with the RT characteristics years before their manifestation, up to the time of CLL diagnosis. The transcriptomic profile of RT is remarkably different from that of CLL. Of particular importance is the overexpression of the OXPHOS pathway, which could be used as a therapeutic vulnerability. Finally, in a second study, the analysis of a case of CLL in a young adult, based on whole genome and single-cell sequencing at different times of the disease, revealed that the founder clone of CLL did not present any somatic driver mutations and was characterized by germline variants in ATM, suggesting its role in the origin of the disease, and highlighting the possible contribution of germline variants or other non-genetic mechanisms in the initiation of CLL

Development and application of methodologies and infrastructures for cancer genome analysis within Personalized Medicine

Programa de Doctorat en Biomedicina / Tesi realitzada al Barcelona Supercomputing Cener (BSC)[eng] Next-generation sequencing (NGS) has revolutionized biomedical sciences, especially in the area of cancer. It has nourished genomic research with extensive collections of sequenced genomes that are investigated to untangle the molecular bases of disease, as well as to identify potential targets for the design of new treatments. To exploit all this information, several initiatives have emerged worldwide, among which the Pan-Cancer project of the ICGC (International Cancer Genome Consortium) stands out. This project has jointly analyzed thousands of tumor genomes of different cancer types in order to elucidate the molecular bases of the origin and progression of cancer. To accomplish this task, new emerging technologies, including virtualization systems such as virtual machines or software containers, were used and had to be adapted to various computing centers. The portability of this system to the supercomputing infrastructure of the BSC (Barcelona Supercomputing Center) has been carried out during the first phase of the thesis. In parallel, other projects promote the application of genomics discoveries into the clinics. This is the case of MedPerCan, a national initiative to design a pilot project for the implementation of personalized medicine in oncology in Catalonia. In this context, we have centered our efforts on the methodological side, focusing on the detection and characterization of somatic variants in tumors. This step is a challenging action, due to the heterogeneity of the different methods, and an essential part, as it lays at the basis of all downstream analyses. On top of the methodological section of the thesis, we got into the biological interpretation of the results to study the evolution of chronic lymphocytic leukemia (CLL) in a close collaboration with the group of Dr. Elías Campo from the Hospital Clínic/IDIBAPS. In the first study, we have focused on the Richter transformation (RT), a transformation of CLL into a high-grade lymphoma that leads to a very poor prognosis and with unmet clinical needs. We found that RT has greater genomic, epigenomic and transcriptomic complexity than CLL. Its genome may reflect the imprint of therapies that the patients received prior to RT, indicating the presence of cells exposed to these mutagenic treatments which later expand giving rise to the clinical manifestation of the disease. Multiple NGS- based techniques, including whole-genome sequencing and single-cell DNA and RNA sequencing, among others, confirmed the pre-existence of cells with the RT characteristics years before their manifestation, up to the time of CLL diagnosis. The transcriptomic profile of RT is remarkably different from that of CLL. Of particular importance is the overexpression of the OXPHOS pathway, which could be used as a therapeutic vulnerability. Finally, in a second study, the analysis of a case of CLL in a young adult, based on whole genome and single-cell sequencing at different times of the disease, revealed that the founder clone of CLL did not present any somatic driver mutations and was characterized by germline variants in ATM, suggesting its role in the origin of the disease, and highlighting the possible contribution of germline variants or other non-genetic mechanisms in the initiation of CLL

ROR1 as a target for cancer immunotherapy

Receptor tyrosine-kinase like orphan receptor (ROR1) is a member of the tyrosine kinase family. Importantly, ROR1 is absent in healthy, critical tissue but overexpressed in various solid and haematological malignancies, including Chronic Lymphocytic Leukaemia (CLL). Moreover, recent studies suggest ROR1 is expressed on cancer stem cell-like cells (CSCs). The overriding aim of this study was therefore to combine the unique features of ROR1 with the exquisite specificity/therapeutic potential of monoclonal antibodies (MAbs) and/or their derivatives. To this end, our group generated a rat hybridoma library and screened >150 anti-ROR1+ clones. I then cloned 16 of our novel antibodies to human IgG1, kappa constant regions, of which 12 recognised ROR1 on different cell types. Based on functional/characterisation data, it was identified that clone SA1 exhibited significant CDC activity on primary CLL cells, whilst clone F was the only MAb able to bind the Frizzled domain of ROR1. Further investigation, however, revealed clone SA1 bound non-specifically to ROR1- cells. Therefore, my investigation focused instead on clone F, which was developed in parallel as a bispecific T-cell engager (BiTE) within our group. Having shown F BiTE elicited potent and specific cytotoxicity of ROR1+ cells on various solid cancer cell lines, including pancreatic cancer (PaCa), ROR1 BiTE was tested on PaCa cell line-derived CSCs using an in vitro tumoursphere model. Immunocytochemistry data confirmed specific elimination of cells expressing both CSC biomarkers and ROR1. As a whole, ROR1 MAb-based immunotherapy, particularly using BiTEs, seems to not only target ROR1+ cells present in the bulk of the tumour but, crucially, it also eliminates ROR1+ CSC subsets in PaCa. This approach represents a relevant and much needed addition to the current options for cancer treatment. Further preclinical and clinical studies will ultimately reveal the true therapeutic potential of ROR1 BiTEs alone and in combination

Residential green and blue spaces and working memory in children aged 6–12 years old. Results from the INMA cohort

Availability of green and blue spaces in the area of residence has been related to various health outcomes during childhood, including neurodevelopment. Some studies have shown that children living in greener and/or bluer areas score better on cognitive tasks although the evidence is inconsistent. These protective effects are hypothesized to occur in part through reductions in air pollution exposure and odds of attention-deficit/hyperactivity disorder (ADHD). This study analysed the effects of residential green and blue spaces on working memory of children in the Spanish INfancia y Medio Ambiente (INMA) birth cohort and the potential joint mediating role of air pollution and ADHD. The study samples were composed of 1738 six-to eight-year-olds (M = 7.53, SD = 0.68, 49% female) and 1449 ten-to twelve-year-olds (M = 11.18, SD = 0.69, 50% female) living in Asturias, Gipuzkoa, Sabadell or Valencia, Spain. Individual Normalized Difference Vegetation Index (NDVI) values in 100-, 300- and 500-m buffers and availability of green and blue spaces &gt;5000 m2 in 300-m buffers were calculated using Geographic Information Systems software. Individual NO2 values for the home environment were estimated using ESCAPE's land use regression models. ADHD diagnosis was reported by participants' parents via a questionnaire. Working memory was measured with numbers and colours (in the younger group only) N-back tests (2- and 3-back d’). Mixed-effects models informed of the beneficial effects of NDVI in a 300-m buffer on numerical working memory in the younger sample although the results were not consistent for all d’ scores considered and failed to detect significant effects through the candidate mediators. Availability of major blue spaces did not predict working memory performance. Provision of green spaces may play a role in children's working memory but further research is required.</p