INTRODUCTION:Understanding the molecular signals associated with the progression of kidney disease is vital for risk stratification and targeted treatment. Recent advances in RNA-sequencing technique have enabled us to characterize extracellular transcriptome profiles for precision diagnostics. METHOD:We evaluated the plasma mRNA profile of participants exhibiting slow (n = 119) and fast (n = 119) decline in estimated glomerular filtration rate (eGFR) among the Chronic Renal Insufficiency Cohort (CRIC) in a nested case control study. The two groups were matched for age, sex, race, baseline eGFR, proteinuria, and diabetes status. The next-generation sequencing data were analyzed using edgeR to identify differentially expressed genes (DEGs) and ingenuity pathway analysis was done to identify the associated pathways. We also compared the top plasma DEGs with gene expression in microdissected human chronic kidney disease (CKD) kidney. RESULTS:We identified fragments from ∼28,000 annotated genes, of which 783 transcripts exhibited differential expression between slow and fast CKD progressors. Among 629 protein coding genes, 469 were overexpressed in slow progressors, while 157 showed increased expression in fast progressors. Expression of GLI2, CUX1, NOTCH1, and LRP1 transcripts were amplified in slow progressors. Pathway analysis linked these DEG to WNT/β-catenin signaling, IL-12 signaling and production in macrophages, Netrin-1 Signaling and Epithelial-Mesenchymal Transition pathways. Many of the plasma DEGs were also upregulated in microdissected human CKD kidney. CONCLUSION:Warranting further validation, circulating levels of aberrantly expressed transcripts hold potential to be used as biomarkers for fast CKD progression.

Pax5 target genes differentially express in p-BALL bearing Pax5+/- mice according to Delogu A. et al 2006, Schebesta A. et al 2007, and Revilla-i-Domingo R. et al 2012.

<p>Supplementary Table 4: Differentially expressed genes in bone marrow proB and preB cells from ETV6-RUNX1 mice compared to wild-type bone marrow proB and preB cells. (12423 genes-probesets with FDR = 0.1).</p>

Differentially expressed genes in bone marrow tumors from Sca1-BCR-ABLp190 mice compared to wild-type B220+ bone marrow B cells. (3692 genes-probesets with FDR = 0.05)

Contents: Supplementary Table 1: Characteristics of the pediatric patients enrolled in whole-genome- and whole-exome-sequencing. Supplementary Table 2: Conventional Facility Health Monitoring Report. Supplementary Table 3: Percentage of hematopoietic subsets in young ETV6-RUNX1 and wild type mice housed in conventional facility. Supplementary Table 5: Differential regulation of histonemodifying genes in ETV6-RUNX1 mice compared to human ETV6-RUNX1 pB-ALL. Supplementary Table 6: Alterations in genes related to histone modification in pB-ALL human samples Supplementary Table 7: Summary of whole genome sequencing statistics. Supplementary Table 8: Summary of exome sequencing statistics. Supplementary Table 9: Validated SNVs in the patient cohort. Supplementary Fig. 1: Distribution of genomic ETV6-RUNX1 breakpoints amongst patients. Supplementary Fig. 2: Development of Sca1-ETV6-RUNX1 mice. Supplementary Fig. 3: pB-ALL development in Sca1-ETV6-RUNX1 mice and B cell development in young Sca1-ETV6-RUNX1 mice housed in SPF facility. Supplementary Fig. 4: Tumor exome sequencing data identify genes mutated in mouse and human. Supplementary Fig. 5: Study of proB cells in young Sca1-ETV6-RUNX1 mice.

Health monitoring report of PAx5+/- held in the SPF and conventional animal facility.

Differentially expressed genes in bone marrow tumors from Sca1-BCR-ABLp190+Pax5+/- mice compared to wild-type B220+ bone marrow B cells. (17032 genes-probesets with FDR = 0.05)

The PDF includes legends for Supplementary Tables S1 - S5 as well as legends for Supplementary Figures S1 - S3. Supplementary Figure S1 indicates pathogens detected in mice held in the SPF and conventional animal facility. Supplementary Figure S2 represents flow cytometric, histological and gene set enrichment characterization in Pax5+/- mice. Supplementary Figure S3 refers to in vivo growth of Ba/F3 cells expressing Jak3V670A and Jak3R653H mutations, respectively.

Total numbers and percentage of hematopoietic subsets in young Pax5+/- and wild type mice.

Supplementary Figure 1: Mouse generation and GSEA enrichment analysis of Sca1-BCR-ABLp190 mice. Supplementary Figure 2: Time course of the percentage of B220+ cells in PB of Sca1-BCR-ABLp190 and Sca1-BCR-ABLp190+Pax5+/- mice. Supplementary figure 3: pB-ALL is transplantable to secondary recipients. Supplementary figure 4: Sca1-BCR-ABLp190+Pax5+/- mice housed in SPF develop similar pB-ALL than Sca1-BCR-ABLp190+Pax5+/- mice housed in CF Supplementary Figure 5: transcriptome analysis of Sca1-BCR-ABLp190+Pax5+/- mice. Supplementary Figure 6: Mouse tumor exome sequencing data identified recurrent Pax5 mutations. Supplementary Figure 7: Polyclonal VDJ recombination events in pre-leukemic B220-positive cells from the BM of Sca1-BCR-ABLp190+Pax5+/- mice Supplementary Figure 8: Altered pathways in leukemic Sca1-BCR-ABLp190+Pax5+/-. Supplementary Figure 9: Quantitative differences of mRNA levels of glycolytic related genes between leukemic Sca1-BCR-ABLp190+Pax5+/-, Sca1-BCR-ABLp190 and wild-type B cells. Supplementary Figure 10: QRT-PCR validation of microarray data. Supplementary Figure 11: Metabolic signature in human pB-ALLs. Supplementary Figure 12: Analysis of ChIP-seq data revealed novel targets of PAX5 in human B lymphocytes. Supplementary Table 1: Percentage and total numbers of hematopoietic progenitor compartments in young Sca1-BCR-ABLp190 and wild type mice. Supplementary Table 3: pB-ALL genotype and phenotype of Sca1-BCR-ABLp190+Pax5+/- and Sca1-BCR-ABLp190 mice.

Supplementary Figures and Table legends as well as supplementary methods

SNVs detected by whole exam sequencing in the three index Pax5+/- pB-ALL.

Differentially expressed genes in bone marrow tumors from Pax5+/- mice compared to wild-type B220+ bone marrow B cells.

In the present study, we further analyzed the data obtained in our previous study, where we investigated the cell-free DNA (cfDNA) of 34 progressive prostate cancer patients via targeted sequencing. Here, we studied the occurrence and prognostic impact of sequence variants according to their clinical pathological significance (CPS) or their functional impact (FI) in 23 DNA damage repair (DDR) genes with a focus on the ATM serine/threonine kinase gene (ATM). All patients had at least one DDR gene with a CPS or FI variant. Kaplan-Meier analysis indicated that the group with a higher number of CPS variants in DDR genes had a shorter time to treatment change (TTC) compared to the group with a lower number of CPS variants (p = 0.038). Analysis of each DDR gene revealed that CPS variants in the ATM gene and FI variants in the nibrin (NBN) gene showed a shorter TTC (p = 0.034 and p = 0.042). In addition, patients with CPS variants in the ATM gene had shorter overall survival (OS; p = 0.022) and disease-specific survival (DSS; p = 0.010) than patients without these variants. Interestingly, patients with CPS variants in seven DDR genes possessed a better OS (p = 0.008) and DSS (p = 0.009), and patients with FI variants in four DDR genes showed a better OS (p = 0.007) and DSS (p = 0.008). Together, these findings demonstrated that the analysis of cfDNA for gene variants in DDR genes provides prognostic information that may be helpful for future temporal and targeted treatment decisions for advanced PCa patients.

Prostate cancer (PCa) is the second most common malignant cancer and is a major cause of morbidity and mortality among men worldwide. There is still an urgent need for biomarkers applicable for diagnosis, prognosis, therapy prediction, or therapy monitoring in PCa. Liquid biopsies, including cell-free DNA (cfDNA) and circulating tumor cells (CTCs), are a valuable source for studying such biomarkers and are minimally invasive. In our study, we investigated the cfDNA of 34 progressive PCa patients, via targeted sequencing, for sequence variants and for the occurrence of CTCs, with a focus on androgen receptor splice variant 7 (AR-V7)-positive CTCs. The cfDNA content was associated with overall survival (OS; p = 0.014), disease-specific survival (DSS; p = 0.004), and time to treatment change (TTC; p = 0.001). Moreover, when considering all sequence variants grouped by their functional impact and allele frequency, a significant association with TTC (p = 0.017) was observed. When investigating only pathogenic or likely pathogenic gene variants, variants of the BRCA1 gene (p = 0.029) and the AR ligand-binding domain (p = 0.050) were associated with a shorter TTC. Likewise, the presence of CTCs was associated with a shorter TTC (p = 0.031). The presence of AR-V7-positive CTCs was associated with TTC (p < 0.001) in Kaplan–Meier analysis. Interestingly, all patients with AR-V7-positive CTCs also carried TP53 point mutations. Altogether, analysis of cfDNA and CTCs can provide complementary information that may support temporal and targeted treatment decisions and may elucidate the optimal choice within the variety of therapy options for advanced PCa patients.

Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post-allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post-allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients' first relapses, which disappeared in the post-allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children's post-allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post-allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.

Constitutional mismatch repair deficiency (CMMRD) is an autosomal recessively inherited childhood cancer susceptibility syndrome caused by biallelic germline mutations in one of the mismatch repair (MMR) genes. The spectrum of CMMRD-associated tumours is very broad and many CMMRD patients additionally display signposting non-neoplastic features, most frequently café-au-lait macules and other pigmentation alterations. We report on a 13-month-old girl suspected of having CMMRD due to a desmoplastic medulloblastoma and a striking skin pigmentation that included multiple café-au-lait macules, hypopigmented areas and Mongolian spots. Whole-exome sequencing revealed homozygosity for MSH2 variant p.(Leu92Val) and MSH6 variant p.(Val809del), both variants of uncertain significance (VUS). Immunohistochemical analysis of the tumour tissue showed expression of all four MMR proteins and gMSI testing was negative. However, functional assays demonstrated that the cells of the patient displayed methylation tolerance and ex vivo microsatellite instability, which unequivocally confirmed the diagnosis of CMMRD. Taken together, the results render the MSH2 variant unlikely to be responsible for the phenotype, while they are compatible with MSH6-associated CMMRD. This case illustrates the diagnostic strategy of confirming CMMRD syndrome in patients with VUS.

Preleukemic clones carrying BCR-ABL(P190) oncogenic lesions are found in neonatal cord blood, where the majority of preleukemic carriers do not convert into precursor B-cell acute lymphoblastic leukemia (pB-ALL). However, the critical question of how these preleukemic cells transform into pB-ALL remains undefined Here, we model a BCR-ABL(P190) preleukemic state and show that limiting BCR-ABL(P190) expression to hematopoietic stem/progenitor cells (HS/PC) in mice (Sca1-BCR-ABL(P190)) causes pB-ALL at low penetrance, which resembles the human disease. pB-ALL blast cells were BCR-ABL-negative and transcriptionally similar to pro-B/pre-B cells, suggesting disease onset upon reduced Pax5 functionality. Consistent with this, double Sca1-BCR- ABI(P190) +Pax5(+/-) mice developed pB-ALL with shorter latencies, 90% incidence, and accumulation of genomic alterations in the remaining wild-type Pax5 allele. Mechanistically, the Pax5-deficient leukemic pro-B cells exhibited a metabolic switch toward increased glucose utilization and energy metabolism. Transcriptome analysis revealed that metabolic genes (IDH1, G6PC3, GAPDH, PGK1, MYC, ENO1, ACO1) were upregulated in Pax5-deficient leukemic cells, and a similar metabolic signature could be observed in human leukemia. Our studies unveil the first in vivo evidence that the combination between Sca1-BCR-ABL(P190) and metabolic reprogramming imposed by reduced Pax5 expression is sufficient for pR-All. development. These findings might help to prevent conversion of BCR-ABL(P190) preleukemic cells. Significance: Loss of Pax5 drives metabolic reprogramming, which together with Scat-restricted BCR-ABL expression enables leukemic transformation. (C) 2018 AACR.

Abstract Preleukemic clones carrying BCR-ABLp190 oncogenic lesions are found in neonatal cord blood, where the majority of preleukemic carriers do not convert into precursor B-cell acute lymphoblastic leukemia (pB-ALL). However, the critical question of how these preleukemic cells transform into pB-ALL remains undefined. Here, we model a BCR-ABLp190 preleukemic state and show that limiting BCR-ABLp190 expression to hematopoietic stem/progenitor cells (HS/PC) in mice (Sca1-BCR-ABLp190) causes pB-ALL at low penetrance, which resembles the human disease. pB-ALL blast cells were BCR-ABL–negative and transcriptionally similar to pro-B/pre-B cells, suggesting disease onset upon reduced Pax5 functionality. Consistent with this, double Sca1-BCR-ABLp190+Pax5+/− mice developed pB-ALL with shorter latencies, 90% incidence, and accumulation of genomic alterations in the remaining wild-type Pax5 allele. Mechanistically, the Pax5-deficient leukemic pro-B cells exhibited a metabolic switch toward increased glucose utilization and energy metabolism. Transcriptome analysis revealed that metabolic genes (IDH1, G6PC3, GAPDH, PGK1, MYC, ENO1, ACO1) were upregulated in Pax5-deficient leukemic cells, and a similar metabolic signature could be observed in human leukemia. Our studies unveil the first in vivo evidence that the combination between Sca1-BCR-ABLp190 and metabolic reprogramming imposed by reduced Pax5 expression is sufficient for pB-ALL development. These findings might help to prevent conversion of BCR-ABLp190 preleukemic cells. Significance: Loss of Pax5 drives metabolic reprogramming, which together with Sca1-restricted BCR-ABL expression enables leukemic transformation. Cancer Res; 78(10); 2669–79. ©2018 AACR.