Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Screening of Chelidonium majus isoquinoline alkaloids reveals berberine and chelidonine as selective ligands for the nuclear receptors RORß and HNF4α, respectively.

The nuclear receptors hepatocyte nuclear factor 4α (HNF4α) and retinoic acid receptor-related orphan receptor-ß (RORß) are ligand-regulated transcription factors and potential drug targets for metabolic disorders. However, there is a lack of small molecular, selective ligands to explore the therapeutic potential in further detail. Here, we report the discovery of greater celandine (Chelidonium majus) isoquinoline alkaloids as nuclear receptor modulators: Berberine is a selective RORß inverse agonist and modulated target genes involved in the circadian clock, photoreceptor cell development, and neuronal function. The structurally related chelidonine was identified as a ligand for the constitutively active HNF4α receptor, with nanomolar potency in a cellular reporter gene assay. In human liver cancer cells naturally expressing high levels of HNF4α, chelidonine acted as an inverse agonist and downregulated genes associated with gluconeogenesis and drug metabolism. Both berberine and chelidonine are promising tool compounds to further investigate their target nuclear receptors and for drug discovery.

Inferring replication timing and proliferation dynamics from single-cell DNA sequencing data.

Dysregulated DNA replication is a cause and a consequence of aneuploidy in cancer, yet the interplay between copy number alterations (CNAs), replication timing (RT) and cell cycle dynamics remain understudied in aneuploid tumors. We developed a probabilistic method, PERT, for simultaneous inference of cell-specific replication and copy number states from single-cell whole genome sequencing (scWGS) data. We used PERT to investigate clone-specific RT and proliferation dynamics in  >50,000 cells obtained from aneuploid and clonally heterogeneous cell lines, xenografts and primary cancers. We observed bidirectional relationships between RT and CNAs, with CNAs affecting X-inactivation producing the largest RT shifts. Additionally, we found that clone-specific S-phase enrichment positively correlated with ground-truth proliferation rates in genomically stable but not unstable cells. Together, these results demonstrate robust computational identification of S-phase cells from scWGS data, and highlight the importance of RT and cell cycle properties in studying the genomic evolution of aneuploid tumors.

Single-cell mtDNA dynamics in tumors is driven by coregulation of nuclear and mitochondrial genomes.

The extent of cell-to-cell variation in tumor mitochondrial DNA (mtDNA) copy number and genotype, and the phenotypic and evolutionary consequences of such variation, are poorly characterized. Here we use amplification-free single-cell whole-genome sequencing (Direct Library Prep (DLP+)) to simultaneously assay mtDNA copy number and nuclear DNA (nuDNA) in 72,275 single cells derived from immortalized cell lines, patient-derived xenografts and primary human tumors. Cells typically contained thousands of mtDNA copies, but variation in mtDNA copy number was extensive and strongly associated with cell size. Pervasive whole-genome doubling events in nuDNA associated with stoichiometrically balanced adaptations in mtDNA copy number, implying that mtDNA-to-nuDNA ratio, rather than mtDNA copy number itself, mediated downstream phenotypes. Finally, multimodal analysis of DLP+ and single-cell RNA sequencing identified both somatic loss-of-function and germline noncoding variants in mtDNA linked to heteroplasmy-dependent changes in mtDNA copy number and mitochondrial transcription, revealing phenotypic adaptations to disrupted nuclear/mitochondrial balance.

Single-cell decoding of drug induced transcriptomic reprogramming in triple negative breast cancers.

BACKGROUND: The encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug-associated transcriptional cell states. RESULTS: We present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure. CONCLUSIONS: Within a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration, and re-treatment strategies.

CDC7 inhibition impairs neuroendocrine transformation in lung and prostate tumors through MYC degradation.

Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.

Single-cell DNA replication dynamics in genomically unstable cancers.

Dysregulated DNA replication is both a cause and a consequence of aneuploidy, yet the dynamics of DNA replication in aneuploid cell populations remains understudied. We developed a new method, PERT, for inferring cell-specific DNA replication states from single-cell whole genome sequencing, and investigated clone-specific DNA replication dynamics in >50,000 cells obtained from a collection of aneuploid and clonally heterogeneous cell lines, xenografts and primary cancer tissues. Clone replication timing (RT) profiles correlated with future copy number changes in serially passaged cell lines. Cell type was the strongest determinant of RT heterogeneity, while whole genome doubling and mutational process were associated with accumulation of late S-phase cells and weaker RT associations. Copy number changes affecting chromosome X had striking impact on RT, with loss of the inactive X allele shifting replication earlier, and loss of inactive Xq resulting in reactivation of Xp. Finally, analysis of time series xenografts illustrate how cell cycle distributions approximate clone proliferation, recapitulating expected relationships between proliferation and fitness in treatment-naive and chemotherapeutic contexts.

Identification of transcriptional programs using dense vector representations defined by mutual information with GeneVector.

Deciphering individual cell phenotypes from cell-specific transcriptional processes requires high dimensional single cell RNA sequencing. However, current dimensionality reduction methods aggregate sparse gene information across cells, without directly measuring the relationships that exist between genes. By performing dimensionality reduction with respect to gene co-expression, low-dimensional features can model these gene-specific relationships and leverage shared signal to overcome sparsity. We describe GeneVector, a scalable framework for dimensionality reduction implemented as a vector space model using mutual information between gene expression. Unlike other methods, including principal component analysis and variational autoencoders, GeneVector uses latent space arithmetic in a lower dimensional gene embedding to identify transcriptional programs and classify cell types. In this work, we show in four single cell RNA-seq datasets that GeneVector was able to capture phenotype-specific pathways, perform batch effect correction, interactively annotate cell types, and identify pathway variation with treatment over time.

Detection of bacterial agents causing prostate infection by culture and molecular methods from biopsy specimens.

Background and Objectives: Prostatitis affects about 16% of men in their lifetime and sometimes leading to prostate cancer. Bacterial infections are the most common causes of prostatitis. Diagnosis of the causative agents of bacterial prostate infections plays an essential role in timely treating and preventing secondary complications. This study isolated bacterial infectious agents in patients' surgical prostate and evaluated them by routine and molecular microbiological methods. Materials and Methods: In this cross-sectional study, 72 prostate biopsy specimens were collected from the Orology Departmen of hospitals of Qazvin University of Medical Sciences. All samples were cultured in aerobic and anaerobic conditions. Antibiotic susceptibility test by Kirby-Bauer standard method was performed for all isolated bacteria. In addition, all isolated bacteria were identified using 16S rDNA PCR and sanger sequencing methods. Also, TaqMan real-time PCR was applied to detect Ureaplasm aurealyticum, Mycoplasma hominins, and Mycoplasma genitalium. Results: In conventional culture method, out of 18 positive samples, 15 samples (83.3%) were Gram-negative bacteria and 3 samples (16.6%) were Gram-positive bacteria, containing Escherichia coli (55.5%), Klebsiella pneumoniae (11.1%), Enterobacter cloacae (5.5%), Pseudomonas aeruginosa (11.1%), Staphylococcus aureus (11.1%), and Enterococcus faecalis (5.5%). The results of molecular identification methods were the same as conventional culture results. Also, four patients were Ureaplasm aurealyticum, and three patients were positive for Mycoplasma hominis. Conclusion: Most bacteria isolated from prostate specimens belonged to the Enterobacteriaceae family, especially Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae. Staphylococcus aureus and Enterococcus faecalis were cocci isolated in the specimens too. Also, Ureaplasma urealyticum, and Mycoplasma hominis were identified in prostatitis.

Accurate determination of CRISPR-mediated gene fitness in transplantable tumours.

Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.

Degradation of azo dye Acid Red 14 (AR14) from aqueous solution using H2 O2 /nZVI and S2 O8 2- /nZVI processes in the presence of UV irradiation.

Azo dyes are mostly toxic and carcinogenic and cause harm to humans and the environment. This study was conducted to investigate the degradation of azo dye acid red 14 (AR14) from aqueous solution using hydrogen peroxide (H2 O2) /nano zerovalent iron (nZVI) and persulfate (S2 O8 2- )/nZVI processes in the presence of ultraviolet (UV) irradiation. This experimental study was carried out in a laboratory-scale batch photoreactor with a useful volume of 1 L. The nZVI was synthesized by the sodium borohydride (NaBH4 ) reduction method. In these processes, the effects of parameters including initial pH, H2 O2 concentration, S2 O8 2- concentration, nZVI dose, concentration of AR14 dye, and reaction time were studied. The results showed that decolorization increased by increasing the nZVI dosage, H2 O2 and S2 O4 2- concentrations, and reaction time, or decreasing dye concentration and pH. However, a too high oxidant concentration (H2 O2 and S2 O4 2- ) could inhibit the degradation. The experimental conditions for degradation of AR14 by UV/S2 O8 2- /nZVI and UV/H2 O2 /nZVI processes were as follows: [H2 O2 ] = 10 mM, [S2 O8 2- ] = 8 mM, AB14 dye = 100 mg/L, pH = 3, and nZVI dose = 0.05 g. Under these conditions, the highest removal efficiencies of AR14, chemical oxygen demand (COD), and total organic carbon (TOC) for the UV/S2 O8 2- /nZVI process were 93.94%, 86.5%, and 81.6%, respectively, while these values were 89.3%, 79.57%, and 72.9% for the UV/H2 O2 /nZVI, respectively. Also, the average oxidation state (AOS) was decreased from 2.93 to 2.14 in the effluent of the UV/S2 O8 2- /nZVI process and from 2.93 to 2.2 for the UV/H2 O2 /nZVI process. The results showed that the ratio of biochemical oxygen demand (BOD5 ) to COD in the effluents of the UV/S2 O8 2 /nZVI and UV/H2 O2 /nZVI processes after 90 min was 0.63 and 0.74, respectively. These findings suggest biodegradability improvement. PRACTITIONER POINTS: Photocatalytic degradation of azo dye Acid Red 14 (AR14) was achieved using H2 O2 /nZVI and S2 O8 2- /nZVI processes in the presence of UV irradiation. Effects of operating parameters on photocatalytic degradation AR14 dye were evaluated in the UV/H2 O2 /nZVI and UV/S2 O8 2- /nZVI processes. Biodegradability and mineralization studies of AR14 dye photocatalytic degradation were performed for the UV/H2 O2 /nZVI and UV/S2 O8 2- /nZVI processes.

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

ddClone: joint statistical inference of clonal populations from single cell and bulk tumour sequencing data.

Next-generation sequencing (NGS) of bulk tumour tissue can identify constituent cell populations in cancers and measure their abundance. This requires computational deconvolution of allelic counts from somatic mutations, which may be incapable of fully resolving the underlying population structure. Single cell sequencing (SCS) is a more direct method, although its replacement of NGS is impeded by technical noise and sampling limitations. We propose ddClone, which analytically integrates NGS and SCS data, leveraging their complementary attributes through joint statistical inference. We show on real and simulated datasets that ddClone produces more accurate results than can be achieved by either method alone.

Transition zone prostate specific antigen density improves prostate cancer detection in Iranian men.

BACKGROUND: Prostate specific antigen (PSA) as a tumor marker has extensively changed the diagnosis of prostate cancer (PCa). With the advent of PSA, the majority of patients are diagnosed with nonpalpable early stage PCa. However, PSA lacks specificity and many patients undergo unnecessary biopsies due to an elevated serum PSA level. OBJECTIVES: This study aimed to assess the sensitivity and specificity of transition zone PSA density (TZPSAD) in detection of PCa. PATIENTS AND METHODS: This study was performed on 1712 men underwent trans-rectal ultrasound guided prostate biopsy in our institution between March 2008 and March 2013. A total of 1120 men with PSA < 20 ng/mL and normal digital rectal exam were selected for evaluation. Transition zone PSA density was calculated in all patients and the receiver operating characteristic (ROC) curve was used to analyze the accuracy of TZPSAD for the diagnosis of PCa. RESULTS: Among 1120 men who were eligible for enrolment, prostate cancer was detected in 265 patients. Mean serum PSA levels were 9.7 ± 4.3 ng/mL and 8.5 ± 3.7 ng/mL in patients with and without PCa, respectively (P < 0.001). Mean value for TZPSAD was 1.18 ± 1.19 ng/mL/mL in patients with PCa, whereas it was 0.55 ± 0.84 ng/mL in men without cancer (P < 0.001). Optimal cut-off value for TZPSAD was 0.32 ng/mL. At this cut-off value, the sensitivity and specificity values for TZPSAD were 85% and 45%, respectively. Applying the TZPSAD for PCa screening decreased 50% of unnecessary biopsies. CONCLUSIONS: Using TZPSAD as an adjunct to PSA may improve the specificity of PSA in the diagnosis of PCa and decrease the number of unnecessary prostatic biopsies in Iranian men with serum PSA level < 20 ng/mL.

Postchemotherapy Retroperitoneal Lymph Node Dissection in Patients With Nonseminomatous Testicular Cancer: A Single Center Experiences.

BACKGROUND: Testicular cancer accounts for about 1 - 1.5% of all malignancies in men. Radical orchiectomy is curative in 75% of patients with stage I disease, but advance stage with retroperitoneal lymph node involvement needs chemotherapy. All patients who have residual masses ≥ 1 cm after chemotherapy should undergo postchemotherapy retroperitoneal lymph node dissection (PC-RPLND). OBJECTIVES: Treatment of advanced nonseminomatous testicular cancer is usually a combination of chemotherapy and surgery. We described our experience about postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) in our center. PATIENTS AND METHODS: In a retrospective cross-sectional study between 2006 and 2011, patients with a history of postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) in Imam Khomeini hospital were evaluated. All patients had normal postchemotherapy serum tumor markers and primary nonseminomatous cancer. We reviewed retrospectively clinical, pathological, and surgical parameters associated with PC-RPLND in our center. RESULTS: Twenty-one patients underwent bilateral PC-RPLND. Mean age was 26.3 years (ranged 16 - 47). Mean size of retroperitoneal mass after chemotherapy was 7.6 cm. Mean operative time was 198 minutes (120 - 246 minutes). Mean follow-up time was 38.6 months. Pathologic review showed presence of fibrosis/necrosis, viable germ cell tumor and teratoma in 8 (38.1%), 10 (47.6%) and 3 (14.28%) patients, respectively. One patient in postoperative period of surgery and three patients in two first years after surgery were expired. Of 17 alive patients, only two (11.8%) had not retrograde ejaculation. CONCLUSIONS: PC-RPLND is one the major operations in the field of urology, which is associated with significant adjunctive surgeries. In appropriate cases, PC-RPLND was associated with good cancer specific survival in tertiary oncology center.