High-throughput sequencing of archival cerebrospinal fluid specimens defines B-lymphoblastic leukemia clonal composition.

Detailed characterization of the B-lymphoblastic leukemia (B-ALL) cells which invade the central nervous system (CNS) has been limited by practical challenges. To test whether the clonal composition of the cerebrospinal fluid (CSF) reflects the primary B-ALL tissue, we applied immunoglobulin (Ig) high-throughput sequencing (HTS) of archival CSF cytospin preparations from six patients with morphologically defined CNS involvement. We discovered that most CSF clones are detectable at some timepoint in the primary tissue, but that shifting clonal abundance is prevalent across tissue sites between diagnosis and relapse. Ig HTS of CSF cytospins may improve understanding of sanctuary site dissemination in B-ALL.

Integrated variant allele frequency analysis pipeline and R package: easyVAF.

Somatic sequence variants are associated with cancer diagnosis, prognostic stratification, and treatment response. Variant allele frequency (VAF), the percentage of sequence reads with a specific DNA variant over the read depth at that locus, has been used as a metric to quantify mutation rates in these applications. VAF has the potential for feature detection by reflecting changes in tumor clonal composition across treatments or time points. Although there are several packages, including Genome Analysis Toolkit and VarScan, designed for variant calling and rare mutation identification, there is no readily available package for comparing VAFs among and between groups to identify loci of interest. To this end, we have developed the R package easyVAF, which includes parametric and nonparametric tests to compare VAFs among multiple groups. It is accompanied by an interactive R Shiny app. With easyVAF, the investigator has the option between three statistical tests to maximize power while maintaining an acceptable type I error rate. This paper presents our proposed pipeline for VAF analysis, from quality checking to group comparison. We evaluate our method in a wide range of simulated scenarios and show that choosing the appropriate test to limit the type I error rate is critical. For situations where data is sparse, we recommend comparing VAFs with the beta-binomial likelihood ratio test over Fisher's exact test and Pearson's χ2 test.

The role of migration in mutant dynamics in fragmented populations.

Mutant dynamics in fragmented populations have been studied extensively in evolutionary biology. Yet, open questions remain, both experimentally and theoretically. Some of the fundamental properties predicted by models still need to be addressed experimentally. We contribute to this by using a combination of experiments and theory to investigate the role of migration in mutant distribution. In the case of neutral mutants, while the mean frequency of mutants is not influenced by migration, the probability distribution is. To address this empirically, we performed in vitro experiments, where mixtures of GFP-labelled ("mutant") and non-labelled ("wid-type") murine cells were grown in wells (demes), and migration was mimicked via cell transfer from well to well. In the presence of migration, we observed a change in the skewedness of the distribution of the mutant frequencies in the wells, consistent with previous and our own model predictions. In the presence of de novo mutant production, we used modelling to investigate the level at which disadvantageous mutants are predicted to exist, which has implications for the adaptive potential of the population in case of an environmental change. In panmictic populations, disadvantageous mutants can persist around a steady state, determined by the rate of mutant production and the selective disadvantage (selection-mutation balance). In a fragmented system that consists of demes connected by migration, a steady-state persistence of disadvantageous mutants is also observed, which, however, is fundamentally different from the mutation-selection balance and characterized by higher mutant levels. The increase in mutant frequencies above the selection-mutation balance can be maintained in small ( N < N c ) demes as long as the migration rate is sufficiently small. The migration rate above which the mutants approach the selection-mutation balance decays exponentially with N / N c . The observed increase in the mutant numbers is not explained by the change in the effective population size. Implications for evolutionary processes in diseases are discussed, where the pre-existence of disadvantageous drug-resistant mutant cells or pathogens drives the response of the disease to treatments.

Patients with polyclonal hepatocellular carcinoma are at a high risk of early recurrence and have a poor recurrence-free survival period.

BACKGROUND/PURPOSE OF THE STUDY: Tumor heterogeneity based on copy number variations is associated with the evolution of cancer and its clinical grade. Clonal composition (CC) represents the number of clones based on the distribution of B-allele frequency (BAF) obtained from a genome-wide single nucleotide polymorphism (SNP) array. A higher CC number represents a high degree of heterogeneity. We hypothesized and evaluated that the CC number in hepatocellular carcinoma (HCC) tissues might be associated with the clinical outcomes of patients. METHODS: Somatic mutation, whole transcriptome, and CC number based on copy number variations of 36 frozen tissue samples of operably resected HCC tissues were analyzed by targeted deep sequencing, transcriptome analysis, and SNP array. RESULTS: The samples were classified into the heterogeneous tumors as poly-CC (n = 26) and the homogeneous tumors as mono-CC (n = 8). The patients with poly-CC had a higher rate of early recurrence and a significantly shorter recurrence-free survival period than the mono-CC patients (7.0 months vs. not reached, p = 0.0084). No differences in pathogenic non-synonymous mutations, such as TP53, were observed between the two groups when targeted deep sequencing was applied. A transcriptome analysis showed that cell cycle-related pathways were enriched in the poly-CC tumors, compared to the mono-CC tumors. Poly-CC HCC is highly proliferative and has a high risk of early recurrence. CONCLUSION: CC is a possible candidate biomarker for predicting the risk of early postoperative recurrence and warrants further investigation.

Analysis of Clonal Composition in Human iPSC and ESC and Derived 2D and 3D Differentiated Cultures.

Human induced pluripotent and embryonic stem cell cultures (hiPSC/hESC) are phenotypically heterogeneous and prone to clonal deviations during subculturing and differentiation. Clonal deviations often emerge unnoticed, but they can change the biology of the cell culture with a negative impact on experimental reproducibility. Here, we describe a computational workflow to profile the bulk clonal composition in a hiPSC/hESC culture that can also be used to infer clonal deviations. This workflow processes data obtained with two versions of the same method. The two versions-epigenetic and transcriptomic-rely on a mechanism of stochastic H3K4me3 deposition during hiPSC/hESC derivation. This mechanism generates a signature of ten or more H3K4me3-enriched clustered protocadherin (PCDH) promoters distinct in every single cell. The aggregate of single-cell signatures provides an identificatory feature in every hiPSC/hESC line. This feature is stably transmitted to the cell progeny of the culture even after differentiation unless there is a clonal deviation event that changes the internal balance of single-cell signatures. H3K4me3 signatures can be profiled by chromatin immunoprecipitation and next-generation sequencing (ChIP-seq). Alternatively, an equivalent PCDH-expression version can be profiled by RNA-seq in PCDH-expressing hiPSC/hESC-derived cells (such as neurons, astrocytes, and cardiomyocytes; and, in long-term cultures, such as cerebral organoids). Notably, our workflow can also distinguish genetically identical hiPSC/hESC lines derived from the same patient or generated in the same editing process. Together, we propose a method to improve data sharing and reproducibility in the hiPSC and hESC fields.

Phenotypic and genotypic characterization of serogroup 6 Streptococcus pneumoniae isolates collected during 10-valent pneumococcal conjugate vaccine era in Bulgaria.

Serogroup 6 remains common in the pneumococcal-conjugated vaccine era in Bulgaria; therefore, we investigated its clonal and serotype dynamics. The antibiotic susceptibilities were assessed by broth microdilution. Strains identified as serogroup 6 with latex agglutination method were subjected to serotype-specific PCRs. Erythromycin-resistant strains were analyzed by PCR for presence of ermB and mefE genes. MLST was performed to define clonal composition of the sequence types (STs). Serogroup 6 was represented by 40 (13.3%) from 301 invasive and non-invasive Streptococcus pneumoniae isolates. Molecular serotyping revealed new emerging serotype 6C (6.6%), not detected in pre-vaccine era. Among unvaccinated patients, mostly we observed serotypes 6А (57.1%) and 6В (28.6%). Serotype 6C was distinctive for vaccinated children (64%), followed by 6A (24%). Penicillin and ceftriaxone non-susceptible serogroup 6 strains were 65% and 5%, respectively; erythromycin- and clindamycin-resistant were 70.0% and 52.5%, respectively. Multidrug-resistant strains were 57.5%. Prevalent genetic determinant for macrolide resistance was ermB gene (75%). MLST revealed 17 STs into 5 clonal complexes and 7 singletons. Predominant genetic lineage was CC386, represented by MDR-6C non-invasive strains. Serotype 6B, principally responsible for invasive diseases in the pre-vaccine era, retreated this position to serotype 6A.

Clonal composition of human ovarian cancer based on copy number analysis reveals a reciprocal relation with oncogenic mutation status.

Intratumoral heterogeneity of cancer cells remains largely unexplored. Here we investigated the composition of ovarian cancer and its biological relevance. A whole-genome single nucleotide polymorphism array was applied to detect the clonal composition of 24 formalin-fixed, paraffin-embedded samples of human ovarian cancer. Genome-wide segmentation data consisting of the log2 ratio (log2R) and B allele frequency (BAF) were used to calculate an estimate of the clonal composition number (CC number) for each tumor. Somatic mutation profiles of cancer-related genes were also determined for the same 24 samples by next-generation sequencing. The CC number was estimated successfully for 23 of the 24 cancer samples. The mean ± SD value for the CC number was 1.7 ± 1.1 (range of 0-4). A somatic mutation in at least one gene was identified in 22 of the 24 ovarian cancer samples, with the mutations including those in the oncogenes KRAS (29.2%), PIK3CA (12.5%), BRAF (8.3%), FGFR2 (4.2%), and JAK2 (4.2%) as well as those in the tumor suppressor genes TP53 (54.2%), FBXW7 (8.3%), PTEN (4.2%), and RB1 (4.2%). Tumors with one or more oncogenic mutations had a significantly lower CC number than did those without such a mutation (1.0 ± 0.8 versus 2.3 ± 0.9, P = 0.0027), suggesting that cancers with driver oncogene mutations are less heterogeneous than those with other mutations. Our results thus reveal a reciprocal relation between oncogenic mutation status and clonal composition in ovarian cancer using the established method for the estimation of the CC number.

Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers.

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM(+) and IgG(+) memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM(+)(IgD(+)) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells--including IgM(+)IgD(+)CD27(+) B cells--are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM(+)(IgD(+)) and class-switched memory B cells.