Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Overactive WASp in X-linked neutropenia leads to aberrant B-cell division and accelerated plasma cell generation.

BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching.

Ultra-low depth sequencing of plasma cell DNA for the detection of copy number aberrations in multiple myeloma.

Cytogenetic abnormalities are powerful prognostic factors in multiple myeloma (MM) and are routinely analyzed by FISH on bone marrow (BM) plasma cells (PC). Although considered the gold standard, FISH experiments can be laborious and expensive. Therefore, array-CGH (aCGH) has been introduced as an alternative approach for detecting copy number aberrations (CNA), reducing the number of FISH experiments per case and yielding genome-wide information. Currently, next generation sequencing (NGS) technologies offer new perspectives for the diagnostic workup of malignant disorders. In this study, we examined ultra-low depth whole genome sequencing (LDS) as a valid alternative for aCGH for the detection of CNA in BM PC in MM. To this end, BM aspirates obtained in a diagnostic setting from 20 MM cases were analyzed. CD138+ cell-sorted samples were subjected to FISH analysis. DNA was extracted for subsequent aCGH and LDS analysis. CNA were detected by aCGH and LDS in all but one case. Importantly, all CNA identified by parallel first generation aCGH analysis were also detected by LDS, along with six additional CNA in five cases. One of these additional aberrations was in a region of prognostic importance in MM and was confirmed using FISH. However, risk stratification in these particular cases was unaffected. Thus, a perfectly concordant prognostication between array-CGH and LDS was observed. This validates LDS as a novel and cost-efficient tool for the detection of CNA in MM.

Ultra-low coverage whole genome sequencing of ccfDNA in multiple myeloma: A tool for laboratory routine?

Multiple myeloma (MM), is a heterogeneous disease in which chromosomal abnormalities are important for prognostic risk stratification. Cytogenetic profiling with FISH on plasma cells from bone marrow samples (BM-PCs) is the current gold standard, but variable infiltration of plasma cells or failed aspiration can hamper this process. Ultra-low coverage sequencing (ULCS) of circulating cell-free DNA (ccfDNA) may offer a minimally invasive alternative for the work-up of these cases. We compared ULCS, aCGH and FISH on selected BM-PCs in a routine setting with ULCS of ccfDNA for the detection of somatic copy number aberrations (CNAs) in MM. METHODS: Purified CD138+ BM-PCs of 23 MM patients at initiation of their treatment were subjected to aCGH, FISH and ULCS. Paired samples of peripheral blood-ccfDNA obtained at diagnosis were analyzed by ULCS and compared to the results found in BM-PCs. RESULTS: Using ULCS of ccfDNA, cytogenetic markers were identified in 18 out of 23 patients; five cases could not be analyzed due to low (≤3%) tumor fraction (TF). High similarity between CNA profiles of BM-PCs and ccfDNA was found. Moreover, 78% of the ccfDNA profiles resulted in the same risk classification as the routine FISH and/or BM-PCs ULCS and aCGH. Chromothripsis was detected in five patients; these had the highest TF values (range 7.1% to 42%) in our series and their profiles showed other high-risk anomalies. CONCLUSION: This proof-of-principle study indicates that ULCS of ccfDNA can reveal CNAs in MM and should be explored further as a cost-efficient alternative, especially in cases where BM-PC purification fails.

The landscape of copy number variations in classical Hodgkin lymphoma: a joint KU Leuven and LYSA study on cell-free DNA.

The low abundance of Hodgkin/Reed-Sternberg (HRS) cells in lymph node biopsies in classical Hodgkin lymphoma (cHL) complicates the analysis of somatic genetic alterations in HRS cells. As circulating cell-free DNA (cfDNA) contains circulating tumor DNA (ctDNA) from HRS cells, we prospectively collected cfDNA from 177 patients with newly diagnosed, mostly early-stage cHL in a monocentric study at Leuven, Belgium (n = 59) and the multicentric BREACH study by Lymphoma Study Association (n = 118). To catalog the patterns and frequencies of genomic copy number aberrations (CNAs), cfDNA was sequenced at low coverage (0.26×), and data were analyzed with ichorCNA to yield read depth-based copy number profiles and estimated clonal fractions in cfDNA. At diagnosis, the cfDNA concentration, estimated clonal fraction, and ctDNA concentration were significantly higher in cHL cases than controls. More than 90% of patients exhibited CNAs in cfDNA. The most frequent gains encompassed 2p16 (69%), 5p14 (50%), 12q13 (50%), 9p24 (50%), 5q (44%), 17q (43%), 2q (41%). Losses mostly affected 13q (57%), 6q25-q27 (55%), 4q35 (50%), 11q23 (44%), 8p21 (43%). In addition, we identified loss of 3p13-p26 and of 12q21-q24 and gain of 15q21-q26 as novel recurrent CNAs in cHL. At diagnosis, ctDNA concentration was associated with advanced disease, male sex, extensive nodal disease, elevated erythrocyte sedimentation rate, metabolic tumor volume, and HRS cell burden. CNAs and ctDNA rapidly diminished upon treatment initiation, and persistence of CNAs was associated with increased probability of relapse. This study endorses the development of ctDNA as gateway to the HRS genome and substrate for early disease response evaluation.