Cloning Analysis Research Articles

Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures.

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.

Abstract B032: Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions

Abstract Chromosomal instability (CIN) is the most common form of genomic instability in cancer. CIN leads to structural and numerical chromosomal aberrations and to heterogeneity of such aberrations inside the same tumor. Together with other forms of genomic and epigenomic instability, CIN underlies the most dangerous feature of cancer cells, namely, ability to evolve, allowing cancer cells to evade immunologic surveillance, to metastasize, to become resistant to drug treatments. Studies of chromosomal instability are significantly impeded by the lack of comprehensive approaches for simultaneous detection of different forms of ongoing chromosomal instability and their outcomes. We developed an approach that allows to analyze the clonal composition and chromosomal heterogeneity in cell lines and cancer samples, as well as rates of different forms of ongoing chromosomal instability in cancer cell lines. At the heart our analysis is a multiplex interphase FISH (miFISH) with seven sequential hybridizations of color probe panels resulting in the assessment of a total of 35 probes per single cell. Each probe panel is designed to include the centromeric probe and probes on the p- and q-arm for a given chromosome that allows for the simultaneous assessment of centromeric probes and gene-specific arm probes. This approach was applied to interphase nuclei, metaphase spreads and bi-nucleated/dividing cancer cells in colorectal and cervical cell lines and cancer samples. miFISH allows for automated acquisition of nuclei and metaphase spreads on a BioView spotcounting system. A gallery provides detailed views for semi-automatic as well as manual analysis of the obtained images. A total of 10000-25000 cell images were collected from each slide. The simultaneous evaluation of our strategically chosen centromeric and gene-specific probes within the same cells allows unprecedented insights in the patterns of distribution of genetic material among cancer cells in cancer cell population, and, especially, in dividing cells. We evaluated clonality, clonal and non-clonal chromosomal heterogeneity, ploidy distributions, and occurrence of endoreplication. Assessment of dividing cells based on equal or unequal distribution of miFISH centromeric and gene-specific signals allowed to estimate and compare the rates and outcomes of different forms of ongoing chromosomal instability such as structural rearrangements and amplifications, loss of chromosomes or chromosomal fragments in micronuclei, non-disjunctions, grossly asymmetrical cell divisions (including multipolar mitoses). Conclusions: Gradual changes in chromosomal/genomic content of cancer cells occur due to structural rearrangements, losses of chromosomes and chromosomal fragments in micronuclei, and by relatively rare single chromosome non-disjunctions. Grossly abnormal cell divisions (massive non-disjunctions and structural rearrangements) is the main source for the high level of chromosomal abnormalities and aneuploidy in evolved cancer cell populations. Citation Format: Anna Roschke. Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B032.

Gut colonization and subsequent infection of neonates caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

The gut microbiota harbors diverse bacteria considered reservoirs for antimicrobial resistance genes. The global emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) significantly contributes to healthcare-associated infections (HAIs). We investigated the presence of ESBL-producing Escherichia coli (ESBL-PEco) and ESBL-producing Klebsiella pneumoniae (ESBL-PKpn) in neonatal patients' guts. Furthermore, we identified the factors contributing to the transition towards ESBL-PEco and ESBL-PKpn-associated healthcare-associated infections (HAIs). The study was conducted from August 2019 to February 2020, in a Neonatal Intensive Care Unit of the Hospital Infantil de México Federico Gómez. Rectal samples were obtained upon admission, on a weekly basis for a month, and then biweekly until discharge from the neonatology ward. Clinical data, culture results, and infection information were gathered. We conducted antimicrobial tests, multiplex PCR assay, and pulsed-field gel electrophoresis (PFGE) to determine the antimicrobial resistance profile and genetic relationships. A comparison between the group's controls and cases was performed using the Wilcoxon and Student t-tests. Of the 61 patients enrolled, 47 were included, and 203 rectal samples were collected, identifying 242 isolates. In 41/47 (87%) patients, colonization was due to ESBL-PEco or ESBL-PKpn. And nine of them developed HAIs (22%, 9/41). ESBL-PEco resistance to cephalosporins ranged from 25.4% to 100%, while ESBL-PKpn resistance varied from 3% to 99%, and both bacteria were susceptible to carbapenems, tigecillin, and colistin. The prevalent bla CTX-M-group-1 gene accounted for 77.2% in ESBL-PEco and 82.2% in ESBL-PKpn, followed by bla TEM 50% and bla OXA-1 43.8% in ESBL-PEco and bla TEM 80.2% and bla SHV 76.2% in ESBL-PKpn. Analysis of clonality revealed identical colonizing and infection isolates in only seven patients. Significant risk factors included hospital stay duration, duration of antibiotic treatment, and invasive device usage. Our findings suggest high ESBL-PEco and ESBL-PKpn rates of colonization often lead to infection in neonates. Attention should be paid to patients with ESBL-PE.

Studies on clinicopathological features of duodenal-type follicular lymphoma of 18 patients

To investigate the clinical and pathological characteristics of duodenal-type follicular lymphoma (D-FL), and to deepen the understanding of Duodenal-type follicular lymphoma. The clinical symptoms, endoscopic features, pathologic features, immunophenotype, molecular pathological features and treatment follow-up of 18 D-FL patients diagnosed in Department of Pathology, Beijing Tiantan Hospital affiliated to Capital Medical University between January 2020 and July 2023 were summarized. A total of 18 patients with D-FL were included, including 10 males and 8 females. The median age was 49 (32-69) years respectively. Most of the patients were found during gastroenteroscopy or presented with the common gastrointestinal symptoms of stomach pain, acid reflux, vomiting and diarrhea. Most endoscopic findings were multiple small gray and white polyposis. In the pathological morphology, the mucous layer and submucous layer showed lymphoid follicular structures with full and strained follicles. The immunophenotype showed that the tumor cells strongly expressed CD20 and BCL2 and had low proliferation activity. Immunoglobulin clonal analysis of 1 case showed IgK monoclonal rearrangement (1/1). FISH showed 1 case of BCL2 gene rearrangement (1/3). All patients did not receive targeted chemotherapy and adopted a wait-and-see strategy. Median follow-up was 12 (2-34) months. This study shows that D-FL is an indolent lymphoma, which tends to occur in the duodenum and has a good prognosis.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

Understanding the cell fate and behavior of progenitors at the origin of the mouse cardiac mitral valve

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.

T-cell receptor gamma gene rearrangement analysis of classic Hodgkin lymphoma using a BIOMED-2 assay: a paraffin-embedded tissue analysis of one hundred cases.

In the new WHO classifications of haematolymphoid tumours (WHO-HAEM5), classic Hodgkin lymphoma (cHL) is categorized into B-cell lymphoid proliferations and lymphomas. Although the majority of Hodgkin Reed-Sternberg (HRS) cells are of germinal center B-cell origin with some defects of B-cell transcription factors, they rarely express T-cell antigens or cytotoxic molecules. Clonality analyses on cHL samples using BIOMED-2 have been reported by several groups; however, those studies were only focused on Ig regions, including IgH, Ig-kappa, and Ig-lambda, and TCR-γ clonality analysis of cHL has not yet been explored. Here, we investigated TCR-γ gene rearrangement for one hundred cases using a PCR-based method. Four of one hundred (4%) cases showed TCR-γ clonal peaks. Of these, three were at an advanced stage and one patient died of the disease. To clarify whether HRS cells showed T-cell clonality or not, we performed PCR analysis using DNAs of microdissected HRS cells. Three samples showed identical clonal peaks with bulk specimens. Our results indicate that cHL is a heterogeneous disease of mainly B-cell and rarely T-cell origin with a special phenotype. Further molecular studies are warranted.

Expression of transcriptional regulator mgrB is enhanced in colistin-resistant Klebsiella pneumoniae: A report from India

Background: Colistin is regarded as the final option for treating Gram-negative bacteria that are resistant to several drugs. A common resistance mechanism for colistin in Klebsiella pneumoniae is mgrB gene inactivation. Inactivation of mgrB activates the PhoPQ which upregulates the PmrAB leading to resistance by lipopolysaccharide modification. Colistin resistance has been reported in Klebsiella pneumoniae, still, studies related to molecular mechanisms of this resistance are lacking. Aim: Role of mgrB in colistin resistance Methods: In this study, eight colistin-resistant clinical Klebsiella pneumoniae were selected, and performed broth microdilution followed by Rapid polymyxin NP test for screening of colistin resistance. PCR was performed for different types of mcr genes. MgrB was amplified and sequenced. Quantitative real-time PCR was performed for mgrB, PmrA, PmrB, PhoP, and PhoQ genes. Further, clonal analysis by ERIC PCR was done. Results: Colistin MIC of 4μg/ml has been shown by the isolates. No mcr gene was detected. Positive amplification for the mgrB gene was found in all the isolates. Sequence analysis of the mgrB gene found no mutation however, higher expression in the mgrB, PmrA, PmrB, PhoP, and PhoQ genes of all the eight colistin-resistant isolates could be observed when the isolates were exposed to colistin and Mg2+ pressure. Clonal analysis exhibited seven different haplotypes of Klebsiella pneumoniae. Conclusion: This study provides firsthand knowledge about molecular mechanisms of colistin resistance in Klebsiella pneumoniae in this geographical area. Enhancement in mgrB expression in the presence of colistin and Mg2+ pressure regardless of any mutation has a remarkable impact in the context of colistin resistance mechanisms in clinical Klebsiella pneumoniae.

Cardiac Progenitor Cells of the First and Second Heart Fields.

The heart forms from the first and second heart fields, which contribute to distinct regions of the myocardium. This is supported by clonal analyses, which identify corresponding first and second cardiac cell lineages in the heart. Progenitor cells of the second heart field and its sub-domains are controlled by a gene regulatory network and signaling pathways, which determine their behavior. Multipotent cells in this field can also contribute cardiac endothelial and smooth muscle cells. Furthermore, the skeletal muscles of the head and neck are clonally related to myocardial cells that form the arterial and venous poles of the heart. These lineage relationships, together with the genes that regulate the heart fields, have major implications for congenital heart disease.

Clinical and epidemiological characteristics of multi-drug resistant Enterobacterales isolated from King Fahad Hospital of the University, AlKhobar, Saudi Arabia.

Multi-drug resistant (MDR) Enterobacterales remain a major clinical problem. Infections caused by carbapenem-resistant strains are particularly difficult to treat. This study aimed to assess the clinical and epidemiological characteristics of MDR Enterobacterales isolates. A total of 154 non-repetitive clinical isolates, including Escherichia coli (n = 66), Klebsiella pneumoniae (n = 70), and other Enterobacterales (n = 18), were collected from the Diagnostic Microbiology Laboratory at King Fahad Hospital of the University. Most E. coli isolates were collected from urine specimens (n = 50, 75.8%) and resistance against the third and fourth-generation cephalosporins (ceftriaxone, ceftazidime, cefixime, and cefepime) and fluoroquinolones (ciprofloxacin and levofloxacin) was assessed. Clonal relatedness analysis using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) revealed two clones (E. coli A and B), each comprising two strains. Most K. pneumoniae samples were collected from respiratory specimens (27.1%, 20 samples), and the strains showed overall resistance to most of the antimicrobials tested (54%‒100%). Moreover, clonal-relatedness analysis using ERIC-PCR revealed seven major clones of K. pneumoniae. These findings suggest nosocomial transmission among some identical strains and emphasize the importance of strict compliance with infection prevention and control policies and regulations. Environmental reservoirs could facilitate this indirect transmission, which needs to be investigated.

Investigating comparative polymerase chain reaction for antigen receptor rearrangement analysis in different types of feline lymphoma samples.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

Staphylococcus epidermidis joint isolates: Whole-genome sequencing demonstrates evidence of hospital transmission and common antimicrobial resistance.

We investigated genetic, epidemiologic, and environmental factors contributing to positive Staphylococcus epidermidis joint cultures. Retrospective cohort study with whole-genome sequencing (WGS). We identified S. epidermidis isolates from hip or knee cultures in patients with 1 or more prior corresponding intra-articular procedure at our hospital. WGS and single-nucleotide polymorphism-based clonality analyses were performed, including species identification, in silico multilocus sequence typing (MLST), phylogenomic analysis, and genotypic assessment of the prevalence of specific antibiotic resistance and virulence genes. Epidemiologic review was performed to compare cluster and noncluster cases. In total, 60 phenotypically distinct S. epidermidis isolates were identified. After removal of duplicates and impure samples, 48 isolates were used for the phylogenomic analysis, and 45 (93.7%) isolates were included in the clonality analysis. Notably, 5 S. epidermidis strains (10.4%) showed phenotypic susceptibility to oxacillin yet harbored mecA, and 3 (6.2%) strains showed phenotypic resistance despite not having mecA. Smr was found in all isolates, and mupA positivity was not observed. We also identified 6 clonal clusters from the clonality analysis, which accounted for 14 (31.1%) of the 45 S. epidermidis isolates. Our epidemiologic investigation revealed ties to common aspirations or operative procedures, although no specific common source was identified. Most S. epidermidis isolates from clinical joint samples are diverse in origin, but we identified an important subset of 31.1% that belonged to subclinical healthcare-associated clusters. Clusters appeared to resolve spontaneously over time, suggesting the benefit of routine hospital infection control and disinfection practices.

High-throughput RNA sequencing of the T cell receptor alpha and beta chains for simultaneous clonality and biological analyses in Sezary syndrome.

Previous investigations pointed out a role for antigen stimulation in Sezary syndrome (SS). High-throughput sequencing of the T cell receptor (TR) offers several applications beyond diagnostic purposes, including the study of T cell pathogenesis. We performed high-throughput RNA sequencing of the TR alpha (TRA) and beta (TRB) genes focusing on the complementarity-determining region 3 (CDR3) in 11 SS and one erythrodermic mycosis fungoides (MF) patients. Five psoriasis patients were employed as controls. Peripheral blood CD4+ cells were isolated and RNA sequenced (HiSeq2500). High-resolution HLA typing was performed in neoplastic patients. Highly expanded predominant TRA and TRB CDR3 were only found in SS patients (median frequency: 94.4% and 93.7%). No remarkable CDR3 expansions were observed in psoriasis patients (median frequency of predominant TRA and TRB CDR3: 0.87% and 0.69%, p < 0.001 compared to SS). CDR3 almost identical to the predominant were identified within each SS patient and were exponentially correlated with frequencies of the predominant CDR3 (R2 = 0.918, p < 0.001). Forty-six different CDR3 were shared between SS patients displaying HLA similarities, including predominant TRA and TRB CDR3 in one patient that were found in other three patients. Additionally, 351 antigen matches were detected (Cytomegalovirus, Epstein-Barr, Influenza virus, and self-antigens), and the predominant CDR3 of two different SS patients matched CDR3 with specificity for Influenza and Epstein-Barr viruses. Besides detecting clonality, these findings shed light on the nature of SS-related antigens, pointing to RNA sequencing as a useful tool for simultaneous clonality and biological analysis in SS.

Multilocus Sequence Typing of Emerging Azole-Resistant Candida auris Isolates from Critically Ill Patients in a New York City Medical Center

Abstract Candida auris has developed into a slow-moving global pandemic since its emergence in 2009, particularly as a source of nosocomial infection amongst immunocompromised and critically-ill patients. According to CDC data, C. auris emergence has accelerated in the US during the COVID-19 pandemic and has garnered particular attention for its high level of resistance to multiple classes of antifungals. C. auris has been categorized into five genetically diverse clades, each with unique antifungal drug resistance patterns. We have observed a sharp increase in cases identified at our tertiary medical center in New York City over the past three years. In this study, we designed a set of C. auris-specific oligonucleotides to determine the clonality of isolates through Multilocus Sequence Typing (MLST) of four genetic loci known to have a high level of divergence amongst Candida species. From 2020 through 2022 we identified 22 patients with C. auris bloodstream infection at our institution by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and performed MLST on isolates that were available from 19 patients. We assessed four genetic loci: ITS1/ITS2, D1/D2, RPB1 and RPB2; ERG11 was included to assess for mutations associated with fluconazole resistance. We found that the number of blood stream infections increased from 2 cases in 2020 to 12 in 2022. Our MLST results showed that all C. auris isolates from blood and body fluids belong to CDC-defined Clade 1c, which carries the ERG11 gene mutation resulting the amino acid substitution K143R in 14-α-Demethylase associated with fluconazole resistance. These isolates also demonstrated fluconazole resistance by in vitro testing (19/19), amphotericin resistance was present in 44% of isolates with available data (7/16), and all isolates were susceptible to micafungin (19/19). Of 19 patients with blood cultures positive for C. auris, 13 (68%) were in a critical care setting at time of collection, 17 (89%) had been in an ICU during the same admission, and 8 (42%) expired within 3 months of C. auris positivity. MLST is considered a relatively fast and inexpensive method of sequence-based strain typing and has recently been applied to assessing the clonality of C. auris isolates. We show that the use of C. auris-specific oligonucleotides for sequencing, rather than those derived from other Candida species, produces high quality data for MLST, as well as the detection of specific mutations involved in antifungal resistance, such as ERG11. Further assessment is needed by the gold standard in sequence typing, Whole Genome Sequencing (WGS), to confirm our analysis of clonality here. MLST may therefore represent a useful tool in understanding the interrelatedness of invasive C. auris infections in hospitalized patients as well as inform clinical decision-making around infection control and empiric antifungal therapy.

Clonality Analysis for the Relationship between the Pulmonary Combined Neuroendocrine Carcinoma and “the So-Called Reported Histologic Transformation”

Histologic transformation (HT) is common following targeted therapy in adenocarcinoma. However, whether the transformed tumor is a new component or a combined neuroendocrine carcinoma (C-NEC) remains controversial. We aimed to explore the relationship between pulmonary C-NEC and HT. Macro-dissection was performed on different components of surgically resected C-NEC samples. Molecular alterations and clonal evolution were analyzed using whole exome sequencing (WES). The gene statuses for TP53 and RB1 were determined using immunohistochemistry (IHC) and WES to analyze the relationship between C-NEC and reported HT. Sixteen combined small-cell lung cancer patients and five combined large-cell neuroendocrine carcinoma patients were enrolled. The frequency of p53 and Rb inactivation, assessed using IHC in NEC and non-NEC components, was 76.2/76.2% and 66.7/61.9%, respectively. The expression consistency between the components was 81.0 and 85.7% for p53 and Rb, respectively. The frequencies of TP53, RB1, and EGFR mutations, assessed using WES in NEC and non-NEC components, were 81.0/81.0%, 28.6/28.6%, and 42.9/42.9%, respectively. The concordance rates for TP53, RB1, and EGFR were 90.5, 71.4, and 90.5%, respectively. The consistency rate between IHC and WES was 81.0 and 61.9% for TP53 and RB1, respectively. The different components had a common clonal origin for the 21 C-NECs in the clonal analysis, consistent with previous studies on HT. Our study shows that IHC is more sensitive for Rb detection and C-NEC, and the reported HT may be due to differences in evaluations between pathologist and clinicians. Assessing the p53/Rb and EGFR status for such cases would help in recognizing potential transformation cases or uncovering potential combined components.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p &amp;lt; 0.0001) and MEP (p &amp;lt; 0.0001), but skewing towards GMP (p&amp;lt;0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p &amp;lt; 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.

Unraveling the Evolutionary Paths in Relapsed Early-Stage Follicular Lymphoma Showing Complete Remission

Background: Follicular lymphoma (FL) develops through a stepwise acquisition of cooperative genetic changes with t(14;18)(q32;q21)/ IGH:: BCL2 occurring early at the pre-B stage of B-cell development. Patients with FL typically show an indolent clinical course, remitting and relapsing, and eventually developing resistance to treatments. Interestingly, the majority of transformed FL do not progress directly from the preceding FL but originate from their clonally related lymphoma precursor cells (CLP). In light of this finding, we wanted to address whether such divergent tumor evolution also occurs in the setting of relapses in early-stage FL in patients who achieved complete remission following initial treatment. Methods: We identified 13 cases of FL with an early clinical stage (stage I = 9; stage II = 4) at the initial diagnosis, who showed complete remission (mean: 5 years; range: 1-11.5 years) following local radiotherapy but subsequently relapsed (≥ 2 in 5). Paired formalin-fixed paraffin-embedded diagnostic and relapsed lymphoma biopsies were obtained in each case. BCL2 translocation status was investigated using interphase fluorescence in-situ hybridization (FISH). Genomic DNA was extracted from the tumor-rich areas in each tissue specimen and targeted next-generation sequencing was performed with a customized gene panel comprising 191 genes/ regions that are implicated in FL, diffuse large B-cell lymphoma (DLBCL), and B-cell biology using the TWIST target enrichment kits and Illumina NextSeq platform. Clonality analysis of IG gene rearrangements was performed by combining the BIOMED-2 PCR assays and Illumina MiSeq sequencing. Sequencing data analyses, variant calling, and filtering were performed using a well-established data analysis pipeline from a previous study (Cucco et al., 2018). Results: Interphase FISH showed BCL2 translocation in 6 cases, BCL2 amplification (up to 8 copies) in 1 case, and BCL2 copy number gain (3-4 copies) in 3 cases. Pathogenic variants were identified in each of the lymphoma biopsies and were typical of those frequently seen in FL and DLBCL. There was no significant difference in the mutational burden between the paired diagnostic (mean: 7.8/sample; range: 1-13) and relapsed FL (7.9/sample; range: 2-13), excluding tFL. Both the diagnostic and relapsed lesions showed similar, frequent mutations in BCL2 (33.3% vs 41.67%), CREBBP (58.3% vs 58.3%), EP300 (41.7% vs 41.7%), KMT2D (50% vs 58.3%), TNFRSF14 (66.7% vs 58.3%), and STAT6 (50% vs 50%). The clonal relationship between the diagnostic FL and relapses was confirmed in 11 cases. In 6 cases, common and distinct variants were seen between the paired diagnostic and relapsed lymphomas, indicating their divergent evolution from a CLP. In 2 cases, distinct B-cell clones were involved in the diagnostic and the relapsed lymphomas, including 1 case involving 2 different BCL2 translocations, indicating independent evolution of the new lymphomas. In the remaining 5 cases, the relapsed lymphomas developed via a linear progression from the preceding FL (n = 5). Conclusion: Both divergent evolution, either from clonally related or unrelated precursors, and linear evolution processes are involved in the development of relapses in patients with an early-stage FL following complete remission, with the former being more frequent. The above findings, together with the evidence of divergent evolution of tFL in the majority of cases, challenge the current therapeutic strategy of early-stage FL, which does not target the premalignant cell population that underpins lymphoma recurrence in the majority of cases. Additionally, the routine diagnostic workup, which includes IG gene clonality and BCL2 translocation analyses, cannot address the evolutionary path in clonally related lymphomas. Our results indicate the significance of mutational profiling at diagnosis as well as at relapse, and the need to comprehensively characterize and target the evasive CLP population to prevent recurrence.

Acquisition of 1pLOH-1qGain Is a Frequent Trajectory in Expanding Relapsed/Refractory Myeloma Subclones

Genetic clonal heterogeneity is common in multiple myeloma (MM). However, the specific drivers of subclonal evolution associated with disease progression under therapeutic selection has not been fully characterized with the resource of sequential samples to accurately observe changes in relation to drug exposures. To address this gap, we generated whole-genome sequencing data for 2-4 sequential samples of 50 UK relapsed/refractory MM (rrMM) patients taken before, during and after acquisition of therapy resistance. These patients were exposed to a real-world range of multi-agent therapy combinations between 2011 and 2020 all of which included IMiDs. We undertook high-resolution subclonal heterogeneity analysis using Battenberg and multi-dimensional DPClust as gold-standard methods [Salcedo et al. 2020, PMID: 31919445] and implemented the Plackett-Luce model of partial rankings to time genomic events based on their cancer cell fraction and identify alternative evolutionary trajectories [Ansari-Pour et al. 2021, PMID: 34836952]. This approach enabled the identification and tracking of recurrent genomic aberrations gaining dominance amongst the evolving subclones of the disease in relation to therapeutic exposure and resistance acquisition, and to our knowledge is the first time that has been applied to analyze multi-timepoint myeloma WGS datasets. We observed recurrent selection of subclones harbouring 17p loss of heterozygosity (LOH) or 1pLOH with disease progression and therapy resistance acquisition in tumour phylogenies. In addition, clonal timing analysis of the longitudinal dataset suggested that sequential acquisition of 1pLOH and 1qGain in the same clone was a common selected trajectory (22% of cases). To validate this finding, the same trajectory analysis was undertaken in a much larger IMiD-refractory rrMM WGS as a validation dataset (N=386, Myeloma Genome Project), albeit with single time-point samples and compared with a set of unrelated newly diagnosed WGS dataset (N=198, IFM 2009) [Ansari-Pour et al. 2023, PMID: 36223594]. Strikingly, 1pLOH was found to be the only event that showed evidence for clonal selection in this case-control analysis (timing rank fold-change = 3.7, P&amp;lt;2.2x10 -16). Interestingly, the 1pLOH-1qGain trajectory (27% of cases; co-occurrence OR = 2.3 (95% CI 1.5-3.8), P=2x10 -4) was also identified in the validation dataset, corroborating the potential driverness of the dual chromosome 1 copy number events in subclonal expansion during therapy resistance acquisition. While this WGS-based analysis of selected evolutionary trajectories in relapsed/refractory myeloma patient tumours needs further confirmation with sequential WGS datasets from uniformly treated cohorts, these findings suggest potential collaborative genetic interaction between 1pLOH and 1qGain in the same cell as emerging co-drivers of resistance/disease progression, conferring a greater selective advantage under therapeutic exposure than either event on its own. If confirmed, these biomarkers may define a new ‘double hit’ in relapsed/refractory myeloma, with biological consequences that will require novel and directed therapeutic approaches.

Single-Cell Genomics and Proteomics Reveals Venetoclax-Resistant Monocytic Differentiation of TP53 LOH Clones in TP53 Mutant AML

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by accumulation of abnormal hematopoietic stem and progenitor cells. TP53 mutations, present in 8 - 30 % of AML, are associated with high risk of relapse and poor prognosis. The mechanisms underlying their persistence are not well understood. Here, we attempt to describe the clonal architecture and immunophenotypes of TP53 mutant ( TP53-mut) clones during therapy. Leveraging advancements in single-cell sequencing and machine learning models, this study integrated single-cell mass cytometry (CyTOF) and single-cell DNA + protein sequencing (seq) profiling of ten paired pre- and post-treatment samples from five TP53-mut AML patients. Methods: Patients 1 and 3 received treatment with CD47AB (Magrolimab), 5'-Azacitidine (Aza), and Venetoclax (Ven); Patient 2 received IMGN632 (CD123-targeting ADC), Aza and Ven. Patient 4 received p97 Inhibitor CB-5339; Patient 5 received CD47 inhibitor (ALX148), Aza, Ven. Consequently, Ven/Aza served as the therapeutic backbone for most patients. For single cell DNA+protein analysis, we sequenced a total of 44,550 cells from these 10 samples. Samples with at least 70% viability were stained with the Total-Seq™-D Human Heme Oncology Cocktail, containing 45 oligo-conjugated antibodies for surface markers relevant to AML. For mutation analysis, we utilized a validated AML custom panel (Morita et al. 2020) covering recurrent mutations in 37 genes. For CyTOF, we multiplexed samples and stained with 50 antibodies characterizing lineage, apoptosis, and signaling pathways. We successfully constructed a machine learning model for integrating both data types using our gradient boosting algorithm established pipelines and were able to predict DNA mutation status in CyTOF data with over 95% accuracy. Results: Analysis of the clonal architecture revealed the presence of TP53 mutations not only in AML blasts but also in lymphocyte subpopulations in all TP53-mut clones from the patients in this study. For example, in patient 1, as a representative case, TP53 mutations were observed in 16.5% of CD4+ T cells, 35.9% of CD8+ T cells, and 19.2% of NK cells. Clonal evolution analysis showed sensitivity of TP53-mut clones with TP53 Loss of heterozygosity ( TP53 mut + LOH) to Ven/Aza and monocytic differentiation in resistant cells, potentially a manifestation of their stem-like nature. Patient 1 displayed three distinct subclones: clone 1 harbored an ETV6 mutation (p.V322S), clone 2, derived from clone 1, acquired additional IDH1 (p.R132L) and TP53 (p.R241G) mutations, clone 3, originating from clone 2 and comprising the highest proportion, exhibited loss of the other TP53 allele (LOH), and decreased from 96.2% to 55.2% post-treatment (Figure 1A). Meanwhile, clone 1 and clone 2 increased from 1.9% to 27.3% and 1.9% to 17.4% respectively. Dissecting the phenotypic changes, clone 3 showed significant pre- and post-treatment shifts in cellular composition. Pre-treatment, it consisted predominantly of 89.7% CD34+ blasts and only 0.2% CD16+ monocytic cells. Post-treatment, CD34+ blasts decreased to 9.3%, while CD16+ monocytic cells increased to 67.8%. CyTOF analysis corroborated our findings (Figure 1B) with trajectory analysis revealing that AML cells had a stemness profile (CD34+CD38- and C-kit+) located at the beginning of the trajectory. Conversely, post-treatment AML cells displayed a loss of stemness and expressed myeloid differentiation markers, including CD33, CD11b, CD14, and CD68. We concluded that Ven-based treatments induce monocytic differentiation in resistant AML cells. CyTOF analysis of the proteomic profiles of resistant cells revealed an increase in Ki67, BCL-XL, and 4EBP1 and a decrease in CD34 and C-KIT, suggesting a potential resistance mechanism involving the upregulation of anti-apoptotic molecules and mTOR pathway. Notably, CD36, a fatty acid transporter, exhibited increased expression in monocytic cells, suggesting an altered metabolism in the survival monocytic cells. Conclusion: This study establishes a genotype-phenotype connection through single-cell proteogenomic profiling of TP53-mutated AML, describing the clonal evolution and immunophenotypic dynamics during treatment while proposing a potential mechanism of resistance.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (&amp;lt;1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (&amp;lt;0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs &amp;lt;10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (&amp;lt;0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.