Clonal Origin Research Articles

BIOM-71. EXPANDING THE SPECTRUM OFTERT PROMOTER NON-CANONICAL SEQUENCE VARIANTS IN CNS TUMORS: A CASE SERIES

Abstract Most TERT promoter (TERTp) clinically relevant sequence variants in CNS tumors are the canonical C228T and C250T mutations. Non-canonical TERTp mutations have been reported mainly in non-CNS tumors. We describe 12 CNS tumors with non-canonical TERTp variants interpreted as clinically relevant. Using clinical targeted NGS panels (2020-2024), we detected c.-100_-79dup (n=2), c.-92_-91insGGCGGCCCCGCCCCTTCCTTTC (n=1), c.-118_-117insTCCCCGGCCCAGCCCCTTCCGGG (n=1), c.-123_-76dup (n=1), c.-110_-89dup (n=2), and c.-57A>C (n=5). Most cases had chromosomal microarray (n=11) and/or methylation array data analyzed using the NCI/Bethesda v.2 classifier (n=9). All non-canonical TERTp variants occurred at a variant allele frequency suggestive of somatic clonal origin (mean, 34%; range, 18-46), and in adults (mean age, 60 years; range, 36-88). Except for the c.-57A>C variant, all non-canonical TERTp variants occurred in tumors with histological and/or molecular diagnostic features of glioblastoma, IDH-wildtype (GBM), often epigenetically aligning to this tumor type. All five tested cases matched to superfamily glioblastoma (score ≥ 0.9), with three to class GBM_RTK_I (>0.9 in 2, 0.86 in 1) and two to GBM_RTK_II (>0.9). The size and sequence of such variants suggest that these may be functionally equivalent to canonical mutations or generate favorable TERTp activation motif conformations like previously reported activating duplications. The c.-57A>C variant is a well-established oncogenic mutation observed in other cancer types but has not been reported in CNS tumors. We describe five cases with the c.-57A>C variant: one GBM matching to methylation class GBM_MES_TYP (0.99); two oligodendroglioma, IDH-mutant and 1p/19q-codeleted, one of which had methylation data matching to class O_IDH (0.99); one diffuse glioma, IDH-mutant, which was partially 1p/19q-codeleted and matched to class O_IDH (0.99); and one anaplastic meningioma, aligning to superfamily meningioma (0.99)/MNG_MAL (0.79). Our findings show that non-canonical TERTp variants typically occur in tumor types that often harbor TERTp canonical mutations, expanding the spectrum of TERTp clinically relevant variants that are molecular biomarkers in CNS tumors.

Evolutionary Characteristics in Primary Aldosteronism Patients.

Primary aldosteronism is predominantly caused by excessive aldosterone production from the adrenal cortex, and the aldosterone-producing structures could take many forms, like adenomas, nodules, micronodules, and so on. Most studies of primary aldosteronism were limited to the hotspot driver genes responsible for autonomous aldosterone production; however, the panoramic genetic architecture and genomic alterations of aldosterone-producing structures and their adjacent hyperplasia glands remain unknown. In this study, whole-exome sequencing and transcriptome sequencing (RNA-seq) analyses were performed using functional nodules and matched hyperplasia tissues, which were microdissected guided by aldosterone synthase immunohistochemistry. Phylogenetic trees were constructed based on the shared and unique mutations, gene mutation spectrums, and clonal characteristics. The rates of mutations represented higher means of functional nodules than hyperplasia samples, and the little mutational overlap was shown between the 2 groups on phylogenetic trees. The mutations of the aldosterone driver gene (KCNJ5 or CACNA1D) were only observed in functional nodules and indicated almost the largest values of cancer cell fraction. Moreover, the functional nodules also harbored some potential variants related to cell proliferation, which were not detected in hyperplasia tissues. Transcriptome analysis suggested that only 25.5% upregulated and 23.3% downregulated genes overlapped between functional nodules and hyperplasia tissues. This study demonstrated a genetic and transcriptome landscape of aldosterone-producing structures and adjacent hyperplasia glands in primary aldosteronism. The results indicated independent clonal origins on functional nodules and hyperplasia tissues, and little mutual evolutionary relationship was found on their phylogenetic trees.

Localized blastic plasmacytoid dendritic cell neoplasm associated with progressive clonal hematopoiesis and myelodysplastic syndrome.

Clonal hematopoiesis is highly prevalent in elderly patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), and around 20% of BPDCN patients have an associated myeloid malignancy. We present a patient with localized BPDCN and concomitant myelodysplastic syndrome (MDS), previously followed for several years due to clonal cytopenia of unknown significance. Compared targeted next-generation sequencing (NGS) of the skin tumor and sequential bone marrow samples showed shared variants in ASXL1 and TET2 with a de novo NRAS variant in both BPDCN and MDS compared to preceding bone marrow samples. These findings illustrate a shared clonal origin of BPDCN and MDS resulting from progressive clonal hematopoiesis.

Diversity of Northwestern and North Caucasian Populations of <i>Puccinia striiformis</i> f. sp. <i>tritici</i> by Virulence and Microsatellite Loci in 2022

Yellow rust (Puccinia striiformis f. sp. tritici, Pst) is a potentially dangerous disease of wheat. Genetic protection of wheat is an environmentally safe method of control. For its successful application, information on the structure of regional populations of the pathogen is needed. The purpose of these studies is to characterize the virulence and molecular polymorphism of Russian Pst populations in 2022. Wheat leaves with Pst urediniopustules were obtained from the North Caucasus (Krasnodar Territory, Dagestan, Kalmykia) and Northwestern (Leningrad region) regions. Virulence analysis was performed on 14 isogenic lines (AvocetNIL) and 15 differentiator varieties. The polymorphism of 20 microsatellite loci was evaluated in molecular analysis. We used SSR markers recommended by the Global Rust Reference Center. Virulence was studied in 74 monopustular isolates: 29 Dagestan, 10 Krasnodar, 5 Kalmyk, 30 northwestern. Resistance to all isolates was shown for lines with genes Yr5, Yr10, Yr15, Yr24, Yr26 and varieties Moro (Yr10, YrMor) and Nord Desprez (Yr3, YrND, Yr+). Isolates virulent to the AvYr17 line were detected for the first time in Dagestan and Krasnodar populations. They were moderately represented in the northwestern population (13%). Significant variation was observed in lines and varieties with the Yr1 and Yr3 genes. There was a decrease in virulence to Yr7 and YrSp compared to 2019–2021. 28 phenotypes (races) were determined in the virulence analysis (15 in Dagestan, 11 in North West, 3 in Krasnodar and 2 in Kalmyk). A common phenotype was noted for three North Caucasian Pst samples. The genetic distances between the identified phenotypes were estimated. In the multidimensional diagram, most of them combined together in a common group, with the exception of three Dagestan phenotypes with the fewest virulence alleles. According to the Fst index, Dagestan and Kalmyk Pst collections were characterized by high similarity; others differed moderately from them. The long-term virulence dynamics (2019–2022) of Pst populations in the Northwestern and North Caucasian regions was assessed. High similarity was determined between all regional population samples in 2019 and 2020. The northwestern and Dagestan populations differed slightly from them in 2021 and 2022. In 2022, the Krasnodar and Kalmyk populations were divided into separate groups, which differed from each other and from the main group. Long-term results of virulence analysis indicate a high dynamic of the structure of Pst populations in Russia. All North Caucasian isolates and 23 Northwestern isolates were used in the SSR analysis. Six loci (RYN3, RYN9, RYN12, WU6, RJO21, RJO24) were monomorphic. Three polymorphic alleles were identified in the RYN13 and RJO27 loci and two in the remaining studied ones. Significant deviations from the Hardy-Weinberg equilibrium are noted for most loci. The observed heterozygosity exceeded the expected one, which indicates an excess of heterozygotes and the clonal origin of the population. The studied collection of isolates was represented by 20 multilocus genotypes (MGs) (Dagestan and Northwestern – 11 each, Krasnodar – 3, Kalmyk – 1). Common genotypes were detected in Dagestan, Krasnodar and Northwestern populations (MG_1); Dagestan, Kalmyk and Northwestern (MG_2); Dagestan and Krasnodar (MG_3, MG_4). The genetic distances between MGs were estimated. In the multidimensional diagram, they are divided into 4 groups. The main group included 80% of MGs. One Dagestan MG, two Northwestern MG and MG_3 common for Dagestan and Krasnodar collections, significantly differentiated from the main group and among themselves. According to the Fst index, most regional Pst collections were moderately differentiated among themselves, with the exception of Dagestan and Kalmyk, which is consistent with the virulence analysis. According to the Mantel test, a moderate correlation was found between the results of virulence analysis and SSR (r = 0.6). This indicates that both analyses can be used in assessing the genetic polymorphism of Pst. The high variability of Russian populations based on virulence and microsatellite loci determines the need for annual monitoring of regional Pst populations in Russia.

Polyploid cancer cells reveal signatures of chemotherapy resistance.

Therapeutic resistance in cancer significantly contributes to mortality, with many patients eventually experiencing recurrence after initial treatment responses. Recent studies have identified therapy-resistant large polyploid cancer cells in patient tissues, particularly in late-stage prostate cancer, linking them to advanced disease and relapse. Here, we analyzed bone marrow aspirates from 44 advanced prostate cancer patients and found the presence of CTC-IGC was significantly associated with poorer progression-free survival. Single cell copy number profiling of CTC-IGC displayed clonal origins with typical CTCs, suggesting complete polyploidization. Induced polyploid cancer cells from PC3 and MDA-MB-231 cell lines treated with docetaxel or cisplatin were examined through single cell DNA sequencing, RNA sequencing, and protein immunofluorescence. Novel RNA and protein markers, including HOMER1, TNFRSF9, and LRP1, were identified as linked to chemotherapy resistance. These markers were also present in a subset of patient CTCs and associated with recurrence in public gene expression data. This study highlights the prognostic significance of large polyploid tumor cells, their role in chemotherapy resistance, and their expression of markers tied to cancer relapse, offering new potential avenues for therapeutic development.

Diversity of Northwestern and North Caucasian Populations of Puccinia striiformis f. sp. tritici by Virulence and Microsatellite Loci in 2022.

Yellow rust (Puccinia striiformis f. sp. tritici (Pst)) is a potentially dangerous disease of wheat. Genetic protection of wheat is an environmentally safe method to control the disease, but its successful application requires information on the structure of regional populations of the pathogen. Virulence and molecular polymorphism were characterized in two Russian Pst populations in 2022. Wheat leaves with Pst urediniopustules were collected from the North Caucasus (Krasnodar Krai, Dagestan, and Kalmykia) and Northwestern Russia (Leningrad Oblast). Virulence was tested on 14 isogenic lines (AvocetNIL) and 15 differentiator cultivars. Polymorphism at 20 microsatellite loci was evaluated in molecular analyses. The SSR markers were as recommended by the Global Rust Reference Center. Virulence was assessed in 74 monopustular isolates, including 29 Dagestan, 10 Krasnodar, 5 Kalmyk, and 30 Northwestern ones. Resistance to all isolates was observed in lines with the genes Yr5, Yr10, Yr15, Yr24, and Yr26 and the cultivars Moro (Yr10, YrMor) and Nord Desprez (Yr3, YrND, Yr+). Isolates virulent to the AvYr17 line were detected for the first time in the Dagestan and Krasnodar populations. Their occurrence was moderate (13%) in the Northwestern population. A significant variation was observed in lines and cultivars with the Yr1 and Yr3 genes. Virulence to Yr7 and YrSp was found to be lower than in 2019 to 2021. In total, 28 phenotypes (races) were determined in the virulence analysis (15 in Dagestan, 11 in the Northwestern region, 3 in Krasnodar, and 2 in Kalmykia). A common phenotype was detected in three North Caucasian Pst samples. Genetic distances between the phenotypes were estimated. Most phenotypes grouped together in a multidimensional diagram, with the exception of three Dagestan phenotypes with the fewest virulence alleles. Based on the Fst index, the Dagestan and Kalmyk Pst collections were high similar to each other and differed moderately from the other collections. Long-term trends in virulence (from 2019 to 2022) were assessed in the Pst populations. A high similarity was observed between all regional population samples in 2019 and 2020. The Northwestern and Dagestan populations slightly differed from the other populations in 2021 and 2022. In 2022, the Krasnodar and Kalmyk populations formed separate groups, which differed from each other and from the main group. The long-term virulence analysis indicated that the structure of Pst populations is highly dynamic in Russia. All North Caucasian isolates and 23 Northwestern isolates were used in the SSR analysis. Six loci (RYN3, RYN9, RYN12, WU6, RJO21, and RJO24) were monomorphic. Three polymorphic alleles were identified in each of the RYN13 and RJO27 loci; two alleles, in each of the remaining loci examined. Significant deviations from Hardy-Weinberg equilibrium were observed for most loci. The observed heterozygosity exceeded the expected one, suggesting a clonal origin of the Pst population. Twenty multilocus genotypes (MGs) were found in the total isolate collection (11 in Dagestan, 11 in the Northwestern region, 3 in Krasnodar, and 1 in Kalmykia). Common MGs were detected in the Dagestan, Krasnodar, and Northwestern populations (MG_1); Dagestan, Kalmyk, and Northwestern populations (MG_2); and Dagestan and Krasnodar populations (MG_3 and MG_4). Genetic distances between MGs were estimated. MGs formed four groups in a multidimensional diagram. A major group included 80% of MGs. One Dagestan MG, two Northwestern MGs, and MG_3 common for the Dagestan and Krasnodar collections were significantly differentiated from the major group and differed from each other. Based on Fst, most regional Pst collections were moderately differentiated from each other, with the exception of the Dagestan and Kalmyk collections. The finding was consistent with the virulence analysis results. The Mantel test detected a moderate correlation between the virulence and SSR data (r = 0.6). This indicates that both analyses can be used to assess genetic polymorphism in Pst. The high variability of the virulence and microsatellite loci warrants annual monitoring of regional Pst populations in Russia.

Clonal Evolution in 207 Cases of Refractory or Relapsed Acute Myeloid Leukemia.

Clonal evolution (CE) is a driving force behind the development and progression of acute myeloid leukemia (AML). Advances in molecular and cytogenetic assays have improved the depth and breadth of detection of CE in AML, which is defined here as a detected change in cytogenetic or molecular profile at relapsed or refractory (RR) disease. In this study, we demonstrate the clinical impact of CE in a cohort of patients with RR AML treated between 2013 and 2023. We discovered CE is significantly more frequent in relapsed disease (58.2%, [46.6%, 69.2%]) than in refractory disease (21.1%, [14.4%, 29.2%], p < 0.001). CE negatively impacts prognosis when detected by conventional karyotyping in refractory disease (4.2 vs. 13.9 months, p < 0.011). In contrast with prior literature, CE had no impact on overall survival if detected in relapsed disease. Surprisingly, those who achieved negative measurable residual disease (MRD) were no more likely to eliminate their original clone than those who did not (p = 1). We found several cytogenetic and molecular signatures which may predispose to CE: aberrations of chromosome 17, trisomy 8, TP53, KRAS, and FLT3-TKD. Finally, physicians were less likely to retreat those with CE with IC after receiving IC as first-line therapy (35.0% vs. 70.9%, p = 0.004). This study illustrates the role of CE in chemotherapy-resistant AML; we identify unique cytogenetic and molecular signatures that define a subset of patients associated with a dismal prognosis. As next-generation sequencing panels expand and new methods to characterize cytogenetic abnormalities emerge, our findings establish a basis for future studies investigating the prognostic and therapeutic impact of CE.

Analysis of the clonal origin and differences in the biological behavior of multifocal papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) exhibits a trend of multifocal growth. However, the clonal origin of multiple cancer foci in the thyroid gland remains an issue of ongoing debate. In order to investigate the clonal origin and biological behavior differences of multifocal PTC (MPTC) from a unique perspective, a combination of dual gene and dual protein detection methods was used. The present study included 52 patients with MPTC. Immunohistochemical staining was used to assess the expression of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and telomerase reverse transcriptase (TERT) proteins, while quantitative PCR and Sanger sequencing were used to identify BRAF and TERT gene mutations. Based on the results, MPTC cases were classified into two clonal origins, namely intraglandular metastatic (71.2%) and independent multicentric origin (28.8%). BRAF protein expression and BRAF gene mutation were significantly higher in the intraglandular metastasis group than in the multicentric cancer group. However, no significant differences in TERT protein expression and TERT gene mutation were observed between the two groups. Sex, central lymph node metastasis rate, Hashimoto's thyroiditis and tumor distribution laterality were not found to differ significantly between the two groups. However, significant differences were detected in age at initial diagnosis, lateral cervical lymph node metastasis rate, tumor capsule invasion rate and maximum tumor diameter. The study found that MPTC predominantly occurs due to intraglandular metastasis, which is associated with stronger tumor invasiveness than cancer foci with multiple independent origins, as it is more likely to exhibit pathogenic gene mutations and abnormal protein expression, cervical lymph node metastasis and capsule invasion. Therefore, it is recommended that the surgical approaches and follow-up strategies for intraglandular metastatic MPTC should be aggressive and individualized.

Multi-omics analysis of SIV-specific CD8+ T cells in multiple anatomical sites.

CD8+ T cells exert immunological pressure against immunodeficiency lentiviruses. In previous studies, we examined the TCR repertoire of CD8+ T cells specific for a single SIV immunodominant epitope, Gag-CM9, throughout SIV infection or after vaccination, and across multiple anatomic sites. We identified both tissue specific TCR sequences and TCRs shared by multiple anatomical sites. Here we use single cell RNA sequencing to evaluate if the tissue localization or TCR sequence of a CM9-specific CD8+ T cell corresponds with unique transcriptomics. CM9-specific CD8+ T cells were sorted from blood, lymph nodes, spleen, and liver from SIV infected rhesus macaques with progressive SIV infection and in animals who spontaneously control SIV replication after cessation of antiretroviral therapy. The cells were processed through a single cell sequencing protocol, creating a TCR amplified library and an RNA gene expression library corresponding to individual cells. Gene set enrichment analysis revealed no distinct transcriptional profiles for CM9 specific CD8+ T cells between different anatomical sites and between cells with shared or tissue specific TCRs. Similarly, no clear transcriptional profiles were associated with clonotypes which were shared across individual animals. However, CM9 specific CD8+ T cells from posttreatment controllers did exhibit enrichment of pathways associated with cellular activation compared to progressively infected animals, suggesting that altered transcription in distinct cellular pathways in antigen specific CD8+ T cells may associate with viral control. Together, these studies represent a thorough analysis of the relationship between anatomical and clonal origin, and the transcriptional profile of antigen specific CD8+ T cells and unravel pathways that may be important for CD8+ T cell mediated control of SIV replication.

Generation of rabbit single-chain variable fragments with different physicochemical and biological properties by complementary determining region-grafting technology

In this study, we have demonstrated a CDR grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the complementary determining region/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of CDR with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L, and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties.

High-level ceftriaxone resistance due to transfer of penA allele 60.001 into endemic gonococcal lineages in Hangzhou, China.

Neisseria gonorrhoeae strains associated with the high-level ceftriaxone-resistant FC428 clone or containing its main resistance determinant, penA allele 60.001, have shown global transmission. In Hangzhou, China, 10% of the isolates were associated with the FC428 clone in 2019. Here, we investigated ceftriaxone resistance and the prevalence of FC428-associated strains in Hangzhou in 2020-22. A total of 209 gonococcal isolates were investigated for antimicrobial susceptibility to ceftriaxone and other antibiotics by agar dilution method. Sequence types and penA alleles were determined by PCR and sequence analysis. Resistance to ceftriaxone (MIC > 0.125 mg/L) was observed for 16% (33/209) of the isolates, whereas 6.7% (14/209) of the isolates displayed high-level ceftriaxone resistance (MIC = 1 mg/L). These 14 high-level ceftriaxone-resistant isolates and another isolate displaying an MIC = 0.25 mg/L contained penA allele 60.001, with eight of these isolates, all from 2020 to 2021 belonging to MLST ST1903, the sequence type commonly associated with the original FC428 clone. Importantly, the six penA allele 60.001-containing isolates from 2022 belonged to MLST ST8123, ST7365 and ST7367, which are among the most frequently encountered sequence types found in China. Therefore, these results indicate that endemic lineages in China have acquired penA allele 60.001. Here, we report continued transmission of gonococcal strains associated with the FC428 clone or containing penA allele 60.001 in Hangzhou. A major concern for public health is the acquisition of penA allele 60.001 by successful endemic lineages, which might enhance the transmission of this high-level ceftriaxone resistance trait.

Polyploid cancer cells reveal signatures of chemotherapy resistance.

Therapeutic resistance in cancer significantly contributes to mortality, with many patients eventually experiencing recurrence after initial treatment responses. Recent studies have identified therapy-resistant large polyploid cancer cells in patient tissues, particularly in late-stage prostate cancer, linking them to advanced disease and relapse. Here, we analyzed bone marrow aspirates from 44 advanced prostate cancer patients and found the presence of circulating tumor cells with increased genomic content (CTC-IGC) was significantly associated with poorer progression-free survival. Single cell copy number profiling of CTC-IGC displayed clonal origins with typical CTCs, suggesting complete polyploidization. Induced polyploid cancer cells from PC3 and MDA-MB-231 cell lines treated with docetaxel or cisplatin were examined through single cell DNA sequencing, RNA sequencing, and protein immunofluorescence. Novel RNA and protein markers, including HOMER1, TNFRSF9, and LRP1, were identified as linked to chemotherapy resistance. These markers were also present in a subset of patient CTCs and associated with recurrence in public gene expression data. This study highlights the prognostic significance of large polyploid tumor cells, their role in chemotherapy resistance, and their expression of markers tied to cancer relapse, offering new potential avenues for therapeutic development.

Comprehensive genomic analysis reveals clonal origin and subtype-specific evolution in a case of sporadic multiple meningiomas.

Meningioma is the most common primary intracranial tumor in adults, with up to 10% manifesting as multiple tumors. Data on the genomic and molecular changes in sporadic multiple meningiomas are scarce, leading to ongoing debates regarding their evolutionary processes. A comprehensive genetic analysis of a large number of lesions, including precursor lesions, is necessary to explore these two possible origins: clonal and independent. In the present study, we performed whole-exome sequencing and analyzed somatic single-nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number alterations (CNAs) in a patient with sporadic multiple meningiomas. These meningiomas included two mass-forming lesions of different histological subtypes (transitional and chordoid) and two small meningothelial nests. Genetic analysis revealed CNAs on chromosomes 22q and Y as common abnormalities in the two largest tumors. Furthermore, we identified SNV/INDELs unique to each focus, with NF2 mutation prevalent in the transitional meningioma and CREBBP mutation in the chordoid meningioma. Loss of chromosome 22 was detected in two small meningothelial nests. Overall, we elucidated the clonal origin and subtype-specific evolution of multiple meningiomas in this case. CNAs may serve as the initial driving event in meningioma development.

Patterns of molecular evolution in a parthenogenic terrestrial isopod (Trichoniscus pusillus).

The "paradox of sex" refers to the question of why sexual reproduction is maintained in the wild, despite how costly it is compared to asexual reproduction. Because of these costs, one might expect nature to select for asexual reproduction, yet sex seems to be continually selected for. Multiple hypotheses have been proposed to explain this incongruence, including the niche differentiation hypothesis, the Red Queen hypothesis, and accumulation of harmful mutations in asexual species due to inefficient purifying selection. This study focuses on the accumulation of mutations in two terrestrial isopods, Trichoniscus pusillus, which has sexual diploid and parthenogenic triploid forms, and Hyloniscus riparius, an obligately sexual relative. We surveyed sex ratios of both species in an upstate New York population and obtained RNA-seq data from wild-caught individuals of both species to examine within- and between-species patterns of molecular evolution in protein-coding genes. The sex ratio and RNA-seq data together provide strong evidence that this T. pusillus population is entirely asexual and triploid, while the H. riparius population is sexual and diploid. Although all the wild T. pusillus individuals used for sequencing shared identical genotypes at nearly all SNPs, supporting a clonal origin, heterozygosity and SNP density were much higher in T. pusillus than in the sexually reproducing H. riparius. This observation suggests this parthenogenic lineage may have arisen via mating between two divergent diploid lineages. Between-species sequence comparisons showed no evidence of ineffective purifying selection in the asexual T. pusillus lineage, as measured by the ratio of nonsynonymous to synonymous substitutions (dN/dS ratios). Likewise, there was no difference between T. pusillus and H. riparius in the ratios of nonsynonymous to synonymous SNPs overall (pN/pS). However, pN/pS ratios in T. pusillus were significantly higher when considering only SNPs that may have arisen via recent mutation after the transition to parthenogenesis. Thus, these recent SNPs are consistent with the hypothesis that purifying selection is less effective against new mutations in asexual lineages, but only over long time scales. This system provides a useful model for future studies on the evolutionary tradeoffs between sexual and asexual reproduction in nature.

Non-small Cell Lung Cancer with Metachronous Mutations of EGFR and ALK Genes: A Case Report and Literature Review

Multiple primary lung cancer (MPLC) refers to patients with two or more primary lesions of lung cancer. It can be divided into synchronous MPLC (sMPLC) and metachronous MPLC (mMPLC) based on the timing of occurrence. In recent years, the detection rate of MPLC has gradually increased. However, considerable controversy exists in distinguishing MPLC from intrapulmonary metastasis (IM), especially when the histopathological types are identical. Given the significant differences in treatment strategies and prognosis in clinical practice currently, accurate diagnosis of MPLC is crucial for personalized precision therapy. Molecular genetics and sequencing technologies offer effective strategies for assessing the clonal origin of tumors. There have been reports of coexisting mutations in the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) fusion genes in non-small cell lung cancer, but case of EGFR mutation following an ALK mutation has not been mentioned. This article accurately diagnoses and retrospectively analyzes the clinical data of a case of ALK mutant adenocarcinoma in a male patient who developed an EGFR mutation with multiple metastases four years after surgery, and reviews the relevant literature. This paper aims to deepen the understanding of mMPLC and provide clinical references for the diagnosis and treatment of such patients. .

Clinical and molecular risk factors for repeat interventions due to symptomatic uterine leiomyomas

BackgroundRepeat leiomyoma occurrence or even reintervention is common after myomectomy. Little is known about the factors related to repeat interventions. ObjectivesThis study aimed to determine the frequency of leiomyoma-related reintervention after an initial laparoscopic or abdominal myomectomy and to analyze both clinical and molecular risk factors for reinterventions. Another objective was to define the frequency of clonally related tumors from repeat operations. Study DesignThis retrospective cohort study included 234 women who had undergone laparoscopic or abdominal myomectomy in 2009-2014. Information on repeat leiomyoma-related interventions as well as on other clinical factors was collected from medical records after a median follow-up time of 11,4 years (range 7,9-13,8 years) after the index procedure. The effect of clinical risk factors on the risk of reintervention was analyzed by the Kaplan-Meier estimator and the Cox proportional hazards model. For molecular analyses, we examined the mutation profiles of 133 formalin-fixed paraffin-embedded leiomyoma samples from 33 patients with repeat operations. We screened the tumors for the three primary leiomyoma driver alterations—MED12 mutations, HMGA2 overexpression, and FH-deficiency—utilizing Sanger sequencing and immunohistochemistry. To further assess the clonal relationship of the tumors, we executed whole-exome sequencing for 52 leiomyomas from 21 patients who exhibited the same driver alteration in tumors obtained from multiple procedures. ResultsReintervention rate at 11,4 years after myomectomy was 20% (46/234). Number of leiomyomas removed at the index myomectomy was a risk factor (hazard ratio 1.21; 95% confidence interval 1.09-1.34). Age at index myomectomy (hazard ratio 0.94; 95% confidence interval 0.89-0.99) and postoperative parity (hazard ratio 0.23; 95% confidence interval 0.09-0.60) were protective factors. Molecular characterization of tumors from index and non-index operations confirmed a clonal relationship of the tumors in 3/33 (9%) patients. None of the leiomyomas harboring a MED12 mutation —the most common leiomyoma driver— were confirmed clonally related. FH-deficiency was detected in repeat leiomyomas from 3/33 (9%) patients. All these patients harbored a germline FH mutation, which is distinctive for the hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. Finally, we identified three (3/33; 9%) patients with two tumors each displaying somatic mutations in a recently identified novel leiomyoma driver gene, YEATS4. All YEATS4 mutations were different and thus the tumors were not clonally related. ConclusionOur study shows that reintervention is common after surgical myomectomy. Uterine leiomyomas typically develop independently, but some share a clonal origin. Repeat leiomyoma occurrence may be due to genetic predisposition, such as a germline FH mutation. Distinct somatic YEATS4 mutations identified in multiple leiomyomas from the same patient indicate a possible role for YEATS4 in repeat leiomyomas.

Disease characterization in cloned foals in Colombia, the effect of placental pathologies on the success and survival of cloned foalsfrom two different cell lines

Cloned foals born in the different cloning programs in the USA, Argentina, and recently in Colombia have experienced several pathological entities during their pregnancy and neonatal period. These entities include abortions, stillbirths, hypoxic-ischemic encephalopathy, large umbilical remanent, and angular deformity on the fore limb. The objectives of this study were to determine whether cloned foals suffer similar diseases and have the same mortality rate as noncloned ones and the differences between cloned foals from two different cell lines (fibroblastic and bone marrow cells) and, to establish whether mares carrying cloned foals from different cell lines have ultrasonographic, paraclinical, and hormonal profile parameters equal to traditional pregnancies and to determine whether there are differences between the cloned foals from the two different cell lines. In the analysis of the continuous variables, significant mean differences were established in the three groups of mares in PCV, hemoglobin, erythrocyte count, MCV, MCH, serum proteins and globulins, and in lymphocyte counts p&lt;0.05. A risk analysis was made for morbidity and mortality associated with the cell origin of the clone and its pregnancy using chi-square sequential tests and logistic regression, and it was considered statistically significant at p&lt; 0.05. A principal component analysis of the paraclinical findings of the mares with the outcome of the foal and the paraclinical parameters of the foals divided by the origin of gestation was performed to evaluate the components of the response variable and their dynamics. Thirty-four pregnancies of clones of fibroblastic origin, 26 of clones of bone marrow origin, and 32 of non-clones were studied. The presentation of placental diseases compatible with placentitis was significantly more frequent (p= 0.026) in mares with cloned foals from fibroblastic cell lines. Neonatal sepsis was determined to be the most frequent disease and was diagnosed more commonly in foals of fibroblastic cell origin—an increase in the risk (OR 37) (p=0.0006). The risk of death was positive and significant with the fibroblast cell line foals (OR: 9.1. P&lt;0.01) compared to the cloned foals of marrow cell origin and non-cloned foals. The risk of death can be predicted in the neonatal period, and some indicators found in this study, such as signs related to placentitis, are associated with increased neonatal mortality and are more frequent in foals of fibroblastic cell origin.

Characterization of driver oncogenic mutations of in-transit melanoma metastases.

9586 Background: In-transit melanoma (ITM) is a distinctive form of melanoma metastasis, marked by aggressive locoregional progression and characterized by the entrapment of tumor cells within the lymphatic channels before reaching regional lymph nodes (LN). The mechanisms leading to lymphatic trapping rather than progression to distant metastasis are not well-understood. Previous research indicates a clonal origin for ITM, suggesting specific early-stage genetic alterations may drive this clinical phenotype. In this study, we sought to identify driver oncogenic mutations specific to ITM that may serve as potential targets. Methods: We analyzed data from the MSK-IMPACT database, which includes targeted sequencing of 341-468 genes for patients with cutaneous melanoma. Patients were stratified into four groups based on the type of sample sequenced: in-transit, primary tumor, regional LN, and distant metastases. We examined the prevalence of driver mutations and genetic variants across these groups, adjusting for multiple comparisons. Results: A total of 528 patients were included, of which 74 were in the ITM, 103 in the primary, 133 in the regional LN, and 218 in the metastatic group. The mean age of the cohort was 59 years (SD = 16) and 67% were male. Among the in-transit samples, 68% were treatment-naïve at the time of sequencing, 36% received anti-PD1 monotherapy, and 12% received anti-PD-1 combination therapy. None of the patients received targeted therapy. NRAS Q61 driver mutations were significantly more common in ITM than all other groups (all p&lt;0.05). NF-1 driver mutations were significantly less common in ITM than distant metastasis (p=0.007) (Table). Comparative analysis across all genes showed a lower incidence of NF-1 mutations in ITM relative to metastatic samples (12% vs 38%, p=0.007). Gene interaction analysis showed mutual exclusivity between NRAS and PAK5 mutations in ITM (p=0.02), but not in other groups (p&gt;0.05). Conclusions: This study reveals distinct driver oncogenic mutations specific to ITM, characterized by a high incidence of NRAS mutations, retained NF-1 function and the novel finding of mutual exclusivity between NRAS and PAK5. Closer examination of the interplay between NRAS, NF-1 and PAK5 interactions in ITM may provide deeper insights into the molecular mechanisms of ITM and offer potential therapeutic targets for ITM. [Table: see text]

Integrative multi-region molecular profiling of primary prostate cancer in men with synchronous lymph node metastasis

Localized prostate cancer is frequently composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing clones that may drive the biological trajectory of the disease. Previous large-scale sequencing efforts have focused on the evolutionary process in metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we perform multi-region, targeted next generation sequencing and construct phylogenetic trees in men with prostate cancer with synchronous LN metastasis to better define the pathologic and molecular features of primary disease most likely to spread to the LNs. Collectively, we demonstrate that a combination of histopathologic and molecular factors, including tumor grade, presence of extra-prostatic extension, cellular morphology, and oncogenic genomic alterations are associated with synchronous LN metastasis.

Generation and maintenance of the circularized multimeric IS26-associated translocatable unit encoding multidrug resistance

In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the “translocatable unit (TU)”, but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.