Circulating tumour DNA analysis to detect clonal evolution in TP53 mutated CLL treated with sequential precision medicines

Circulating tumour DNA (ctDNA) is increasingly being investigated not only for the early detection of treatment failure but also for the assessment of clonal evolution during treatment, facilitating a more comprehensive assessment of tumour burden compared to existing techniques. Here, we describe the case of a male patient with TP53 mutated CLL, diagnosed in 2000,initially treated with 2 lines of immune-chemotherapy prior to relapse in 2012, when he received sequential treatment with the selective BTKi tiraburitinib followed by the selective BCL2 inhibitor, venetoclax. Using serial ctDNA analysis we studied the dynamics of clonal evolution during treatment with tirabrutinib and venetoclax prior to the development of Richter’s transformation(RT). To elucidate clonal dynamics leading to therapeutic resistance and subsequent RT, we carried out paired whole exome sequencing(WES), deep targeted sequencing using a customised 72 gene panel and digital droplet PCR (ddPCR) on sequential peripheral blood mononuclear cells (PBMCs) and ctDNA. ctDNA was derived from plasma samples obtained pre-, during and post-treatment with tirabrutinib and venetoclax. Tumour tissue was obtained at the point of diagnosis of RT. This patient initially responded for 31 months on a potentially non-saturating dose of 40mg OD of tirabrutinib prior to clinicalrelapse. Analysis of ctDNA did not detect either a BTK or PLCG2 mutation at initial progression and therefore the dose of tirabrutinib was increased to 600mg OD, the maximal dose permitted on study. A further clinical response was seen with a resultant secondary BTKi induced lymphocytosis. The patient remained in partial remission for an additional 12 months. Two months prior to further clinical relapse, the presence of a BTK p.L528W mutation was detected by ctDNA analysis, a mutation disrupts the BTKi binding site which has been previously described in refractory CLL (Sharma et al., 2016). On WES of PBMC, mutations were found in TP53p.V216M at 73% variant allele frequency (VAF) and MSH2 p.E572_Q574del at 29.6% VAF. Analysis of IGHV further demonstrated a single productive IGH clonotype of IGHV2-5*06.Following initiation of venetoclax, ctDNA levels of BTK p.L562W and TP53 p.V216M rapidly became undetectable, correlating with clinical response. The patient remained in remission for 13 months before developing rapidly progressive RT resistant to R-CHOP.WES of the RT tumour showed additional mutations, deletions and chromosomal translocations. However, deep-targeted sequencing uncovered a low level BCL2 p.A113G (1.8% VAF) undetected by WES. This mutation was also detected by ctDNA analysis at this timepoint demonstrating ctDNA offers a non-invasive rout to RT detection. The IGHV clonotype matched the pre-RT PBMC, indicating RT was clonally related to the CLL clone. A striking increase in the frequency of frameshift mutations in post-MSH2 aberration (119%, compared to sequenced samples pre-MSH2 acquisition) was observed, suggestive of chromosomal instability, which likely contributed to treatment resistance. The increase in VAF of MSH2 through sequential relapses in treatment, further suggests an association with RT development (tirabrutinib(16.7%), second relapse (29.6%), and RT (45.3%)).Here we have shown that mutational profiling of ctDNA is an effective tool for detecting clonal resistance to precision medicines prior to the development of clinical or radiological evidence of progression.</p

Successful treatment of primary cutaneous diffuse large B cell lymphoma leg type with single agent venetoclax

Successful treatment of primary cutaneous diffuse large B cell lymphoma leg type with single agent venetocla

Computational modeling of DLBCL predicts response to BH3-mimetics

In healthy cells, pro- and anti-apoptotic BCL2 family and BH3-only proteins are expressed in a delicate equilibrium. In contrast, this homeostasis is frequently perturbed in cancer cells due to the overexpression of anti-apoptotic BCL2 family proteins. Variability in the expression and sequestration of these proteins in Diffuse Large B cell Lymphoma (DLBCL) likely contributes to variability in response to BH3-mimetics. Successful deployment of BH3-mimetics in DLBCL requires reliable predictions of which lymphoma cells will respond. Here we show that a computational systems biology approach enables accurate prediction of the sensitivity of DLBCL cells to BH3-mimetics. We found that fractional killing of DLBCL, can be explained by cell-to-cell variability in the molecular abundances of signaling proteins. Importantly, by combining protein interaction data with a knowledge of genetic lesions in DLBCL cells, our in silico models accurately predict in vitro response to BH3-mimetics. Furthermore, through virtual DLBCL cells we predict synergistic combinations of BH3-mimetics, which we then experimentally validated. These results show that computational systems biology models of apoptotic signaling, when constrained by experimental data, can facilitate the rational assignment of efficacious targeted inhibitors in B cell malignancies, paving the way for development of more personalized approaches to treatment

Targeting intermediary metabolism enhances the efficacy of BH3 mimetic therapy in haematological malignancies.

BH3 mimetics are novel targeted drugs with remarkable specificity and potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of Navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we have developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-XL (A-1331852) or MCL-1 (A-1210477), in relevant haematological cancer cell lines. In these models, resistance could be attributed neither to consistent changes in expression levels of the anti-apoptotic proteins nor interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we report that targeting of glutamine uptake and its downstream signalling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mTOR signalling result in marked sensitisation of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy

Limitations of Monitoring Disease Progression Using Circulating Tumor DNA in Lymphoma: An Example from Primary Cutaneous DLBCL Leg-type

Chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL) remains a significant clinical problem. The ability to predict patients likely to have poor outcomes with conventional therapies may facilitate rational use of alternative, targeted treatment approaches before fulminant relapse. The utility of circulating tumor DNA (ctDNA) in DLBCL is currently being investigated. Initial studies have reported high levels of sensitivity for detection of residual disease, outperforming imaging techniques such as 18FDG-PET/CT scans.1 For example, Alizadeh et al recently demonstrated the feasibility of CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) approach in DLBCL; the mean elapsed time between the first ctDNA-positive time point and radiological relapse was 188 days.2 Kurtz et al detected pretreatment ctDNA in 98% of patients with DLBCL receiving treatment with frontline or salvage immunochemotherapy.3 Early and major molecular responses after 1 and 2 cycles of treatment, resulted in superior outcome at 24 months. However, only 9% and 23% of patients within these validation sets had stage I and II disease, respectively. Limitations to the use of ctDNA to detect early stage malignancy have been identified; for example, in lung, where low-volume disease cannot be reliably detected using mutation profiles in ctDNA.4 Furthermore, some specific subtypes of disease (lung adenocarcinoma) do not shed detectable ctDNA into the peripheral blood.5</p

CUDC-907 blocks multiple pro-survival signals and abrogates microenvironment protection in CLL.

CUDC-907, a dual PI3K/HDAC inhibitor, has been proposed to have therapeutic potential in hematopoietic malignancies. However, the molecular mechanisms of its effects in chronic lymphocytic leukaemia (CLL) remain elusive. We show that CLL cells are sensitive to CUDC-907, even under conditions similar to the protective microenvironment of proliferation centres. CUDC-907 inhibited PI3K/AKT and HDAC activity, as expected, but also suppressed RAF/MEK/ERK and STAT3 signalling and reduced the expression of anti-apoptotic BCL-2 family proteins BCL-2, BCL-xL, and MCL-1. Moreover, CUDC-907 downregulated cytokines BAFF and APRIL and their receptors BAFFR, TACI, and BCMA, thus blocking BAFF-induced NF-κB signalling. T cell chemokines CCL3/4/17/22 and phosphorylation of CXCR4 were also reduced by CUDC-907. These data indicated that CUDC-907 abrogates different protective signals and suggested that it might sensitize CLL cells to other drugs. Indeed, combinations of low concentrations of CUDC-907 with inhibitors of BCL2, BTK, or the NF-κB pathway showed a potent synergistic effect. Our data indicate that, apart from its known functions, CUDC-907 blocks multiple pro-survival pathways to overcome microenvironment protection in CLL cells. This provides a rationale to evaluate the clinical relevance of CUDC-907 in combination therapies with other targeted inhibitors

Efficacy of venetoclax monotherapy in patients with relapsed, refractory mantle cell lymphoma post BTK inhibitor therapy.

Mantle cell lymphoma (MCL), an aggressive B-cell malignancy accounting for 6% of non-Hodgkin lymphomas, remains incurable with standard therapy. Despite the approval of bortezomib,1 temsirolimus,2 lenalidomide,3 ibrutinib4 and acalabrutinib,5 patients with relapsed, refractory MCL have a survival of 2 years

Long-term follow-up of patients with CLL treated with the selective Bruton's tyrosine kinase inhibitor ONO/GS-4059.

[First paragraph] The inhibitor of Bruton’s tyrosine kinase (BTK) ibrutinib has transformed the treatment of chronic lymphocytic leukemia (CLL); many patients with previously untreatable disease may now enter durable remissions.1,2 Nevertheless, the kinome of ibrutinib is broad, resulting in toxicities including bleeding, arthralgia, diarrhea, hypertension, and atrial fibrillation.3-6 Up to 20% of patients discontinue ibrutinib due to toxicity.7-9 More selective BTK inhibitors (BTKis) include ONO/GS-4059, acalabrutinib, and BGB-3111. Preliminary data indicate that these drugs have comparable activity to ibrutinib, but with reduced toxicities.10-12 However, long-term follow-up and response data have not yet been reported. We provide an updated, 3-year follow-up of treatment efficacy, safety, and laboratory correlates, including baseline mutational profiling of CLL patients in the phase 1 ONO/GS-4059 extension study

DNA methylation profiling of hepatosplenic T-cell lymphoma.

[First paragraph] Hepatosplenic T–cell lymphoma (HSTL) is a malignancy with an unfavorable outcome mainly affecting young adults. To discover genes showing altered DNA-methylation in HSTL we performed array-based DNA methylation profiling of HSTL cells from 11 patients and compared the findings to those obtained from purified non-neoplastic ab-positive and gd-positive T cells. The procedure identified 1,339 hypermethylated and 2,774 hypomethylated CpG-loci in HSTL compared to controls. DNA methylation changes in HSTL were enriched for regulatory elements, like enhancers. Considering the top 100 differentially-methylated CpGs from various subset comparisons, we identified eight consistently hypermethylated genes (BCL11B, CD5, CXCR6, GIMAP7, LTA, SEPT9, UBAC2, UXS1) and four consistently hypomethylated genes (ADARB1, NFIC, NR1H3, ST3GAL3) in HSTL

Phase 1b study of venetoclax-obinutuzumab in previously untreated and relapsed/refractory chronic lymphocytic leukemia.

This single-arm, open-label, phase 1b study evaluated the maximum tolerated dose (MTD) of venetoclax when given with obinutuzumab and its safety and tolerability in patients with relapsed/refractory (R/R) or previously untreated (1L) chronic lymphocytic leukemia. Venetoclax dose initially was escalated (100-400 mg) in a 3+3 design to define the MTD combined with standard-dose obinutuzumab. Patients received venetoclax (Schedule A) or obinutuzumab (Schedule B) first to compare safety and determine dose/schedule for expansion. Venetoclax-obinutuzumab was administered for 6 cycles, followed by venetoclax monotherapy until disease progression (R/R) or fixed-duration 1 year of treatment (1L). 50 R/R and 32 1L patients were enrolled. No dose-limiting toxicities were observed. Safety, including incidence of tumor lysis syndrome (TLS), did not differ between schedules (2 laboratory TLS per schedule). Schedule B and 400 mg dose of venetoclax was chosen for expansion. The most common grade 3-4 adverse event was neutropenia (R/R, 58% of patients; 1L, 53%). Rates of grade 3-4 infections were 29% (R/R) and 13% (1L); no fatal infections occurred in 1L. All infusion-related reactions were grade 1-2, except for 2 grade 3 events. No clinical TLS was observed. Overall best response rate was 95% (CR/CRi, 37%) in R/R and 100% (CR/CRi, 78%) in 1L patients. Rate of undetectable (<10-4) minimal residual disease (MRD) in peripheral blood for R/R and 1L patients respectively was 64% and 91% ≥3 months after last obinutuzumab dose. Therapy with venetoclax and obinutuzumab had an acceptable safety profile and elicited durable responses and high rates of undetectable MRD. The study is registered to https://clinicaltrials.gov as NCT01685892