Clonal Patterns Research Articles

A207 CLONAL PATTERNS BETWEEN POUCH NEOPLASIA AND PRIOR COLORECTAL NEOPLASIA IN INFLAMMATORY BOWEL DISEASE PATIENTS: AN EXPLORATORY COHORT STUDY

Abstract Background Inflammatory bowel disease (IBD) patients with an ileo-anal pouch anastomosis (IPAA) bear an increased risk of pouch neoplasia, with prior colorectal neoplasia as the strongest predictor. It is unknown if pouch neoplasia develops independently or is derived from prior colorectal neoplasia. Purpose We aimed to assess potential clonality between prior colorectal neoplasia and pouch neoplasia in IPAA patients with IBD. Method In this explorative study we used the Dutch Nationwide Pathology Databank to identify IBD patients with both pouch neoplasia and colorectal neoplasia prior to colectomy. Clonality was assessed on colonic tissue of the lesion with shallow whole genome sequencing based copy number aberration (CNA) analysis and validated with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Result(s) We included 13 patients who fulfilled the inclusion criteria. Three patients showed matching clonality CNA profiles between prior colorectal neoplasia and pouch neoplasia, validated with matching IHC and FISH for p19 and HER2. Patients with matching clonal samples also showed on retrospective review concordant histology of the neoplastic lesion pre- and post IPAA, positive resection margins, metastasized disease or a short interval (<2 years) between colorectal and pouch neoplasia diagnoses. Image Conclusion(s) Three patients showed matching clonality patterns of neoplastic lesions, confirmed by clinical and histological data. Most pouch neoplasia in our cohort were molecularly different from their prior colorectal neoplasia. CNA provides a feasible method for clonality assessment in patients with colorectal neoplasia and subsequent pouch neoplasia. Disclosure of Interest None Declared

Abstract P5-13-01: TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS

Abstract INTRODUCTION: Breast cancer are the main cause of related deaths cancer among women, corresponding to 25% of new cases each year. When diagnosed at early stages, it has an overall five-year survival rate of up to 90%. However, in more advanced stages, survival is reduced to about 24%, with 90% of women in stage IV dying as a result of complications related to metastases. Considering that brain metastasis is an unfavorable prognostic site, and the identification of genetic-molecular profiles in primary tumors and in metastatic sites are a subject poorly described in the literature, we understand that the identification of mutational profiles may contribute to elucidate the genetic-molecular mechanisms associated with tumor progression. AIM: The aim of this study was to identify clonal and subclonal driver mutations that lead to evolution of metastatic clones from a breast cancer progression model. MATERIAL AND METHODS: For tumor progression model, automated extraction of DNA from buffy coat and paraffin samples of breast tumors and paired brain metastases (n=9) was performed. In the present work, we used a subclonal reconstruction model based on the combination of machine learning and population genetics concepts. This proposal is based on the frequency spectrum of each somatic mutation (SNVs or indels), considering VAF (Variant Allele Frequency - ratio of mapped reads of the mutant allele) in relation to the coverage of variant locus, as known as CCF (Cancer Cell Fraction). CCF is defined as the proportion of neoplastic cells that have a certain set of mutations and then is normalized considering the sample purity and the segments with changes in number of DNA copies (Copy Number Alterations). Then, a statistical model based on finite Dirichlet mixtures with mixed distributions is applied. In this model, Beta components capture clonal expansions and population genetics concepts were applied to mutant alleles in each population considering principles of cancer evolution. Finally, confidence was computed using both parametric and non-parametric bootstraps. The functions for building the model are implemented at https://caravagnalab.github.io/mobster/, and for visualization and construction of graphs, packages in R statistical-mathematical environment were used, such as ggplot2, sads, cli, clisymbols, cowplot, crayon, ctree, dndscv, dplyr, magrittr, reshape2, and tidyr. RESULTS: With this model was possible to identify the distribution of clones according to somatic alterations in patients with breast cancer and brain metastasis. It was possible to observe common patterns, such as alterations of the SF3B1 gene as a common ancestral clone in both conditions and the frequency of the AKT1 gene in a subclonal condition. Other genes relevant to breast cancer carcinogenesis, such as PIK3CA and TP53, are found in a different clonal hierarchical pattern between the two conditions. CONCLUSION: This data suggests a model of clonal evolution capable to identify which drivers clones and subclones are involved in the metastatic process. Citation Format: Muriele B. Varuzza, Adriane F. Evangelista, Cristiano P. Souza, Márcia C. Marques. TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-01.

Therapy-Acquired Clonal Mutations in Thiopurine Drug-Response Genes Drive Majority of Early Relapses in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Forty pediatric (0-12 years) B-ALL DNA samples (20 paired Diagnosis-Relapse) and an additional six B-ALL DNA samples (without relapse at 3 years post treatment), as the non-relapse arm, were retrieved from the biobank for advanced genomic analysis. Deep sequencing (1050-5000X; mean 1600X) was performed using a custom NGS panel of 74 genes incorporating unique molecular barcodes. A total 47 major clones (>25% VAF) and 188 minor clones were noted in 40 cases after bioinformatic data filtering. Of the forty-seven major clones, eight (17%) were diagnosis-specific, seventeen (36%) were relapse-specific and 11 (23%) were shared. In the control arm, no pathogenic major clone was noted in any of the six samples. The most common clonal evolution pattern observed was therapy-acquired (TA), with 9/20 (45%), followed by M-M, with 5/20 (25%), m-M, with 4/20 (20%) and unclassified (UNC) 2/20 (10%). The TA clonal pattern was predominant in early relapses 7/12 (58%), with 71% (5/7) having major clonal mutations in the NT5C2 or PMS2 gene related to thiopurine-dose response. In addition, 60% (3/5) of these cases were preceded by an initial hit in the epigenetic regulator, KMT2D. Mutations in common relapse-enriched genes comprised 33% of the very early relapses, 50% of the early and 40% of the late relapses. Overall, 14/46 (30%) of the samples showed the hypermutation phenotype, of which the majority (50%) had a TA pattern of relapse. Our study highlights the high frequency of early relapses driven by TA clones, demonstrating the need to identify their early rise during chemotherapy by digital PCR.

Spatial genomic diversity associated with APOBEC mutagenesis in squamous cell carcinoma arising from ovarian teratoma.

Although the gross and microscopic features of squamous cell carcinoma arising from ovarian mature cystic teratoma (MCT-SCC) vary from case to case, the spatial spreading of genomic alterations within the tumor remains unclear. To clarify the spatial genomic diversity in MCT-SCCs, we performed whole-exome sequencing by collecting 16 samples from histologically different parts of two MCT-SCCs. Both cases showed histological diversity within the tumors (case 1: nonkeratinizing and keratinizing SCC and case 2: nonkeratinizing SCC and anaplastic carcinoma) and had different somatic mutation profiles by histological findings. Mutation signature analysis revealed a significantly enriched apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) signature at all sites. Intriguingly, the spread of genomic alterations within the tumor and the clonal evolution patterns from nonmalignant epithelium to cancer sites differed between cases. TP53 mutation and copy number alterations were widespread at all sites, including the nonmalignant epithelium, in case 1. Keratinizing and nonkeratinizing SCCs were differentiated by the occurrence of unique somatic mutations from a common ancestral clone. In contrast, the nonmalignant epithelium showed almost no somatic mutations in case 2. TP53 mutation and the copy number alteration similarities were observed only in nonkeratinizing SCC samples. Nonkeratinizing SCC and anaplastic carcinoma shared almost no somatic mutations, suggesting that each locally and independently arose in the MCT. We demonstrated that two MCT-SCCs with different histologic findings were highly heterogeneous tumors with clearly different clones associated with APOBEC-mediated mutagenesis, suggesting the importance of evaluating intratumor histological and genetic heterogeneity among multiple sites of MCT-SCC.

P199 Clonal patterns between pouch neoplasia and prior colorectal neoplasia in Inflammatory Bowel Disease patients: an exploratory cohort study

Abstract Background Inflammatory Bowel Disease (IBD) patients with an ileo-anal pouch anastomosis (IPAA) bear an increased risk of pouch neoplasia, with prior colorectal neoplasia as the strongest predictor. It is unknown if pouch neoplasia develops independently or is derived from prior colorectal neoplasia. Therefore, we aimed to elucidate the pathogenesis of pouch neoplasia by assessment of clonality between prior colorectal neoplasia and pouch neoplasia in IPAA patients with IBD. Methods In this explorative study we used the Dutch Nationwide Pathology Databank to identify IBD patients with both pouch neoplasia and prior colorectal neoplasia. Clonality was assessed on tissue with shallow whole genome sequencing based copy number aberration (CNA) analysis and validated with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Results Three out of 13 included patients showed clonally matching CNA profiles between colorectal neoplasia and pouch neoplasia, validated with matching IHC and FISH for p16 and HER2. Patients with clonal samples corresponded with concordant histology pre- and post IPAA, positive resection margins, metastasized disease or a short interval (<2 years) between colorectal and pouch neoplasia diagnoses. Conclusion CNA provides a feasible method for clonality assessment in patients with colorectal neoplasia and subsequent pouch neoplasia. Most pouch neoplasia in our cohort were molecularly different from their prior colorectal neoplasia. This may indicate a multifactorial pathogenesis of pouch neoplasia.

Genetic trajectory and clonal evolution of multiple primary lung cancer with lymph node metastasis

Multiple primary lung cancer (MPLC) with lymph node metastasis (LNM) is a rare phenomenon of multifocal lung cancer. The genomic landscapes of MPLC and the clonal evolution pattern between primary lung lesions and lymph node metastasis haven’t been fully illustrated. We performed whole-exome sequencing (WES) on 52 FFPE (Formalin-fixed Paraffin-Embedded) samples from 11 patients diagnosed with MPLC with LNM. Genomic profiling and phylogenetic analysis were conducted to infer the evolutional trajectory within each patient. The top 5 most frequently mutated genes in our study were TTN (76.74%), MUC16 (62.79%), MUC19 (55.81%), FRG1 (46.51%), and NBPF20 (46.51%). For most patients in our study, a substantial of genetic alterations were mutually exclusive among the multiple pulmonary tumors of the same patient, suggesting their heterogenous origins. Individually, the genetic profile of lymph node metastatic lesions overlapped with that of multiple lung cancers in different degrees but are more genetically related to specific pulmonary lesions. SETD2 was a potential metastasis biomarker of MPLC. The mean putative neo-antigen number of the primary tumor (646.5) is higher than that of lymph node metastases (300, p = 0.2416). Primary lung tumors and lymph node metastases are highly heterogenous in immune repertoires. Our findings portrayed the comprehensive genomic landscape of MPLC with LNM. We characterized the genomic heterogeneity among different tumors. We offered novel clues to the clonal evolution between MPLC and their lymphatic metastases, thus advancing the treatment strategies and preventions of MPLC with LNM.

Single-cell resolved ploidy and chromosomal aberrations in nonalcoholic steatohepatitis-(NASH) induced hepatocellular carcinoma and its precursor lesions

Nonalcoholic steatohepatitis (NASH)-induced hepatocellular carcinoma (HCC) and its precursor, nonalcoholic fatty liver disease (NAFLD) are an unmet health issue due to widespread obesity. We assessed copy number changes of genes associated with hepatocarcinogenesis and oxidative pathways at a single-cell level. Eleven patients with NASH-HCC and 11 patients with NAFLD were included. Eight probes were analyzed using multiplex interphase fluorescence in situ hybridization (miFISH), single-cell imaging and phylogenetic tree modelling: Telomerase reverse transcriptase (TERT), C-Myc (MYC), hepatocyte growth factor receptor tyrosine kinase (MET), tumor protein 53 (TP53), cyclin D1 (CCND1), human epidermal growth factor receptor 2 (HER2), the fragile histidine triad gene (FHIT) and FRA16D oxidoreductase (WWOX). Each NASH-HCC tumor had up to 14 distinct clonal signal patterns indicating multiclonality, which correlated with high tumor grade. Changes frequently observed were TP53 losses, 45%; MYC gains, 36%; WWOX losses, 36%; and HER2 gains, 18%. Whole-genome duplications were frequent (82%) with aberrant tetraploid cells evolving from diploid ancestors. Non-tumorous NAFLD/NASH biopsies did not harbor clonal copy number changes. Fine mapping of NASH-HCC using single-cell multiplex FISH shows that branched tumor evolution involves genome duplication and that multiclonality increases with tumor grade. The loss of oxidoreductase WWOX and HER2 gains could be potentially associated with NASH-induced hepatocellular carcinoma.

Molecular response patterns in relapsed/refractory AML patients treated with selinexor and chemotherapy

Relapse in patients with acute myeloid leukemia (AML) is common and is associated with a dismal prognosis. Treatment options are limited and the understanding of molecular response patterns is still challenging. We analyzed the clonal response patterns of 15 patients with relapsed/refractory AML treated with selinexor in a phase II trial (SAIL). DNA was analyzed at three time points and showed a decline of mutated alleles in FLT3, SF3B1, and TP53 under SAIL treatment. Overall survival (OS) was similar between patients with declining versus persisting clones. We show an interesting long-term course of a patient who relapsed after allogeneic stem cell transplantation (alloHCT) with SF3B1- and SRSF2-mutated AML and received selinexor as maintenance treatment for 4 years. Measurable residual disease (MRD) remained detectable for 2 weeks after donor lymphocyte infusion (DLI) in this patient and then remained negative under selinexor maintenance treatment. Selinexor was tolerated well and was stopped after 4 years of SAIL treatment. We present an exploratory study and identify subclonal patterns of patients treated with selinexor.

Expanded NK cells used for adoptive cell therapy maintain diverse clonality and contain long-lived memory-like NK cell populations

Multiple clinical trials exploring the potential of adoptive natural killer (NK) cell therapy for cancer have employed exvivo expansion using feeder cells to obtain large numbers of NK cells. We have previously utilized the rhesus macaque model to clonally track the NK cell progeny of barcode-transduced CD34+ stem and progenitor cells after transplant. In this study, NK cells from barcoded rhesus macaques were used to study the changes in NK cell clonal patterns that occurred during exvivo expansion using culture protocols similar to those employed in clinical preparation of human NK cells including irradiated lymphoblastoid cell line (LCL) feeder cells or K562 cells expressing 4-1BBL and membrane-bound interleukin-21 (IL-21). NK expansion cultures resulted in the proliferation of clonally diverse NK cells, which, at day 14 harvest, contained greater than 50% of the starting barcode repertoire. Diversity as measured by Shannon index was maintained after culture. With both LCL and K562 feeders, proliferation of long-lived putative memory-like NK cell clones was observed, with these clones continuing to constitute a mean of 31% of the total repertoire of expanded cells. These experiments provide insight into the clonal makeup of expanded NK cell clinical products.

1189. Clonal Recovery Pattern of Staphylococcus aureus during Sham-controlled, Interventional, Crossover Trial on the Effectiveness of Pulsed Xenon Ultraviolet Light (PX-UV) in Reducing Healthcare-associated Infections

Abstract Background PX-UV has shown great effectiveness at reducing Staphylococcus aureus (S. aureus) on healthcare surfaces. S. aureus is one of the most prevalent causes of healthcare-associated infections (HAIs). Here we assess the clonal recovery pattern and the disruption caused by PX-UV on S. aureus collected from patients, using Whole Genome Multilocus Sequence Type (wgMLST) analysis. Methods During a 3-year period, 2017 to 2020, a prospective sham-controlled double-blinded interventional crossover trial designed to assess the effectiveness of post-discharge PX-UV room disinfection on HAIs in 2 Detroit hospitals. The interventional group (Q) and the control group (W) was treated with PX-UV and sham UV light after terminal cleaning respectively, both treatments lasting 12 months. Crossover occurred after a 6-month washout period (R) during which no UV light was used. Whole Genome Sequencing (WGS) of the 291 S. aureus samples collected was performed using the Nextseq 550 (Illumina). Following de novo assembly, construction of minimum spanning tree (MST) and wgMLST analysis was completed using BioNumerics calculation engine (v7.6). Results A total of 20 sequence types (STs) were found throughout the trial, the most common being ST5 and ST8 with roughly 20 to 50 each found in all 3 treatment groups (Table 1. The other 18 STs were only found 7 times or less in each treatment group. ). During intervention (Q) 9 STs were found, however 11 were not recovered after PX-UV (ST1, ST1508, ST22, ST231, ST3287, ST3462, ST398, ST59, ST6790, ST840, ST97). During the sham treatment (W) 15 STs were found while 7 were found during the washout period (R). The minimum spanning tree (MST) shows clusters of related ST groups color coded by treatment type (Figure 1). Fig. 1:Minimum spanning tree (MST) for S. aureusTable 1:Total number of each sequence type (ST) per treatment group Conclusion The difference between the STs recovered during intervention and during sham UV treatment was inconsequential because many factors influence the amount of STs recovered (treatment, number of patients, sampling bias, and terminal cleaning methods). There was no clonality noted among any of the intervention group (Fig. 1). Both dominant strains (ST5, ST8) have been characterized as hypervirulent and predominant previously, consistent with our data. Our data does not demonstrate any notable change in the clonal prevalence of S. aureus after PX-UV use. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

1190. Effect of Pulsed Xenon Ultraviolet Light (PX-UV) on Clonal Recovery of Escherichia coli in a Prospective, Sham-controlled, Double-blinded, Interventional, Crossover Trial Conducted in Two Detroit Hospitals

Abstract Background Healthcare-associated infections (HAIs) can be caused by some multidrug-resistant Escherichia coli, most commonly due to production of an extended spectrum beta-lactamase (ESBL), and lead to increased morbidity and mortality. Pulsed Xenon Ultraviolet light (PX-UV), in combination with terminal cleaning, has been shown to improve disinfection and has the potential to lower HAIs by reducing the horizontal spread of infections in hospitals. Here, we assess the effect of PX-UV on the clonal recovery pattern of several E. coli sequence types (STs) using Whole Genome Sequencing (WGS). Methods A prospective, sham-controlled, double-blinded, interventional, crossover trial was conducted to compare standard terminal cleaning with PX-UV (intervention, Group Q) and standard terminal cleaning with sham UV (control, Group W) in 2 Detroit hospitals from 2017 to 2020. Treatments lasted 12 months before crossover, with a 6-month washout period in between (Group R) during which UV was not used. A total of 67 E. coli samples were collected. WGS of the isolates was performed using the Nextseq 550 (Illumina). After de novo assembly, BioNumerics calculation engine (v7.6) was used to complete Whole Genome Multilocus Sequence Type (wgMLST) analysis, assembly free and assembly-based call, and construction of minimum spanning tree (MST). Results The total number of different STs found for the intervention UV device group (Q) and the sham UV device group (W) was 6 while the washout group (R) was 5. Out of the 9 total STs obtained, the most common was ST131 (Table 1). In Group Q, 11 of ST131 were found; 15 were found in Group W. During the washout period (R) 17 of ST131 were found. All other STs had 3 or less circulating clusters. After UV treatment ST1193, ST399, and ST7394 were not recovered. Fig. 1:Minimum spanning tree (MST) for E. coliTable 1:Total number of each sequence type (ST) per treatment group Conclusion During UV intervention 3 different STs (ST1193, ST399, ST7394) were not recovered, but there were negligible changes to the frequency of recovery of the other 6 STs as compared to sham UV treatment. ST131 was the dominant E. coli ST found in Detroit, which is consistent with previously published data stating ST131 as the predominant strain. While PX-UV has previously demonstrated effectiveness on decreasing bioburden, our data does not indicate any remarkable change in clonality and prevalence of E. coli STs after PX-UV use. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

1187. Analysis of the Clonal Recovery Pattern of Enterococcus faecium to Evaluate the Efficacy of Pulsed Xenon Ultraviolet Light (PX-UV) in Bioburden Reduction

Abstract Background Enterococcus faecium (E. faecium) is a common hospital-associated infection (HAI) that can lead to increased costs, morbidity, and mortality. Pulsed Xenon Ultraviolet light (PX-UV) has been shown to reduce bacterial bioburden levels on surfaces. This study aims to assess the effect of the addition of PX-UV to terminal cleaning on the clonal recovery of E. faecium sequence types (STs) using Whole Genome Sequencing (WGS) on patient isolates. Methods During 2017 to 2020 a prospective, randomized, double-blinded, sham-controlled, interventional, crossover trial in 2 separate Detroit hospitals (H1 and H2) compared HAI counts after the addition of either PX-UV or a non-UV sham device to terminal cleaning methods. The trial consisted of a total of 16 units randomized to have either the treatment of PX-UV (Group Q) or the sham control device with no UV (Group W) for 12 months. A washout period (Group R) of 6 months followed and the trial concluded with a 12-month crossover of treatments. A total of 60 E. faecium samples were collected, then WGS was performed by the Illumina Nextseq 550 instrument. de novo assembly was preformed using the SPAdes program. Whole Genome Multilocus Sequence Type (wgMLST) analysis was performed by BioNumerics (v7.6) to construct minimum spanning tree (MST). Results A total of 7 STs were obtained across the 2 hospitals (H1 and H2). ST117, ST17, and ST80 all had more than 10 total recovered isolates, ST117 being the most frequent with 24 isolates. Less than 3 isolates were recovered for all other STs. For all STs, Group Q (PX-UV) had 14, Group R (washout period) had 22, and Group W (non-UV sham) had 24. ST18 was only found in Group Q. ST412, ST584, and ST736 were not found in Group W. The data shows that the intervention PX-UV group had a reduction of clonal recovery by 10 STs as compared to the sham UV group. Table 1:Total number of each sequence type (ST) in H1 and H2 Conclusion The overall reductions in the number of isolates in the real UV units was driven by reductions in the ST117 a predominant strain in a hospital environment reported previously in Detroit and ST80. There were negligible differences in recovery of other ST between the groups. The reduction in clonal recovery of E. faecium isolates in Group Q as compared to Group R might be related to use of PX-UV following terminal room cleaning. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1month post-transplant, replaced by multilineage clones by 2 to 3months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9months; this clonal pattern disappeared at 13months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.

Clinical Significance of IGH Clonality Analysis with RNA Sequencing in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common pediatric malignancy. Using minimal residual disease (MRD) and genetic subtypes, risk stratification algorithms have considerably improved clinical outcomes. However, despite being in a better prognostic group, a non-negligible number of relapses occur, and prognostic markers for these patients are required for more effective treatment strategies. RNA sequencing (RNA-seq) is a useful tool for detecting patient-specific immunoglobulin heavy chain (IGH) junctional sequences (Li et al., Leukemia. 2020), which can be informative for evaluating the nature of leukemic clones. Here, we analyzed IGH clonality of pediatric BCP-ALL using RNA-seq data and revealed its validity for evaluating disease characteristics and impact on prognosis. Methods: We analyzed RNA-seq data of 136 pediatric BCP-ALL samples at diagnosis and 46 samples at relapse from the TARGET ALL Expansion Phase 2 cohort. Using MiXCR (Bolotin et al., Nat Methods 2015) and Vidjil (Duez et al., PLoS One 2016), we detected IGH complete VDJ and incomplete DJ rearrangements. Then, we estimated the expected number of IGH rearrangements corresponding to each read count (E-value) by power-law model. Finally, we identified leukemic "disease clones” with E-values less than 0.01 (Fig. 1a). Results: First, we detected a total of 55,518 IGH clones, of which 48,555 were complete rearrangements and 6,963 were incomplete. In total, 351 disease clones (median 2, range 0-8 per sample) were identified by power-law model: 193 clones were complete rearrangements, and 158 clones were incomplete. Interestingly, the numbers and compositions (complete or incomplete rearrangements) of disease clones varied significantly between samples. Thus, we defined three clonality patterns and classified each sample based on identified disease clones: "Undetectable disease clones (UDC)” if the sample had no disease clone (n=18); "Incomplete Disease Clones (IDC)” if there were one or more disease clones with incomplete rearrangements (n=72); and "Complete Disease Clones (CDC)” if all disease clones of that sample have complete rearrangements (n=46). Gene Set Enrichment Analysis (GSEA) indicated a strong enrichment for hematopoietic stem cell (HSC) and multipotent progenitor (MPP) signatures in UDC and pro-B cell signatures in IDC. Similarly, CIBERSORTx (Newman et al., Nat Biotechnol 2019) showed higher proportions of HSCs, MPPs, and pre-pro B cells in UDC and pro-B cells in IDC. Furthermore, there was a significant enrichment in pathways promoting cell cycle progression in IDC. Of note, in high hyperdiploid (HHD) cases, which are typically associated with a favorable prognosis, event-free survival (EFS) was significantly lower in UDC and IDC (p=0.01) (Fig. 1b). Moreover, even in MRD-negative HHD patients, those with UDC and IDC had significantly worse EFS (p=0.01) and overall survival (p=0.03) (Fig. 1b). Finally, we compared the primary and relapse clones in 46 patients. As a result, 37 patients had one or more disease clones at diagnosis, and 29 (78%) had these primary disease clones at relapse. On the other hand, 9 (24%) patients showed relapse-specific disease clones, which should lead to relapse in these cases. The other 9 primary patients with UDC had no disease clone also at relapse. Discussions: We identified three clonality patterns, UDC, IDC, and CDC based on RNA-seq analysis of the IGH rearrangement status. According to the findings of GSEA and CIBERSORTx, leukemic cells of UDC, IDC, and CDC patients have nature of progenitors to pre-proB cells, pro-B cells, and more mature cells, respectively. These findings are consistent with the knowledge that IGH rearrangement occurs during pro-B to pre-B maturation in normal B-cells. Patients with immature clonality status, UDC and IDC, were associated with poor prognosis in high hyperdiploid subtype and could be a potential prognostic marker in MRD-negative patients. In addition, we revealed that primary disease clones contributed to relapse in 78% of cases, while relapse-specific clones were implicated in relapse in 24% of cases. Additionally, UDC clonality pattern was carried over at relapse. These findings shed light on the mechanisms of leukemic clonal evolution and relapse, and suggest that the status of IGH rearrangement may serve as a novel prognostic marker in BCP-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetically Undetectable and Negative Minimal Residual Disease after Induction Therapy Are Equally Crucial for Survival in Multiple Myeloma

Introduction Cytogenetic abnormalities (CA) identified by interphase fluorescence in situ hybridization (iFISH) has been served as the cornerstone for risk stratification at diagnosis. However, iFISH analyses were generally performed in newly diagnosed and relapsed multiple myeloma (MM) patients, but CA architecture has received little attention in patients after induction therapy. Since it is not the be-all and end-all for everyone to achieve minimal residual disease (MRD)-negativity. iFISH assay may be a good tool to explore the nature of residual plasma cells (PCs) and has proven to be a reliable complementary technique for MRD detection. We thus performed iFISH analyses after proteasome inhibitor (PI) or immunomodulatory drugs (IMiDs) induction in our prospective clinical trial. Methods A total of 396 patients with NDMM between January 2014 and March 2020 who had cytogenetic analysis by iFISH both at diagnosis and within 6 months from the start of induction therapy were included in this study. All of the participants in this study were enrolled in the prospective, non-randomized clinical trial (BDH 2014/03), which was a part of the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199). A cohort of 269 patients with iFISH results after induction therapy were analyzed, with 252 of them were immunophenotyped by multiparameter flow cytometry. The median follow-up time was 41.5 months (25th/75th, 26.5/61.4 months) for the 269 patients who underwent post-induction iFISH analyses. Kaplan-Meier method was used to plot survival curves and compared by the 2-sided log-rank test. We also investigated the interaction between post-induction flow-MRD and iFISH status concerning patient outcomes. Multivariable Cox proportional hazard regression was used to assess the impact of variables on PFS and OS. Results Persistent high frequencies of CA were observed in residual PCs, survival of patients decreased with the increasing clonal size of unique CA. iFISH-positive (iFISH+) was observed in 118 (43.9%) patients whose any clonal size was more than 10%. FISH-positive patients or MRD-positive (MRD+) patients experienced significantly inferior survival than those without CA or undetectable (< 10-4) MRD. Patients were clustered into four groups according to the iFISH and flow-MRD status: (1) iFISH-negative (iFISH-) & MRD-negative (MRD-) (100/252, 39.7%), (2) iFISH+ & MRD- (42/252, 16.6%), (3) iFISH- & MRD+ (45/252, 17.9%) and (4) iFISH+ & MRD+ (65/252, 25.8%). iFISH+ & MRD- patients achieved deeper response depth than iFISH- & MRD+ patients, but this did not translate into better survival, regardless of response depth or treatment regimens. An adjusted survival analysis was performed and showed that the survival outcomes of the 4 clusters in the adjusted analysis and the crude analysis were identical, and that outcomes were similar between patients with iFISH+ & MRD- and those with iFISH- & MRD+ (mPFS: 49.49 months vs. 41.49 months; mOS: 75.33 vs. 70.74 months). Follow-up suggested that persistent CA in residual PCs facilitated the conversion of MRD- to MRD+. The MRD- conversion rate was slightly higher in patients with iFISH+ & MRD- than iFISH- & MRD- (49% vs. 36%, p = 0.139), and the duration of MRD- was also shortened by persistent CA. Longitudinal iFISH examinations were available for 21 patients with MRD- conversion at 3 time points: at diagnosis, post-induction, and at relapse. Notably, a more adverse clonal evolution pattern was observed among patients with iFISH+ & MRD-, with 5 cases of linear evolution at relapse. Conclusions In summary, our study underscores the importance of tumor cell biological characteristics examinations post-induction among patients with NDMM. We demonstrated that patients continued to have high frequencies of CA after therapy. iFISH examination after induction therapy not only provided a complementary tool to flow-MRD but also deciphered the cytogenetic profiles of residual PCs based on the quantification of these residual PCs. This comprehensive approach of combining flow-MRD and iFISH examination leverages common tests. Therefore, it may be practical to adapt in clinical practice to help identify the resistant clones and guide risk-adapted treatment strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pediatric-Specific Patterns of Clonal Evolution Arising from Acquired Aplastic Anemia

Introduction Acquired aplastic anemia (aAA) occurs in both children and adults, though pathophysiology and clinical outcomes differ significantly by age. While clinical testing for paroxysmal nocturnal hemoglobinuria (PNH) clones is routine, targeted next generation sequencing (NGS) for myelodysplastic syndrome (MDS) associated variants has only recently been implemented. Furthermore, single nucleotide polymorphism array (SNP-A) assessing for copy neutral loss of heterozygosity in the human leukocyte antigen (HLA) region on chromosome 6p (6p CN-LOH) and HLA NGS assessing for allele specific loss of function (LoF) mutations, which together are seen in up to 25% of aAA cases, are only now being considered for clinical use. Recent studies have reported on the frequency and overlap of these 3 types of clonal evolution (PNH, MDS-associated, HLA-targeting) in adult aAA cohorts, but none focus specifically on pediatric patients, whose MDS-associated clonal evolution patterns are distinct. Delineating patterns of clonal hematopoiesis in pediatric aAA to determine whether evolution driven by immune escape (PNH and HLA loss) affects likelihood of MDS development is critical for personalizing therapeutic options. Thus, we aimed to describe the frequency, clinical significance, and overlap of clonal evolution including PNH, HLA loss, and MDS-associated gene mutations in a pediatric cohort of aAA patients. Methods This study identified 85 pediatric aAA patients treated at Children's Hospital of Philadelphia (CHOP) and enrolled on the IRB-approved CHOP/Penn BMF Registry and Repository in whom clinical testing for all patterns of clonal hematopoiesis was available or could be performed with biorepository samples. PNH flow cytometry was performed clinically in all cases; subjects with PNH > 0.05% in the granulocyte fraction were considered as having a PNH clone. Peripheral blood and bone marrow samples were used for SNP-A, HLA-NGS, and an institutional somatic mutation NGS panel for hematological malignancies encompassing 118 genes. Descriptive and contingency statistical analyses along with Fisher's Exact Test were performed using SAS (Cary, NC). Results Demographic composition of the 85 pediatric subjects with aAA included a median age of 10.7 years (1.7-20.8), male/female ratio of 59%/41%, and a representative race/ethnicity distribution (Table 1). In total, 66% displayed at least one clonal event. As expected, PNH clones were most common, present in 45% (38/85) of the cohort. An additional 16 (19%) exhibited acquired HLA loss by 6p CN-LOH (n=6), allele specific LoF mutations (n=6), or both (n=4). Fifteen (18%) had clonal hematopoiesis associated with MDS, of whom 3 had cytogenetic lesions that progressed to clinical MDS (3.5%), and 12 developed no morphologic features of MDS but demonstrated a total of 14 mutations in MDS-associated genes (BCOR/BCORL1 n=6, KMT2C/KMT2D n=3, RUNX1 n=1, ASXL1 n=1, SRSF2 n=1, PRPF8 n=1, CALR n=1). Critically, pediatric subjects with aAA who developed HLA loss did not develop MDS-associated mutations, with no patient demonstrating both subtypes of clonal events (Figure 1). In contrast, PNH and MDS-associated clonal events overlapped in 11% of the overall cohort. In total, 60% of patients with MDS-associated clonal hematopoiesis also possessed PNH clones, and 23% of patients with detectable PNH clones demonstrated at least one MDS-associated clonal event. Only 3.53% (n=3) of the cohort demonstrated both PNH and HLA loss, and PNH clone size was < 1% in all 3 patients. Furthermore, patients with HLA loss were significantly less likely to have PNH clones than those without HLA somatic alterations (19% vs 51%, p = 0.026). Correlations between patterns of clonal hematopoiesis and clinical outcomes will be presented. Concalusions Pediatric aAA patients treated with IST are at risk for developing MDS and hemolytic PNH. Presence and evolution of clonal hematopoiesis influenced decisions to pursue IST versus alternative donor transplant. Importantly, our study demonstrates that occurrence of clonal hematopoiesis by HLA loss does not increase and may even decrease the risk of developing MDS-associated or PNH clones. This finding strongly suggests that treatment decisions for aAA patients whose only clonal event is HLA loss need not be considered distinctly from patients with no clonal changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Durable Response to CD19 CAR T Cell Therapy Is Associated with Cytotoxicity and Clonotypic Expansion of the Native T Cell Repertoire

Background: Axicabtagene ciloleucel (Axi-cel), a CD19 directed CAR T cell therapy, results in durable remission in a subset of patients with relapsed refractory large B cell lymphoma (LBCL) in the absence of persistent circulating CAR T cells. Aim: We postulated that long-term efficacy of CAR T therapy depends on the downstream triggering of native T cell immunity. To investigate this hypothesis, we performed single cell transcriptomic analysis of peripheral blood samples obtained at serial time points from patients participating in the ZUMA 1 Axi-cel study. We interrogated the T cell repertoire in patients who achieved durable remission compared to those experiencing disease relapse with respect to the expression of activated, cytotoxic, and inhibitory markers and clonotypic expansion of critical T cell subsets. Methods: Single cell immunoprofiling (expression + V(D)J sequencing) was performed using the 10x Genomics single cell 5' assay on PBMC samples from ZUMA-1 patients (N=32), collected at leukapheresis, 4 weeks, 6 months, and 12 months post Axi-cel. Based on clinical response, patients were divided into 3 groups: non-responders (N=8), relapsed within 1 year from CAR T infusion (N=9), and long-term responders (N=12). Three participants did not consent to the release of clinical outcomes. Results: A total of 405,775 cells passed quality check, capturing 73 cellular populations (Fig. 1a). T cells comprised the largest compartment with 28 different populations and 264,059 cells. Long-term responders demonstrated a distinct T cell landscape compared to relapsed patients, notable for a higher CD8/CD4 T cell ratio and increased expression of cytotoxic genes at all time points. Accordingly, activated CD8 cytotoxic effector memory T cell (Tem) populations were significantly increased in long-term responders compared to relapsed patients; especially two Tem populations, one characterized by high expression of CD44, GZMB and TGFB1 (Tem/TGFB1+), and a second highly expressing cytotoxic markers such as GZMA, GZMB, NKG7, CTS7, and CTSW (Tem/NKG7+). Following CAR T cell infusion, relapsed patients exhibited increased proportions of T regs and of T reg-like Th17 cells highly expressing FOXP3, IL2RA, and CTLA4. In contrast to all other CD4 populations, which were more abundant in the relapsed group, long-term responders presented higher abundance of CD4 cytotoxic T lymphocytes (CD4 CTL), characterized by high expression of both CD4-related and cytotoxic markers, including granzymes, and GNLY. Comparison of the TCR repertoire pre- and post-CAR T demonstrated a greater clonal expansion (TCRα/β-clonotype frequency > 1) of cytotoxic T cell populations in the long-term responders compared to relapsed and non-responder patients. In particular, higher clonotypic expansion was observed in the Tem/TGFB1+, Tem/NKG7+, and CD4 CTL populations (Fig. 1b). In contrast to our observations for the cytotoxic T cell populations, T regs were identified as more clonally expanded over time in the relapsed patients compared to the long-term responders. Conclusion: The application of single-cell immunoprofiling on longitudinal samples from CAR T cell treated LBCL patients from the ZUMA-1 trial demonstrated striking differences in cellular populations and clonal expansion patterns among patients who demonstrated long term remission compared to those who relapsed within 1 year following initial response. Reduced cytotoxic T cell expansion and increased proportions of immunomodulatory T cell populations were noted in relapsed patients compared to long term responders. These data provide insight into crucial factors that can be leveraged both to predict patient response and to further our understanding of long-term remission following CAR T cell treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparing the Impact of NHEJ-CRISPR/Cas9 and Base Gene Editing on Rhesus Macaque Hematopoietic Stem Cell Clonal Behavior and Integrity

Despite recent transformative advancements in gene editing tools and hematopoietic stem and progenitor cell (HSPC) gene therapies, there are still unanswered questions about the impact of CRISPR/Cas9 and base editing technologies on HSPC clonal behavior, function, and integrity in vivo. Better understanding of the impact of available gene therapy modalities on HSCs in a relevant animal model should help safely move these therapies forward for clinical use and provide information regarding their relative advantages and risks. CRISPR/Cas9 editing generates double strand DNA breaks (DSBs) at targeted genomic locations and potentially at off-target sites, resulting in potential HSC genotoxicity and loss. To counter DSB-related consequences, base editors (BEs) have been developed with the ability to produce single-nucleotide conversion without DSBs, avoiding potentially deleterious DNA damage repair pathways. CD33 is expressed on all myeloid cells downstream of the common myeloid progenitor and is used as both a diagnostic marker and a therapeutic target for acute myeloid leukemia. Our past work demonstrated successful Cas9/NHEJ-mediated CD33 KO in rhesus macaque (RM) HSPCs with no functional impact (Kim et al, Cell, 2018). In the current study, we first genetically barcoded RM HSPCs, and then knocked out the CD33 locus with the intent of comparing the clonal impact of CRISPR/Cas9 editing via non-homologous end joining (NHEJ) to base editing in RM HSPCs in vitro by myeloid differentiation assays and in vivo following autologous HSPC transplantation. Highly diverse DNA barcodes were introduced into RM HSPCs via transduction with a lentiviral vector also expressing GFP. 24 hours later, the cells were electroporated with either Cas9/NHEJ or BE complexes targeted to disrupt CD33. Cells were further cultured in myeloid differentiation media for 8 days in vitro for differentiation assays (IVD). Cell viability and proliferation were analyzed over time during IVD. DNA was collected on day 8 for editing efficiency and barcode clonal analysis. In addition, GFP and CD33 expression were analyzed by flow cytometry to assess CD33 KO efficiency and examine whether the addition of gene editors impacted the clonality of the HSPCs as assessed by barcode retrieval, and whether the initial lentiviral transduction impacted editing efficiency. HSPC colony-forming unit (CFU) assays were carried out to assess differentiation potential of the transduced and/or edited HSPCs and check for biallelic versus monoallelic editing status at a clonal level. Throughout the 8 days of IVD, cell numbers increased overtime and maintained high cell viability (80-95%) in all test groups, which indicates barcoded lentiviral transduction and gene editing procedures had minimal impact on cell proliferation and viability. Base editing resulted in 80-85% CD33 KO efficiency, regardless of barcode lentivirus transduction prior to gene editing. However, Cas9/NHEJ was significantly less efficient, with CD33 KO efficiency of 10-20%, requiring additional optimization (Figure 1A). Variant allele frequency analysis confirmed the relative levels of CD33 locus editing by both editing methods. CFU assays indicated the lentiviral transduction and gene editing methods do not alter the HSPC differentiation potential as assessed in vitro. Barcode clonal analysis revealed both NHEJ and BE test groups demonstrated retention of high clonal diversity as assessed by Shannon Diversity Index, all > 8 regardless of gene editing methods (Figure 1B). In summary, lentiviral barcode transduction and editing procedures had no negative impact on cell viability and proliferation. Base editing allowed very high gene editing efficiency in HSPCs and can serve as a powerful editing tool. Importantly, both editing methods resulted highly diverse polyclonal clonal patterns in vitro long term, with no significant differences compared to the non-edited group. Combining genetic barcode technology and the BE or Cas9/NHEJ editing in an in vivo RM autologous HSPC transplantation model to track the hematopoietic clonal reconstitution of the edited HSPCs over time and compare the impact of BE or Cas9/NHEJ editing on engrafting HSPCs clonal behavior is ongoing. Therefore, these clinically relevant findings can determine which gene editing method is more suitable for developing human HSPC gene therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Barcode clonal tracking of tissue-resident immune cells in rhesus macaque highlights distinct clonal distribution pattern of tissue NK cells.

Tissue resident (TR) immune cells play important roles in facilitating tissue homeostasis, coordinating immune responses against infections and tumors, and maintaining immunological memory. While studies have shown these cells are distinct phenotypically and functionally from cells found in the peripheral blood (PB), the clonal relationship between these populations across tissues has not been comprehensively studied in primates or humans. We utilized autologous transplantation of rhesus macaque hematopoietic stem and progenitor cells containing high diversity barcodes to track the clonal distribution of T, B, myeloid and natural killer (NK) cell populations across tissues, including liver, spleen, lung, and gastrointestinal (GI) tract, in comparison with PB longitudinally post-transplantation, in particular we focused on NK cells which do not contain endogenous clonal markers and have not been previously studied in this context. T cells demonstrated tissue-specific clonal expansions as expected, both overlapping and distinct from blood T cells. In contrast, B and myeloid cells showed a much more homogeneous clonal pattern across various tissues and the blood. The clonal distribution of TR NK was more heterogenous between individual animals. In some animals, as we have previously reported, we observed large PB clonal expansions in mature CD56-CD16+ NK cells. Notably, we found a separate set of highly expanded PB clones in CD16-CD56- (DN) NK subset that were also contributing to TR NK cells in all tissues examined, both in TR CD56-CD16+ and DN populations but absent in CD56+16- TR NK across all tissues analyzed. Additionally, we observed sets of TR NK clones specific to individual tissues such as lung or GI tract and sets of TR NK clones shared across liver and spleen, distinct from other tissues. Combined with prior functional data that suggests NK memory is restricted to liver or other TR NK cells, these clonally expanded TR NK cells may be of interest for future investigation into NK cell tissue immunological memory, with implications for development of NK based immunotherapies and an understanding of NK memory.

Spatial genomics maps the structure, nature and evolution of cancer clones

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour1–3. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive4,5. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.