Clonal Origin Research Articles

Blood Count Parameters As Early Predictors of Clonal Cytopenia of Undetermined Significance (CCUS) Using a Diverse Ethnoracial Data Set

Background: Hematologic malignancies are a spectrum of disease that frequently have an insidious onset and may be difficult to diagnose. Improved screening using basic complete blood count parameters are needed to enhance early detection of these processes. Aging results in an expansion of hematopoietic clones that may predispose individuals to subsequent development of a hematologic malignancy. Recent studies with next generation sequencing (NGS) and whole-exome sequencing have identified certain somatic mutations known as DTA mutations that are associated with aging (e.g., DNMT3A, TET2, and ASXL1). These mutations, although more commonly seen in myeloid malignancies, are initially benign but may develop into overt hematologic malignancy and are collectively termed clonal hematopoiesis of indeterminate potential (CHIP). Similarly, individuals with cytopenia and clonal mutations are designated as clonal cytopenia of undetermined significance (CCUS). However, these mutations carry a risk of progression to hematologic malignancy. Animal models have shown that clonal hematopoiesis can accelerate cardiovascular mortality due to increased atherogenesis by causing inflammation and endothelial injury via upregulation of inflammasome pathways. Also, it has already been shown that clonal hematopoiesis with increasing number of clonal mutations, higher variant allele frequency and mutations in known driver genes have a higher risk of progression to hematopoietic malignancy (acute myeloid leukemia, AML). Certain acute phase reactants, like C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR) have been used in prior studies as markers of inflammation and are strongly associated with variations in red cell size as determined by the red cell distribution width (RDW), reflecting chimeric clonal origins. We therefore propose that RDW and mean corpuscular volume (MCV) with or without other blood count parameters (red cell count, white blood cell count and platelet count) might be used as important predictors of progression of myeloid neoplasms, especially myelodysplastic syndromes (MDS), clonal cytopenias (CCUS) and acute myeloid leukemias (AML). Methods: Utilizing Epic's self-service reporting tool SlicerDicer, cohort discovery was performed on the Caboodle Patients data model. Cohort criteria included individuals with the cytopenia diagnostic grouper that had undergone blood or bone marrow myeloid panel next generation sequencing, that also had concurrent or historic complete blood count (CBC) results. Patients with known acute leukemias were excluded. Once the cohort was identified, a data pull was performed that captured CBC values, age, gender, race, and ethnicity, in addition to the NGS results. The NGS results were ranked into 3 classes from 0 to 2. Class 0 included results with no apparent disease, while class 2 results included high grade myeloid mutations (i.e. mutations in NRAS, KRAS, TP53, RUNX1 or ASKL1). Class 1 mutations were considered low grade and featured mutations not seen in class 2. The collated data was analyzed using SAS software. Results: Overall 72 patients met inclusion criteria. Patients ranged in age from 23 to 92 years, 48% identified as female and 52% identified as male with a mixture of race and ethnicity (Table 1). Abnormalities in several CBC components including MCH, MCHC, RBC count, and hemoglobin concentration were associated with increased odds of identifying those with malignant myeloid processes (Figure 1). It is notable that no mutations in FLT3 or KIT were observed in this cohort. Discussion: Based on our preliminary data, these cost-effective parameters can be added to an algorithm for risk assessment of hematologic neoplasms and better predict which patients will further benefit from targeted gene testing. Also, additional data will be helpful and applying this data to modern machine learning techniques will further the clinical application of these findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Association of the interaction between mosaic chromosomal alterations and polygenic risk score with the risk of lung cancer: an array-based case-control association and prospective cohort study.

Mosaic chromosomal alterations (mCAs) detected from blood-derived DNA are large structural alterations of clonal haematopoietic origin and are associated with various diseases, such as haematological malignancies, infections, and solid cancers. We aimed to investigate whether mCAs contribute to the risk of lung cancer and modify the effect of polygenic risk score (PRS) on lung cancer risk prediction. The blood-derived DNA of patients with lung cancer and cancer-free controls with Chinese ancestry from the Nanjing Lung Cancer Cohort (NJLCC) study were genotyped with a Global Screening Array, and mCAs were detected with the Mosaic Chromosomal Alterations (MoChA) pipeline. mCA call sets of individuals with European ancestry were obtained from the prospective cohort UK Biobank (UKB) study, including documented incident lung cancer. All patients with lung cancer from the NJLCC study (aged 15 years or older at diagnosis) were histopathologically confirmed as new lung cancer cases by at least two pathologists and were free of chemotherapy or radiotherapy before diagnosis. Participants with incident lung cancer (aged 37-73 years at assessment) diagnosed after recruitment to the UKB were identified through linkage to national cancer registries. Logistic regression and Cox proportional hazard models were applied to evaluate associations between mCAs and risk of lung cancer in the NJLCC (logistic regression) and UKB (Cox proportional hazard model) studies. The NJLCC study included 10 248 individuals (6445 [62·89%] men and 3803 [37·11%] women; median age 60·0 years [IQR 53·0-66·0]) with lung cancer and 9298 individuals (5871 [63·14%] men and 3427 [36·86%] women; median age 60·0 years [52·0-65·0]) without lung cancer recruited from three sub-regions (north, central, and south) across China between April 15, 2003, and Aug 18, 2017. The UKB included 450 821 individuals recruited from 22 centres across the UK between March 13, 2006, and Nov 1, 2010, including 2088 individuals with lung cancer (1075 [51·48%] men and 1013 [48·52%] women; median age 63·0 years [IQR 59·0-66·0]), and 448 733 participants were free of lung cancer (204 713 [45·62%] men and 244 020 [54·38%] women; median age 58·0 years [IQR 50·0-63·0]). Compared with non-carriers of mosaic losses, carriers had a significantly increased risk of lung cancer in the NJLCC (odds ratio [OR] 1·81, 95% CI 1·43-2·28; p=6·69 × 10-7) and UKB (hazard ratio [HR] 1·40, 95% CI 1·00-1·95; p=0·048) studies. This increased risk was even higher in patients with expanded cell fractions of mCAs (ie, cell fractions ≥10% vs cell fractions <10%) in the NJLCC (OR 1·61 [95% CI 1·26-2·08] vs 1·03 [0·83-1·26]; p for heterogeneity test=6·41 × 10-3). A significant multiplicative interaction was observed between PRS and mosaic losses on the risk of lung cancer in both the NJLCC (interaction p value=0·030) and UKB (p=0·043). Compared with non-carriers of mosaic loss abnormalities with low genetic risk, participants with expanded mosaic losses (cell fractions ≥10%) and high genetic risk had around a six-times increased risk of lung cancer in the NJLCC study (OR 6·40 [95% CI 3·22-12·69]), and an almost four-times increased risk of lung cancer (HR 3·75 [95% CI 1·86-7·55]) in the UKB study. The additive interaction also contributed a 3·67 (95% CI 0·49-6·85) relative excess risk of developing lung cancer in the NJLCC study, and a 2·15 (0·12-4·19) relative excess risk in the UKB study. mCAs act as a new endogenous indicator for the risk of lung cancer and might be jointly used with PRS to optimise personalised risk stratification for lung cancer. National Natural Science Foundation of China, Outstanding Youth Foundation of Jiangsu Province, Natural Science Foundation of Jiangsu Province, and Postdoctoral Science Foundation of China. For the Chinese translation of the abstract see Supplementary Materials section.

Aestuariirhabdus haliotis sp. nov., isolated from abalone viscera and emended description of the genus Aestuariirhabdus.

Two novel strains, Z083T and Z084, were isolated from the viscera of abalone, Haliotis discus hannai, sampled in Weihai, PR China. The phenotypic, chemotaxonomic and genomic characteristics of the two strains were studied. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 99.8 and 98.9 %, respectively, suggesting that the two strains belonged to the same species. The 16S rRNA gene sequence analysis showed 99.8 % similarity between the two strains, while the genome analysis indicated that they were not from one clonal origin. Phylogenetic analysis of 16S rRNA gene sequences showed the two strains belonged to the genus Aestuariirhabdus and Aestuariirhabdus litorea JCM 32043T was the closest strain (97.5 %). Genomic analysis, including calculations of ANI, dDDH, amino acid identity (AAI) and percentage of conserved proteins (POCP), between Z083T, Z084 and A. litorea JCM 32043T clearly separated those two strains from A. litorea JCM 32043T as the values were below the thresholds for species delineation. The genome size of strains Z083T and Z084 were approximately 4.16 and 4.23 Mbp, respectively, and the DNA G+C contents of both strains were 51.8 mol%. According to the phenotypic, chemotaxonomic and phylogenetic characterizations and the results of genome analysis, Z083T and Z084 could be identified as belonging to a novel species of the genus Aestuariirhabdus, for which the name Aestuariirhabdus haliotis sp. nov., is proposed, with Z083T (=MCCC 1H00501T=KCTC 92006T) as the type strain.

Determination of ERG(+), EZH2, NKX3.1, and SPINK-1 subtypes to evaluate their association with clonal origin and disease progression in multifocal prostate cancer.

The prognostic relevance of prostate cancer (PCa) molecular subtypes remains controversial, given the presence of multiple foci with the possibility of different subtypes in the same patient. To determine the clonal origin of heterogeneity in PCa and its association with disease progression, SPOP, ERG(+), EZH2, NKX3.1, and SPINK-1 subtypes were analyzed. A total of 103 samples from 20 PCa patients were analyzed; foci of adjacent non-tumor prostate tissue, HGPIN, GL3, GL4, GL5, and LN were examined to determine the presence of the TMPRSS2-ERG fusion and ERG, EZH2, NKX3.1, and SPINK-1 expression levels, using RT-PCR. Mutations in exons 6 and 7 of the SPOP gene were determined by sequencing. The presence of subtypes and molecular patterns were identified by combining all subtypes analyzed. To establish the clonal origin of multifocal PCa, molecular concordance between different foci of the same patient was determined. Association of these subtypes with histopathological groups and time to biochemical recurrence (BCR) was assessed. No mutation was found in SPOP in any sample. The ERG(+) subtype was the most frequent. The molecular pattern containing all four PCa subtypes was only detected in 3 samples (4%), all LN, but it was the most frequent (40%) in patients. Molecular discordance was the predominant status (55%) when all analyzed molecular characteristics were considered. It was possible to find all subtypes, starting as a preneoplastic lesion, and all but one LN molecular subtype were ERG(+) and NKX3.1 subtypes. Only the expression of the NKX3.1 gene was significantly different among the histopathological groups. No association was found between BCR time in patients and molecular subtypes or molecular concordance or between clinicopathological characteristics and molecular subtypes of ERG, EZH2, and SPINK-1. The predominance of molecular discordance in prostatic foci per patient, which reflects the multifocal origin of PCa foci, highlights the importance of analyzing multiple samples to establish the prognostic and therapeutic relevance of molecular subtypes in a patient. All the subtypes analyzed here are of early onset, starting from preneoplastic lesions. NKX3.1 gene expression is the only molecular characteristic that shows a progression pattern by sample.

Characterization of Staphylococcus epidermidis clinical isolates from hospitalized patients with bloodstream infection obtained in two time periods.

BackgroundIn recent years Staphylococcus epidermidis has been considered an important and frequent causative agent of health care-associated infections (HAIs), increasing the costs of hospitalization, morbidity, and mortality. Antibiotic resistance and biofilm formation are the most important obstacles in the treatment of infections caused by this microorganism. The aim of this work was to determine the most prevalent STs, as well as the antibiotic resistance profile and biofilm formation of S. epidermidis clinical isolates obtained from hospitalized patients in two hospitals in Acapulco, Guerrero in two time periods.MethodsTwenty methicillin-resistant S. epidermidis strains isolated from patients with bacteremia in two hospitals in two time periods were analyzed. Identification and antibiotic susceptibility were performed using the Vitek automated system. Molecular confirmation of the identification and methicillin resistance was performed by duplex PCR of the mecA and nuc genes. Biofilm production was analyzed, and the clonal origin was determined by multilocus sequence typing (MLST).ResultsWe identified 14 antibiotic resistance profiles as well as 13 sequence types (ST), including the new ST761. We also found that ST2 and ST23 were the most prevalent and, together with ST59, were found in both time periods. Seventeen of our clinical isolates were multidrug-resistant, but all of them were sensitive to linezolid and vancomycin, and this was not related to biofilm production. Additionally, we standardized a duplex PCR to identify methicillin-resistant S. epidermidis strains. In conclusion, S. epidermidis STs 2, 23, and 59 were found in both time periods. This study is the first report of S. epidermidis ST761. The clinical isolates obtained in this work showed a high multidrug resistance that is apparently not related to biofilm production.

ALL-503 Lineage Switch Upon Relapse of Acute Leukemia: A Rare Event Which Heralds Poor Clinical Outcomes

Lineage switch in acute leukemias (AL) is a rarely described entity. Several hypotheses have been proposed to explain it, notably the expansion of a coexisting subclone after suppression of the leukemic clone apparent at diagnosis by chemotherapy, alterations brought by chemotherapy upon the original clone, by suppressing differentiation programs, thus inducing a shift in phenotypic expression, or high fate plasticity of the hematopoietic progenitor cells, regulated by microenvironmental signals and transcription factors. We report the case of a patient diagnosed with B-cell acute lymphoblastic leukemia, who relapsed after twelve months, with morphological and immunophenotypic findings consistent with acute monocytic leukemia. An 18-year-old female presented to our clinic with a month-long history of fever, fatigue and menorrhagia. Upon clinical examination, hepatosplenomegaly and unilateral axillary adenopathy were found. Laboratory studies revealed severe pancytopenia with a white blood cell count of 1.6×109/L, hemoglobin value of 2.8 g/dL and platelet count of 30×109/L. Bone marrow (BM) examination showed an 80% lymphoblasts infiltrate, while the immunophenotypic analysis indicated B-cells positive for CD19, cCD79a, CD22, CD24, CD10, CD34, CD38, CD58, with dim expression of CD45, CD20, nuTdT, CD13, CD33, negative for myeloperoxidase (MPO) or markers of monocytic differentiation. No chromosomal or genetic abnormality was detected, including MLL gene rearrangement at 11q23, previously associated with lineage switch in AL. She was treated according to the GRAALL-2003 protocol and achieved complete remission. One year after the primary diagnosis, the BM aspirate showed a hypercellular marrow with 70% blasts, while flow cytometry identified expression of MPO, CD13, CD33 (bright), HLA-DR, CD14, CD15, CD56, CD38. She received chemotherapy with fludarabine, cytarabine and filgrastim, with failure in attaining remission, then followed a clofarabine, cytarabine and idarubicin regimen. At day +23, the BM aspirate showed normocellular marrow with 2-3% blast cells and a sibling hematopoietic stem-cell transplantation was planned, but three weeks later, the peripheral blood smear showed 43% blasts with monocytoid morphology. She is currently undergoing treatment with azacytidine and venetoclax. Despite tremendous advancements in the understanding of AL, many questions regarding the molecular mechanisms driving lineage switch need to be addressed. This rare phenomenon is associated with therapy resistance and poor clinical outcomes.

Malignant clonal evolution drives multiple myeloma cellular ecological diversity and microenvironment reprogramming

BackgroundMultiple myeloma (MM) is a heterogeneous disease with different patterns of clonal evolution and a complex tumor microenvironment, representing a challenge for clinicians and pathologists to understand and dissect the contribution and impact of polyclonality on tumor progression.MethodsIn this study, we established a global cell ecological landscape of the bone marrow (BM) from MM patients, combining single-cell RNA sequencing and single-molecule long-read genome sequencing data.ResultsThe malignant mutation event was localized to the tumor cell clusters with shared mutation of ANK1 and IFITM2 in all malignant subpopulations of all MM patients. Therefore, these two variants occur in the early stage of malignant clonal origin to mediate the malignant transformation of proplasmacytes or plasmacytes to MM cells. Tumor cell stemness index score and pseudo-sequential clonal evolution analysis can be used to divide the evolution model of MM into two clonal origins: types I and IX. Notably, clonal evolution and the tumor microenvironment showed an interactive relationship, in which the evolution process is not only selected by but also reacts to the microenvironment; thus, vesicle secretion enriches immune cells with malignant-labeled mRNA for depletion. Interestingly, microenvironmental modification exhibited significant heterogeneity among patients.ConclusionsThis characterization of the malignant clonal evolution pattern of MM at the single-cell level provides a theoretical basis and scientific evidence for a personalized precision therapy strategy and further development of a potential new adjuvant strategy combining epigenetic agent and immune checkpoint blockade.

Patterns of metastases progression- The linear parallel ratio.

Linear and parallel are the two leading models of metastatic progression. In this study we propose a simple way to differentiate between them. While the linear model predicts accumulation of genetic and epigenetic alterations within the primary tumor by founder cells before spreading as waves of metastases, the parallel model suggests preclinical distribution of less advanced disseminated tumor cells with independent selection and expansion at the ectopic sites. Due to identical clonal origin and time of dispatching, linear metastases are expected to have comparable diameters in any specific organ while parallel metastases are expected to appear in variable sizes. Retrospective revision of chest CT of oncological patients with lung metastases was performed. Metastasis number and largest diameters were recorded. The sum number of metastases with a similar diameter (c) and those without (i) was counted and the linear/parallel ratio (LPR) was calculated for each patient using the formula (∑c-∑i)/(∑c+∑i). A LPR ratio of 1 implies pure linear progression pattern and -1 pure parallel. 12,887 metastases were measured in 503 patients with nine malignancy types. The median LPR of the entire group was 0.71 (IQR 0.14-0.93). In carcinomas of the pancreas, prostate, and thyroid the median LPR was 1. Median LPRs were 0.91, 0.65, 0.60, 0.58, 0.50 and 0.43 in renal cell carcinomas, melanomas, colorectal, breast, bladder, and sarcomas, respectively. Metastatic spread of thyroid, pancreas, and prostate tumors is almost exclusively by a linear route. The spread of kidney, melanoma, colorectal, breast, bladder and sarcoma is both linear and parallel with increasing dominance of the parallel route in this order. These findings can explain and predict the clinical and genomic features of these tumors and can potentially be used for evaluation of metastatic origin in the individual patient.

Genomic landscape of multiple Bowen's disease using whole-exome sequencing.

The genomic landscape of Bowen's disease (BD), with multiple manifestations, has not yet been determined. This study aimed to investigate the genomic alterations in multiple BD. We performed whole-exome sequencing of BD lesions (n=9) and matched germlines collected from three patients with multiple (≥3) BD to detect somatic and germline mutations. We found a median of 64 somatic mutations in each sample (range 20-267). UV-signature mutations accounted for 64.9% (median, range 26.0%-82.1%) of point mutations. Putative driver mutations were found in five BDs (RB1 p.R445*, ARID2 p.R274*, TP53 p.Y163D/p.Y205D/p.R342*, KMT2C p.R4549C) but not in the other four lesions. Somatic mutations were not shared between multiple BD lesions collected from the same patient, indicating a different clonal origin. We also found no known pathogenic germline mutations in cancer-related genes. The mutational signature analysis revealed that UV signatures (SBS7a/7b) and age-related signatures (SBS1/5) were the main active signatures. Copy number alterations (CNAs) were found in two BDs: one with extensive CNA regions (21.7% of the genome), including driver genes (PIK3CA/SOX2/TP63 and MYC gain, and CDKN2A loss), and the other with 1q gain. Our study revealed that multiple BD lesions harbor distinct genomic landscapes, suggesting that they have different risks of malignant progression.

Mutational signature and clonal relatedness of recurrent urothelial carcinomas with aristolochic acid.

Urothelial carcinomas (UCs) are malignant tumors that arise from the lower and upper urinary tract and are characterized by multiple recurrences. Aristolochic acid (AA) is a potent nephrotoxin and human carcinogen associated with UC. East Asian populations with a high UC prevalence have an unusual genome-wide AA-induced mutational pattern. To address the genomic differences and clonal relatedness between primary and recurrent tumors in the UCs with AA pattern, we investigated the genomic differences and tumor microenvironment (TME) of AA and non-AA UCs. 17 UC patients were recruited, with nine documented AA exposure. Eleven of them showed recurrence. After-surgery tissues of primary and paired recurrent tumors were collected. Capture-based targeted deep sequencing was performed using a commercial panel consisting of 520 cancer-related genes. Tumor-infiltrating lymphocytes (TILs) were identified with an immunofluorescence-based microenvironment analysis panel (MAP). Hierarchical clustering based on the COSMIC signatures confirmed two significant subtypes: AA Sig and non-AA Sig. AA Sig was associated with AA-containing herbal drug intake, recurrence, and higher tumor mutation burden (TMB). The clonal architecture of UCs revealed three types of clonal evolution patterns. Non-AA Sig cohort showed shared clonal origin of primary and recurrent tumors. AA Sig showed heterogeneity and had multiple independent origins. Recurrent tumors as second primary tumors in AA Sig showed immunoreactive TME, indicating a better response with immune checkpoint inhibitor therapy. The AA mutational signature and unique immune profiles are helpful molecular markers to distinguish AA exposure from other carcinogens. These results also provide new insights into the origin of recurrent UCs that could affect treatment strategies.

An in vitro model of glucose transporter 1 deficiency syndrome at the blood-brain barrier using induced pluripotent stem cells.

Glucose is an important source of energy for the central nervous system. Its uptake at the blood-brain barrier (BBB) is mostly mediated via glucose transporter 1 (GLUT1), a facilitated transporter encoded by the SLC2A1 gene. GLUT1 Deficiency Syndrome (GLUT1DS) is a haploinsufficiency characterized by mutations in the SLC2A1 gene, resulting in impaired glucose uptake at the BBB and clinically characterized by epileptic seizures and movement disorder. A major limitation is an absence of in vitro models of the BBB reproducing the disease. This study aimed to characterize an in vitro model of GLUT1DS using human pluripotent stem cells (iPSCs). Two GLUT1DS clones were generated (GLUT1-iPSC) from their original parental clone iPS(IMR90)-c4 by CRISPR/Cas9 and differentiated into brain microvascular endothelial cells (iBMECs). Cells were characterized in terms of SLC2A1 expression, changes in the barrier function, glucose uptake and metabolism, and angiogenesis. GLUT1DS iPSCs and iBMECs showed comparable phenotype to their parental control, with exception of reduced GLUT1 expression at the protein level. Although no major disruption in the barrier function was reported in the two clones, a significant reduction in glucose uptake accompanied by an increase in glycolysis and mitochondrial respiration was reported in both GLUT1DS-iBMECs. Finally, impaired angiogenic features were reported in such clones compared to the parental clone. Our study provides the first documented characterization of GLUT1DS-iBMECs generated by CRISPR-Cas9, suggesting that GLUT1 truncation appears detrimental to brain angiogenesis and brain endothelial bioenergetics, but maybe not be detrimental to iBMECs differentiation and barriergenesis. Our future direction is to further characterize the functional outcome of such truncated product, as well as its impact on other cells of the neurovascular unit.

Rosai-Dorfman-Destombes disease - histiocytic disorder with inflammatory manifestation.

Rosai-Dorfman-Destombes disease (RDD) is a rare histiocytosis characterized by accumulation of activated histiocytes within affected tissues. Although the immunophenotype of this disease was described, the pathophysiology of this disease is still not sufficiently understood. Recent studies have found NRAS, KRAS, MAP2K1, and ARAF mutations in RDD lesions, raising the possibility of a clonal origin in some forms of RDD while in other cases reactive origin or association with other malignant and autoimmune disease is supposed. RDD is a widely heterogeneous entity with a range of clinical phenotypes occurring in some patients in association with autoimmune or malignant diseases. Its therapy should reflect the localization of the disease. Monotherapy with glucocorticoids is sufficient only in limited disease. In patients with advanced disease, combined nodal and extranodal forms of RDD need more intensive therapy. In older publications, antimetabolites, vinca alkaloids and prednisone were used; in recent publications, remissions after cladribine, rituximab, sirolimus, thalidomide, lenalidomide and cobimetinib were described. This text summarizes current knowledge about this rare disease and reviews the therapeutic options.

Whole genome sequencing reveals the independent clonal origin of multifocal ileal neuroendocrine tumors

BackgroundSmall intestinal neuroendocrine tumors (SI-NETs) are the most common neoplasms of the small bowel. The majority of tumors are located in the distal ileum with a high incidence of multiple synchronous primary tumors. Even though up to 50% of SI-NET patients are diagnosed with multifocal disease, the mechanisms underlying multiple synchronous lesions remain elusive.MethodsWe performed whole genome sequencing of 75 de-identified synchronous primary tumors, 15 metastases, and corresponding normal samples from 13 patients with multifocal ileal NETs to identify recurrent somatic genomic alterations, frequently affected signaling pathways, and shared mutation signatures among multifocal SI-NETs. Additionally, we carried out chromosome mapping of the most recurrent copy-number alterations identified to determine which parental allele had been affected in each tumor and assessed the clonal relationships of the tumors within each patient.ResultsAbsence of shared somatic variation between the synchronous primary tumors within each patient was observed, indicating that these tumors develop independently. Although recurrent copy-number alterations were identified, additional chromosome mapping revealed that tumors from the same patient can gain or lose different parental alleles. In addition to the previously reported CDKN1B loss-of-function mutations, we observed potential loss-of-function gene alterations in TNRC6B, a candidate tumor suppressor gene in a small subset of ileal NETs. Furthermore, we show that multiple metastases in the same patient can originate from either one or several primary tumors.ConclusionsOur study demonstrates major genomic diversity among multifocal ileal NETs, highlighting the need to identify and remove all primary tumors, which have the potential to metastasize, and the need for optimized targeted treatments.

Plasma cells arise from differentiation of clonal lymphocytes and secrete IgM in Waldenström macroglobulinemia

SummaryWaldenström macroglobulinemia (WM) is characterized by bone marrow infiltration with malignant lymphoplasmacytic cells (LPCs), a smaller population of plasma cells (PCs), and hypersecretion of IgM monoclonal protein. Here, we show that CD45low, CD38+, and CD138+ PCs and CD45high, CD38−, CD138-, CD19+, and CD20+ LPCs carry a heterozygous L265P mutation in the Toll-like receptor signaling adaptor MYD88. Both PCs and LPCs express the same auto-reactive IgHV sequences, suggesting a similar clonal origin and role for auto-antigens in WM cell survival. PCs are primarily responsible for IgM production even without substantial cell proliferation. When cultured in isolation, LPCs give rise to more differentiated PCs and secrete less IgM. Our analyses suggest that malignant PCs arise from the clonal LPC population, and are primarily responsible for IgM secretion in WM. Targeting malignant PCs may have therapeutic benefits in the treatment of WM and improve the duration of response and potentially, survival.

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography

We developed affinity chromatographic resins that immobilized rabbit single-chain Fv antibodies (scFvs). By biopanning using antigen-coupled multilamellar vesicles (Ag-MLVs), 152 types of original scFv clones that specifically bind to human IgG were isolated and identified. Apparent dissociation rate constants, appkoff, of six different candidates were less than 10-3 s-1 and their dissociation constants, KDs, were ranged from 5.56× 10-10 to 4.04× 10-8M. Consequently, the clones, R1-27, R2-18, and R3-26 were further investigated for use in affinity purification of human IgG. Both the clones, R1-27 and R3-26 maintained more than 40% of antigen-binding activities on the surface of affinity resins. Especially, R3-26 had a relatively high alkaline resistance. The direct separation of human IgG from 10% FBS-D-MEM by use of the column with R1-27 achieved 97.2% purity, while the column with R3-26 showed almost 100% recovery. The affinity resins at the densities between 4.32 and 15.19mg-scFv/cm3 exhibited maximum binding amount of human IgG, while the highest ligand utilization was achieved by use of the resin at approximately 9mg-scFv/cm3. The resin exhibited 7.69mg/cm3 of equilibrium binding capacity (EBC) in affinity chromatography. It was expected that the EBC of affinity resins was strongly dependent on the specific surface area as well as the pore volume of the base resin. Therefore, the strategies to develop affinity ligands will be beneficial for development of on-demand affinity columns with higher affinity/selectivity, chemical resistance, while optimization of pore size and pore volume for scFv-coupled resins will further improve the EBC.

Clonal reconstruction from co-occurrence of vector integration sites accurately quantifies expanding clones in vivo

High transduction rates of viral vectors in gene therapies (GT) and experimental hematopoiesis ensure a high frequency of gene delivery, although multiple integration events can occur in the same cell. Therefore, tracing of integration sites (IS) leads to mis-quantification of the true clonal spectrum and limits safety considerations in GT. Hence, we use correlations between repeated measurements of IS abundances to estimate their mutual similarity and identify clusters of co-occurring IS, for which we assume a clonal origin. We evaluate the performance, robustness and specificity of our methodology using clonal simulations. The reconstruction methods, implemented and provided as an R-package, are further applied to experimental clonal mixes and preclinical models of hematopoietic GT. Our results demonstrate that clonal reconstruction from IS data allows to overcome systematic biases in the clonal quantification as an essential prerequisite for the assessment of safety and long-term efficacy of GT involving integrative vectors.

Transcription factor-based direct conversion of human fibroblasts to functional astrocytes.

SummaryAstrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.

P1221: DIFFUSE LARGE B-CELL LYMPHOMA RICHTER SYNDROME IN CHRONIC LYMPHOCYTIC LEUKEMIA – A RETROSPECTIVE ANALYSIS OF TREATMENT OUTCOMES IN POLISH ADULT LEUKEMIA STUDY GROUP

Background: Richter syndrome (RS) is a recognized uncommon manifestation and evolution of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with a dismal prognosis. It occurs in 2-10% of CLL patients. It is associated with a clonal evolution and progression, generally with a transformation of the original CLL clone to diffuse large B-cell lymphoma (DLBCL) or, less frequently, to Hodgkin Lymphoma (HL). Despite the advances in understanding pathogenesis of the disease, the DLBCL variant of RS (DLBCL-RS) is associated with a dismal prognosis with a median survival < 1 year. The treatment options and risk factors of worse outcome for RS still need to be further expanded. Aims: To study the characteristic and treatment outcomes of patients with DLBCL-RS. Methods: The retrospective analysis of 99 patients diagnosed with DLBCL-RS treated in hematology centers of the Polish Adult Leukemia Study Group (PALG) between 2011 and 2021. Results: Clinical data of 124 patients with Richter syndrome was collected. Ninety-nine patients were diagnosed with DLBCL-RS (79.8%) and further analyzed. Median time since CLL diagnosis to Richter transformation was 43.0 months (range 0-252.0). At the time of transformation 31.3% patients had CLL Rai stage IV, 74.7% were ECOG< 2, while the median Cumulative Illness Rating Scale (CIRS) was 4 (range 0-16). Thirty-seven patients transformed during CLL treatment, 56.7% of them were receiving novel agents (Brutons tyrosine kinase inhibitor or BCL2 inhibitor). Median time of observation after transformation was 10.7 months (range 0-98.6). Median overall survival (OS) after DLBCL-RS diagnosis was 17.3 months (95% Cl 11.9-46.8) and median progression-free survival (PFS) 13.5 months (95% Cl 9.6-22.7). Seventy-six patients (76.8%) had RS treated with intensive chemotherapy protocols, mainly R-CHOP-like regimens for the first line treatment. Only 42.3% had response to the treatment (25/76 complete responses and 11/76 partial responses). Worth mentioning is that in the group of patients with response two patients had ibrutinib monotherapy and one had rituximab-ibrutinib. In forty patients RS was primarily resistant to administered treatment and 65% of them died due to progression or treatment related complications. In the primary resistant DLBCL-RS p53 pathway aberration was noted in 47.5% of patients (17p deletion in 13 patients or TP53 mutation in 6 patients). Better results were achieved for patients who proceeded to autologous hematopoietic stem cell transplantation (auto-HSCT; n=8) and allogeneic HSCT (allo-HSCT; n=10). The median OS in auto-HSCT recipients was 46.1 months, while in allo-HSCT the median OS reached 18.3 months (3 patients died due to allo-HCST-related complications). Significantly lower OS (3.4 months, 95% Cl 14.13-not reached) was observed for RS in heavily pretreated CLL patients (>3 lines of therapy preceding transformation) compared to those treated with up to 3 lines of therapy (23.7 months) (p=0.0002). Median OS for treatment naïve patients at DLBCL-RS diagnosis was not reached. Other factors identified as predictors of poor OS in univariate analysis were ECOG ≥2 (p=0.0014), hemoglobin level <10 g/dl (p=0.017) and platelet count <100x109/L (p=0.0065). Summary/Conclusion: Our study represents the largest dataset of DLBCL-RS patients in Polish hematology centers and confirms the poor outcomes and prognosis associated with RS. The worst treatment results were observed in RS receiving >3 lines of CLL therapy preceding transformation. Only proceeding to hematopoietic stem cell transplantation may improve the prognosis.

Abstract 6198: Genetic analysis of synchronous or metachronous multiple pancreatic cancers

Abstract [Introduction] The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 17 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] We analyzed 17 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 10 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 37.7 months (12 - 85 months). In addition, we analyzed 5 adjacent pancreatic intraepithelial neoplasia (PanIN) in one of these cases. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=16), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. [Conclusions] In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Takeuchi Yasuhide, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamiguchi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa. Genetic analysis of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6198.

Abstract 46: Independent clonal origins of synchronous primary tumors in multifocal ileal neuroendocrine tumor patients

Abstract Background: Small intestinal neuroendocrine tumors (SI-NETs) are the most common neoplasms of the small bowel, accounting for 40% of all small intestinal malignancies. Previous high-throughput sequencing studies have shown that SI-NETs have a low somatic mutation rate. Loss of heterozygosity (LOH) at chromosome 18 is the most frequent genomic alteration identified, occurring in ~60% of tumors, and the only recurrent mutations reported to date are loss-of-function mutations in CDKN1B in ~10% of tumors. Most previous studies have focused on sequencing a single primary tumor from each SI-NET patient, even though up to 50% of the patients are diagnosed with multiple synchronous tumors. Thus, the mechanisms by which multifocal SI-NETs arise remain elusive. A better understanding of their tumorigenesis is essential for the optimal treatment of the patients. Materials and Methods: We performed a comprehensive genomic profiling of 75 de-identified synchronous primary tumors, 15 metastases, and 18 normal ileal mucosa and/or whole blood specimens from 13 patients with multifocal ileal NETs using whole genome sequencing. Results: We observed lack of shared somatic variation among the multifocal ileal NET patients and between the synchronous primary tumors within each patient, indicating that these tumors develop independently. Although recurrent copy-number alterations were identified, additional chromosome mapping revealed that tumors from the same patient can gain or lose different parental alleles. In addition to the previously reported CDKN1B loss-of-function mutations, we identified potential driver gene alterations in TNRC6B in a subset of ileal NETs. We also observed two different metastatic dissemination patterns among patients with multiple metastases. Metastases were either clonal or independent, originating from one or several primary tumors. Conclusions: Our study demonstrates notable genomic diversity among multifocal ileal NETs, which corroborates previous literature suggesting that these tumors originate independently. We identified potential loss-of-function mutations in TNRC6B, a candidate tumor suppressor gene in a small subset of ileal NETs. Identification of different metastatic dissemination patterns has an important clinical implication, highlighting the need to carefully remove all synchronous primary tumors of the entire jejunum and ileum at the time of surgery. Lastly, our results indicate that the tumorigenesis of multifocal ileal NETs is not exclusively driven by genomic alterations, suggesting that SI-NETs could be also driven by epigenetic mechanisms, or arise from morphologically normal small intestine as a result of field cancerization. Also, the tumor microenvironment in the ileum may play a role in the growth and development of SI-NETs. Citation Format: NETTA MÄKINEN, Meng Zhou, Zhouwei Zhang, Yosuke Kasai, Elizabeth Perez, Grace E. Kim, Chrissie Thirlwell, Eric Nakakura, Matthew Meyerson. Independent clonal origins of synchronous primary tumors in multifocal ileal neuroendocrine tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 46.