Alexander R. Vartanov

Evolution and Impact of Subclonal Mutations in Chronic Lymphocytic Leukemia

Clonal evolution is a key feature of cancer progression and relapse. We studied intratumoral heterogeneity in 149 chronic lymphocytic leukemia (CLL) cases by integrating whole-exome sequence and copy number to measure the fraction of cancer cells harboring each somatic mutation. We identified driver mutations as predominantly clonal (e.g., MYD88, trisomy 12, and del(13q)) or subclonal (e.g., SF3B1 and TP53), corresponding to earlier and later events in CLL evolution. We sampled leukemia cells from 18 patients at two time points. Ten of twelve CLL cases treated with chemotherapy (but only one of six without treatment) underwent clonal evolution, predominantly involving subclones with driver mutations (e.g., SF3B1 and TP53) that expanded over time. Furthermore, presence of a subclonal driver mutation was an independent risk factor for rapid disease progression. Our study thus uncovers patterns of clonal evolution in CLL, providing insights into its stepwise transformation, and links the presence of subclones with adverse clinical outcomes.

SF3B1 and Other Novel Cancer Genes in Chronic Lymphocytic Leukemia

BACKGROUND The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood. METHODS We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease. RESULTS Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing. CONCLUSIONS Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

Somatic mutation as a mechanism of Wnt/β-catenin pathway activation in CLL.

One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. Although many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with nonrecurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared with leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that nonrecurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.

Association of Advanced Leukemic Stage and Skin Cancer Tumor Stage With Poor Skin Cancer Outcomes in Patients With Chronic Lymphocytic Leukemia

IMPORTANCE Although it has been well established that patients with chronic lymphocytic leukemia (CLL) have an increased risk of developing skin cancer, few studies have investigated the effect of CLL stage on the risk of poor skin cancer outcomes. The present study of CLL staging assesses outcomes of melanoma, squamous cell carcinoma, and Merkel cell carcinoma in this high-risk population. OBJECTIVE To determine if progression of CLL measured by advanced Rai stage (III or IV) is associated with worse skin cancer outcomes. DESIGN, SETTING, AND PARTICIPANTS Twenty-year retrospective study at 2 academic centers in Boston, Massachusetts, of adults with CLL and either melanoma, squamous cell carcinoma, or Merkel cell carcinoma. MAIN OUTCOMES AND MEASURES Hazard ratios (HRs) for the development of poor skin cancer outcomes (local recurrence, nodal metastasis, distant metastasis, or death from skin cancer). RESULTS In total, 133 patients with 377 primary skin cancers and a median follow-up of 41 months were included. Squamous cell carcinoma predominated (92.0%). The risk of death from skin cancer was equivalent to the risk of death from CLL (13.5%). On multivariate analysis, advanced Rai stage (III or IV) at the time of the first skin cancer diagnosis (HR, 4.5; 95% CI, 2.3-8.9) and a high skin cancer tumor (T) stage (HR, 4.9; 95% CI, 2.2-10.8) were associated with poor skin cancer outcomes. Those with both a low skin cancer T stage and a low Rai stage (n = 265) had a low risk (5.3%; 95% CI, 3.2%-8.7%) of poor skin cancer outcomes. Those with a low T stage and a high Rai stage (n = 89) had a significantly higher risk of poor skin cancer outcomes (16.9%; 95% CI, 10.9%-26.0%). The 23 patients with a high T stage had high risks of poor outcomes regardless of CLL status (27.3% if a low Rai stage and 50.0% if a high Rai stage, with wide 95% CIs). CONCLUSIONS AND RELEVANCE In patients with CLL and non-basal cell carcinoma skin cancer, mortality is as high from skin cancer as from CLL. The Rai stage and skin cancer T stage should be considered when risk-stratifying patients with skin cancer. Regular communication between dermatologists and oncologists will help facilitate the identification of patients with CLL who are at high risk of having poor skin cancer outcomes.

Controversial fluorescence in situ hybridization cytogenetic abnormalities in chronic lymphocytic leukaemia: new insights from a large cohort

The significance of rarer cytogenetic abnormalities in chronic lymphocytic leukaemia (CLL) remains controversial. We performed fluorescence in situ hybridization (FISH) prior to initial therapy on 618 CLL patients seen at our centre between 2005 and 2012. With a median follow‐up of 5·6 years, we found that 55 patients harbouring 14q32 rearrangements without t(14;18) had a shorter time to first treatment (TTFT) (median 26 months, P = 0·03) than patients with t(14;18) (median not reached). Patients with mono‐ or bi‐allelic del(13q) as a sole abnormality had a similarly long TTFT (median not reached). Those patients who harboured 3 or more FISH abnormalities without del(17p) had a short TTFT (4·6 months), comparable to patients with del(17p) (8 months); however, the overall survival for patients with 3 or more FISH abnormalities was longer than for patients with del(17p) with 0 or 1 additional abnormalities (median not reached vs. 54 months). FISH cytogenetics remains a useful genetic tool in the clinic, even in the era of next generation sequencing and, as such, our data provide valuable new insights for counselling patients.

Germline copy number variation associated with Mendelian inheritance of CLL in two families

Germline copy number variation associated with Mendelian inheritance of CLL in two families

Ofatumumab plus high dose methylprednisolone followed by ofatumumab plus alemtuzumab to achieve maximal cytoreduction prior to allogeneic transplantation for 17p deleted or TP53 mutated chronic lymphocytic leukemia

Abstract We hypothesized that ofatumumab with sequential methylprednisolone – alemtuzumab would be an effective and tolerable regimen for patients with high-risk chronic lymphocytic leukemia (CLL) with TP53 dysfunction. Thirty CLL patients with TP53 dysfunction (15 treatment naive (TN), 15 relapsed/refractory (R/R)) were enrolled in this phase II study. Therapy included ofatumumab with methylprednisolone for 2–4 monthly cycles, then ofatumumab with alemtuzumab for 4–24 weeks, then allogeneic transplantation or maintenance. The rate of overall response, complete response, marrow minimal residual disease (MRD) negativity, 3-year progression-free survival and overall survival were 80, 13, 80, 53, and 66%, respectively, in TN patients and 68, 0, 54, 25, and 53%, respectively, in R/R patients. Notable grade 3/4 toxicities included neutropenia and infection in 43 and 40% of patients, respectively. At median follow-up of 45 months, 13 patients died, and 10 patients are alive posttransplant. Overall, we observed high rates of MRD-negativity and acceptable tolerability in high-risk CLL.