Clonal Origin Research Articles

Clonal origin of KMT2A wild-type lineage-switch leukemia following CAR-T cell and blinatumomab therapy

Children with acute lymphoblastic leukemia (ALL) undergoing anti-CD19 therapy occasionally develop acute myeloid leukemia (AML). The clonal origin of such lineage-switch leukemias1–4 remains unresolved. Here, we reconstructed the phylogeny of multiple leukemias in a girl who, following multiply relapsed ALL, received anti-CD19 cellular and antibody treatment and subsequently developed AML. Whole genome sequencing unambiguously revealed the AML derived from the initial ALL, with distinct driver mutations that were detectable before emergence. Extensive prior diversification and subsequent clonal selection underpins this fatal lineage switch. Genomic monitoring of primary leukemias and recurrences may predict therapy resistance, especially regarding anti-CD19 treatment.

The expression of tuftelin 1 as a new theranostic marker in early diagnosis and as a therapeutic target in hepatocellular carcinoma.

Currently, many challenges are associated with hepatocellular carcinoma (HCC) as the failure of early diagnosis, and the lack of effective therapy. This study aimed to investigate the possible role of tuftelin 1 (TUFT 1) in the early diagnosis of HCC and evaluate the potential contribution of the TUFT 1/Ca+2 /phosphinositol 3 kinase (PI3K) pathway in dantrolene sodium (Dan) therapeutic outcomes. The study was performed on two sets of rats, the staging (30 rats) and treatment sets (80 rats). HCC was induced by a single dose of diethylnitrosamine (DENA). The hepatic content of TUFT 1 protein was assayed via western blot and immunohistochemistry (IHC), while PI3K, vascular endothelial growth factor (VEGF), Cyclin D1, and matrix-metalloproteinase-9 (MMP-9) contents were assessed using enzyme-linked immunosorbent assay. Hepatic and serum calcium were measured colorimetrically. Furthermore, the nuclear proliferation marker, (Ki-67), (Kiel [Ki] where the antibody was produced in the University Department of Pathology and the original clone number is 67)-expression was assessed by IHC. TUFT 1/Ca+2 /PI3K signaling pathway was progressively activated in the 3 studied stages of HCC with subsequent upregulation of angiogenesis, cell cycle, and metastasis. More interestingly, Dan led to TUFT 1/Ca+2 /PI3K pathway disruption by diminution of the hepatic contents of TUFT 1, calcium, PI3K, VEGF, Cyclin D1, and MMP-9 in a dose-dependent pattern. TUFT 1 can serve as a theranostic biomarker in HCC. Moreover, Dan exerted an antineoplastic effect against HCC via the interruption of TUFT 1/Ca+2 /PI3K pathway.

Rapid development of cefiderocol resistance in a carbapenem-resistant Pseudomonas aeruginosa isolate associated with mutations in the pyoverdine biosynthesis pathway

Here we report the in vivo development of cefiderocol resistance within 11 days after therapy initiation in a critically ill patient with bloodstream infection, infection of peri-anal fistula, and pneumonia caused by a VIM-2 harbouring, carbapenem-resistant Pseudomonas aeruginosa. Compared to a cefiderocol-naïve P. aeruginosa blood culture isolate, agar diffusion susceptibility testing found a reduced cefiderocol inhibition zone diameter in a P. aeruginosa recovered from peri-anal abscess tissue cultures after initiation of cefiderocol therapy. Subsequent whole-genome sequencing suggested that both isolates were of clonal origin. Comparison of genomes found an accumulation of missense mutations within pvdP, pvdE, pvdJ, and pvdD (i.e. genes associated with biosynthesis of pyoverdine), the main siderophore produced by P. aeruginosa. Quantification of pyoverdine production under iron-depleted conditions showed a significantly (P=0.0003) higher pyoverdine production by the cefiderocol-resistant isolate. While pyoverdine quantity alone appears not to be decisive for cefiderocol resistance, the reported case highlights the potentially rapid emergence of cefiderocol resistance in P. aeruginosa and points towards a potential involvement of iron up-take systems in this process.

The different clonal origins of metachronous and synchronous metastases

BackgroundMetastases are the leading cause of mortality in cancer patients. Linear and parallel are the two prominent models of metastatic progression. Metastases can be detected synchronously along with the primary tumor or metachronously, following treatment of localized disease. The aim of the study was to determine whether synchronous metastases (SM) and metachronous metastases (MM) differ only in lead-time or stem from different biological processes.Materials and methodsWe retrospectively studied the chest CTs of 791 patients inflicted by eleven malignancy types that were treated in our institution in the years 2010–2020. Patient’s population included 396 with SM and 395 with MM. The diameter of 15,427 lung metastases was measured. Clonal origin was deduced from the linear/parallel ratio (LPR)-a computerized analysis of metastases diameters. LPR of 1 suggests pure linear dissemination and − 1 pure parallel.ResultsPatients with MM were significantly older (average of 62.9 vs 60.7 years, p = 0.02), and higher percentage of them were males (58.7% vs 51.1%, p = 0.03). Median overall survival of patients with MM and SM was remarkably similar (23 months and 26 months respectively, p = 0.774) when calculated from the time of metastases diagnosis. Parallel dissemination (LPR ≤ 0) was found in 35.4% of patients with MM compared to only 19.8% of the patients with SM (p < 0.00001).ConclusionPatients with SM and MM differ in demography and in clonal origin. Different therapeutic approaches may be considered in these two conditions.

Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer.

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinical oncology research method. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scid IL2Rgammanull (NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. The immunophenotype of lymphoproliferations arising in the lung TRACERx PDX pipeline were characterised. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview). Lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma with plasma cell features. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest that B cell clones with lymphoproliferative potential are present within primary NSCLC tumours, and that these are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines.

Paternity pseudo-exclusion caused by tetragametic chimerism in a gestational surrogacy case

Chimerism is a condition when an organism is composed of two or more populations of genetically distinct cells. Chimerism often produces curious results of medical and genetic investigations and could be a major cause of false negative conclusions in parentage testing. Here we describe a paternity pseudo-exclusion due to tetragametic chimerism in a gestational surrogacy case originated in a fertility clinic. Initial analysis using a buccal swab from the child and a peripheral blood sample from the father showed paternity exclusion at 6 STR loci. To find out the reason for observed paternal discrepancy father's semen sample used for IVF and samples from other tissues were genotyped. Buccal swabs, semen, hair follicles, nail clippings and cerumen showed identical mixed autosomal STR profiles originated from two genetically different cell lines and for all the 24 informative loci contain paternal obligate alleles. Results of Y-STR profiling of all paternal sample types showed a DNA profile originated from a single man. The mixed profiles obtained for different tissue types suggest that two genetically different cell lines contributed to formation of both the endoderm and the ectoderm of the father. The mesoderm seems to be monoclonal having originated from a genetically homogenous cell line as evidenced by the STR profile of peripheral blood. Such allelic pattern for various tissues suggests that the clonal origin happened at the very early stage of embryonic development. Approaches to minimise the rate of false exclusions in DNA parentage tests due to chimerism are discussed.

Abstract PR008: Molecular determinants of prostate cancer lineage plasticity

Abstract Androgen deprivation therapy (ADT) has been an effective treatment for advanced prostate cancer (PCa) for decades because PCa retains the lineage specific dependence of normal prostate tissue on androgen receptor (AR) signaling. Unfortunately, nearly all patients will progress through ADT with castrate resistant prostate cancer (CRPC). CRPC is typically associated with reactivation of AR signaling despite ADT. However with widespread clinical deployment of potent AR signaling inhibitors, an increasing fraction of CRPCs (~20) are now observed to lose AR signaling activity and show gene expression patterns consistent with alternative lineage states. These lineage variants share clonal origin with the pre-existing adenocarcinoma, so some PCa cells have sufficient plasticity to reprogram to an AR independent lineage state like neuroendocrine prostate cancer (NEPC) that is recalcitrant to available therapies. The molecular determinants of prostate cancer lineage plasticity are incompletely defined, and this knowledge gap has hindered development of diagnostic and therapeutic approaches necessary to identify and treat prostate cancer lineage plasticity. We present results from experiments testing the contribution of EZH2 and NOTCH signaling to prostate cancer lineage plasticity and NEPC reprogramming. EZH2 expression is elevated in advanced PCa, particularly NEPC, suggesting it drives lineage reprogramming. However, using genetically engineered mice and organoids we find that EZH2 is not required for NEPC reprogramming that occurs in some PCa cells in the absence of the RB1 and TP53 tumor suppressor genes. EZH2 deficiency does alter multi-lineage prostate cancer plasticity, biasing development towards lineage variants observed clinically including amphicrine and double negative prostate cancer. In contrast, data from clinical specimens indicates NOTCH signaling activity declines in NEPC compared to AR+ CRPC. NOTCH signaling suppresses NEPC development in both organoids and genetically engineered mice, and loss of NOTCH signaling is required to maintain the NEPC lineage state. Constitutive NOTCH signaling biases PCa development towards unique lineage variants including AR positive metastatic PCa with luminal differentiation. These results identify EZH2 and NOTCH signaling as molecular determinants of PCa lineage plasticity and NEPC reprogramming with implications for ongoing efforts to target these molecules for PCa therapy. Citation Format: Kristine Wadosky, Sheng-Yu Ku, Yanqing Wang, Xiaojing Zhang, Jie Wang, Jianmin Wang, Himisha Beltran, David W. Goodrich. Molecular determinants of prostate cancer lineage plasticity [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR008.

Precise microdissection of gastric mixed adeno-neuroendocrine carcinoma dissects its genomic landscape and evolutionary clonal origins

Gastric mixed adenoneuroendocrine carcinoma (MANEC) is a clinically aggressive and heterogeneous tumor composed of adenocarcinoma (ACA) and neuroendocrine carcinoma (NEC). The genomic properties and evolutionary clonal origins of MANEC remain unclear. We conduct whole-exome and multiregional sequencing on 101 samples from 33 patients to elucidate their evolutionary paths. We identify four significantly mutated genes, TP53, RB1, APC, and CTNNB1. MANEC resembles chromosomal instability stomach adenocarcinoma in that whole-genome doubling in MANEC is predominant and occurs earlier than most copy-number losses. All tumors are of monoclonal origin, and NEC components show more aggressive genomic properties than their ACA counterparts. The phylogenetic trees show two tumor divergence patterns, including sequential and parallel divergence. Furthermore, ACA-to-NEC rather than NEC-to-ACA transition is confirmed by immunohistochemistry on 6 biomarkers in ACA- and NEC-dominant regions. These results provide insights into the clonal origin and tumor differentiation of MANEC.

The genetics of transformation from myelodysplastic syndrome to secondary acute myeloid leukemia.

e19073 Background: Gene mutations play key role in the transformation from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (s-AML). The aim of this study was to explore the clonal progression pattern from MDS to s-AML. Methods: A total of 663 patients diagnosed with MDS (n = 291), s-AML (n = 86), and primary AML (p-AML) (n = 286, M3 excluded) were enrolled in this study from May 2018 to December 2021, and the median age was 57 year-old (range 1~88). The diagnosis was established according to the WHO criteria. The MDS patients were divided into low-risk MDS ( n = 158) and high-risk MDS (n = 133) according to 2012 IPSS-R. Next generation sequencing was performed to detect the mutations of entire coding regions of 112 genes related with hematologic tumors in all patients. Results: It was found that mutations from 31 genes occurred in both MDS and AML, including DNA methylation ( DNMT3A, TET2, IDH1, IDH2, WT1), chromatin modification ( SETBP1, ASXL1, EZH2, PHF6), RNA splicing ( U2AF1, SF3B1, SRSF2, ZRSR2), transcription regulation ( TP53, CEBPA, RUNX1, ETV6, GATA2, BCOR, BCORL1), signal transduction pathways ( NRAS, KRAS, PTPN11, NOTCH1, FLT3, JAK2, KIT), and others ( NPM1, FAT, STAG2, DDX41). By enrichment analysis, those mutations were classified into two categories: type A mutations ( FLT3, IDH1, NPM1, NRAS, PTPN11, WT1, CEBPA) and type B mutations ( IDH2, KRAS, U2AF1, ZRSR2, SRSF2, STAG2, DNMT3A, EZH2, RUNX1, ETV6, TP53). The type A genes were significantly enriched in p-AML than in MDS, while some type B mutations, particularly RNA splicing, were significantly enriched in MDS than in p-AML. The VAFs of type A mutations were significantly lower than those of type B mutations. The longitudinal analysis of 10 patients with s-AML revealed two main models of clonal evolution, linear evolution and clone sweeping. Although the two models differed in the disease development, new sub-clones appeared in 9/10 patients. Interestingly, many of the new mutations were type A mutations (n = 7, 7/10), and 7 out of 10 patients carried type B mutations when they were at MDS state. The VAF data also showed that the preexisting mutations were generally higher than the newly emerging genetic alterations. Finally, an individual case was used to delineate the clone evolution. The patient was initially diagnosed with 5q syndrome and U2AF1 mutation (type B) was identified. After treatment, the patient experienced remission, and developed a new ETV6 mutation while the clonal size of U2AF1 decreased. When the patient relapsed, the clone size of ETV6 decreased while U2AF1 mutated clone size were amplified, a typical “clone sweeping” pattern. When the patient eventually transformed to s-AML, three type A mutations ( NRAS, KRAS and PTPN11) appeared simultaneously. Conclusions: This study revealed that the type A mutations are mainly responsible for the progression of MDS to AML, and the type B mutations are related to the clonal origin of MDS.

Whole-exome sequencing reveals the metastatic potential of hepatocellular carcinoma from the perspective of tumor and circulating tumor DNA.

Relapse of hepatocellular carcinoma (HCC) due to vascular invasion is common, but the genomic mechanisms remain unclear, and molecular determinants of high-risk relapse cases are lacking. We aimed to reveal the evolutionary trajectory of microvascular invasion (MVI) and develop a predictive signature for relapse in HCC. Whole-exome sequencing was performed on tumor and peritumor tissues, portal vein tumor thrombus (PVTT), and circulating tumor DNA (ctDNA) to compare the genomic profiles between 5 HCC patients with MVI and 5 patients without MVI. We conducted an integrated analysis of exome and transcriptome to develop and validate a prognostic signature in two public cohorts and one cohort from Zhongshan Hospital, Fudan University. Shared genomic landscapes and identical clonal origins among tumor, PVTT, and ctDNA were observed in MVI ( +) HCC, suggesting that genomic changes favoring metastasis occur at the primary tumor stage and are inherited in metastatic lesions and ctDNA. There was no clonal relatedness between the primary tumor and ctDNA in MVI ( - ) HCC. HCC had dynamic mutation alterations during MVI and exhibited genetic heterogeneity between primary and metastatic tumors, which can be comprehensively reflected by ctDNA. A relapse-related gene signature named RGSHCC was developed based on the significantly mutated genes associated with MVI and shown to be a robust classifier of HCC relapse. We characterized the genomic alterations during HCC vascular invasion and revealed a previously undescribed evolution pattern of ctDNA in HCC. A novel multiomics-based signature was developed to identify high-risk relapse populations.

Genomics, biofilm formation and infection of bladder epithelial cells in potentially uropathogenic Escherichia coli (UPEC) from animal sources and human urinary tract infections (UTIs) further support food-borne transmission

Escherichia coli is the main cause of urinary tract infections (UTI). While genomic comparison of specific clones recovered from animals, and human extraintestinal infections show high identity, studies demonstrating the uropathogenicity are lacking. In this study, comparative genomics combined with bladder-cell and biofilm formation assays, were performed for 31 E. coli of different origins: 7 from meat (poultry, beef, and pork); 2 from avian-farm environment; 12 from human uncomplicated UTI, uUTI; and 10 from human complicated UTI, cUTI. These isolates were selected based on their genetic uropathogenic (UPEC) status and phylogenetic background. In silico analysis revealed similar virulence-gene profiles, with flagella, type 1 and curli fimbriae, outer-membrane proteins (agn43, ompT, iha), and iron-uptake (iutA, entA, and fyuA) associated-traits as the most prevalent (>65%). In bladder-cell assays, moderate to strong values of association (83%, 60%, 77.8%) and invasion (0%, 70%, 55.5%) were exhibited by uUTI, cUTI, and animal-derived isolates, respectively. Of interest, uUTI isolates exhibited a significantly lower invasive capacity than cUTI isolates (p < 0.05). All isolates but one produced measurable biofilm. Notably, 1 turkey meat isolate O11:H6-F-ST457, and 2 cUTI isolates of the pandemic lineages O83:H42-F-ST1485-CC648 and O25b:H4-B2-ST131, showed strong association, invasion and biofilm formation. These isolates showed common carriage of type 1 fimbriae and csg operons, toxins (hlyF, tsh), iron uptake systems (iutA, entA, iroN), colicins, protectins (cvaC, iss, kpsM, traT), ompT, and malX. In summary, the similar in vitro behaviour found here for certain E. coli clones of animal origin would further reinforce the role of food-producing animals as a potential source of UPEC. Bladder-cell infection assays, combined with genomics, might be an alternative to in vivo virulence models to assess uropathogenicity.

THE INFLUENCE OF CLIMATE CHANGES ON THE CONDITION AND REPRODUCTIVE PROCESSES OF PINE OF THE COMMON FINNISH ORIGIN IN THE CONDITIONS OF VINNYTSIA REGION

According to research results, it was established that all clones were characterized by intensive formation of microstrobils and pollination. Tree analysis showed that clones of Finnish origin entered the pollen maturation phase earlier compared to local origin. Externally, the anthers of the local population were much larger. The analysis of photographs showed that the clones were characterized by different intensity of pollen grain germination and also had a different proportion of pollen grains with abnormalities. A general review of the images revealed that most of the clones of Finnish origin and the local population are characterized by fairly high viability. It was determined that the average number of germinated pollen grains for the studied clones was 89, of which about half (44) had anomalies. According to the obtained research results, we determined that the largest number of germinated grains is 150 pcs. was noted in clone No. 13 (E 618). The minimum number was recorded in clone No. 21 (E1881) – 13 pcs. Accordingly, the same clones recorded the maximum and minimum number of ungerminated pollen grains. In particular, clone No. 10 had 218 units, and clone No. 21 had 223 units. The most pollen grains with anomalies were found in clone No. 10 – 95 pcs. It was found that the highest percentage of pollen without germination was characteristic of clone No. 21 and was about 94.2%. At the same time, the lowest share of such pollen was recorded in clone No. 13 – 40.5%. As a result of regular records of the preservation of clones on the plantation, a high level of preservation was noted, which is 94.8%. Out of the initial number of 413 clones, 22 died. The total loss of clones in the section of representative offices was 1-4 trees. The highest dropout was characteristic of clones: No. E2312 (33.3%), E2257 (20.0%) K801 (20.0%). According to the correlation analysis, a weak correlation was found between preservation and state of clones and preservation and fruiting (r = - 0.21 and 0.19, respectively). The analysis of correlation dependences indicates that the deterioration of the condition of clones is not the main reason for their death. It was established that the average intensity of the formation of cones during the last years fluctuated within the range of 1.3-2.0 points. The best level of seed bearing was noted in 2015 and 2018. Along with the local control, the formation of cones was more intense in clones E 80 and E 709 and averaged more than 2.0 points. As a result of conducting field research, it was determined that the average condition of clones has slightly deteriorated in recent years. In particular, there is an increase in the average status category from 1.3 to 1.7. The best condition, along with local control, is characterized by clones E 80 and E 2125 (condition category 1,2). Clones E 1591, E 2257, K 684, K 801, K 818 are in much worse condition.

Progress on the molecular mechanism of portal vein tumor thrombosis formation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, with poor prognosis and high mortality. Early-stage HCC has no obvious clinical symptoms, and most patients are already at an advanced stage when they are diagnosed. Portal vein tumor thrombus (PVTT) is the most common complication and a poor prognostic factor for HCC, which frequently leads to portal vein hypertension, ascites, gastrointestinal bleeding, and tumor metastasis. The formation of PVTT is related to the complex structure and hemodynamic changes of the portal vein and is closely related to changes at the cellular and molecular levels. The differentially-expressed genes (DEGs) between PVTT and primary tumor (PT) suggest that the two tissues may have different clonal origins. Epigenetic and proteomic analyses also suggest complex and diverse mechanisms for the formation of PVTT. In addition, the tumor microenvironment and energy metabolism pathways are interrelated in regulating the invasion and progression of PVTT. Aerobic glycolysis and the tumor immune microenvironment have been the focus of recent studies on PVTT. In this review, we summarize the mechanism of PVTT formation at the cellular and molecular levels to provide information to guide better prevention and treatment of PVTT in the clinic.

Genomic Profiling of Urothelial Carcinoma in Situ of the Bladder

BACKGROUND AND AIMS Urothelial carcinoma in situ (CIS) of the bladder is an intra-epithelial, high grade, malignant neoplasm characterised by flat (non-papillary) growth, with high probability of disease progression.1,2 CIS has histologic features similar to invasive cancer and is often a precursor to and associated with invasive cancer. Molecularly, bladder cancer can be broadly categorised into luminal and basal subtypes.1-7 However, the molecular features that are unique to CIS, as compared to other high-grade lesions, including papillary Ta and T1 tumours, are underexplored. Bulk RNA expression profiling is a standard for molecularly characterising bladder cancer, and RNA-sequencing protocols utilising formalin-fixed paraffin-embedded (FFPE) tumours are well established. However, CIS presents challenges for RNA sequencing due to the inability to reliably detect, the low incidence rate, and the quality of RNA derived from FFPE samples. While enhanced cystoscopy and narrow band imaging have improved the ability to detect CIS, sampling acquisition techniques still limit the optimisation of RNA sequencing. Furthermore, the authors determined that established dual nucleic acid extraction protocols for FFPE samples were not feasible due to small biopsy sample sizes, and that protocols should be optimised for either RNA or DNA. This study attempts to overcome above stated challenges to understand the molecular landscape of CIS by contrasting against papillary tumours and normal urothelium using RNA-sequencing on FFPE samples, as well as whole exome sequencing, and immunohistochemical and immunofluorescent analyses, with an intent to identify unique molecular signatures associated with CIS. MATERIALS AND METHODS The authors performed whole transcriptome profiling with RNA sequencing of FFPE specimens from 15 CIS, nine high-grade papillary Ta/T1 tumours, and eight normal urothelial samples (Cohort A). Wilcoxon test was used to filter differentially expressed genes and The Cancer Genome Atlas (TCGA) single sample classifier was used to assign molecular subtypes. Whole exome sequencing was performed for 19 patients with matched CIS and papillary tumour samples (Cohort B). Using multiplex immunofluorescence and immunohistochemistry analyses, 24 samples from 15 patients were analysed for the presence of cytotoxic T cells, T helper cells, regulatory T cells, B cells, M1 and M2 macrophages, and programmed cell death protein 1 and programmed death-ligand 1-expressing cells. RESULTS The authors performed molecular subtyping applying the UROMOL classification and as previously shown for CIS, the majority were Class 2a and 2b, with four Class 3 and one Class 1. They applied the TCGA single patient classifier and the majority were luminal with a breakdown of two luminal, five luminal infiltrated, seven luminal papillary, three basal, and one neuronal subtype (Figure 1). A 46-gene signature of differentially expressed genes in CIS samples was identified and included known druggable targets that were selectively upregulated (MTOR, TYK2, AXIN1, CPT1B, GAK, and PIEZO1) or downregulated (BRD2 and NDUFB2; p&lt;0.05). An independent dataset was used to assess the robustness of these markers. High expression of MTOR, GUSBP11, KMT2D, and URB1 was significantly associated with CIS in this independent dataset.Additionally, mutational analysis of 34 matched CIS and 33 papillary tumours revealed a clonal origin of the lesions with mutations shared between both synchronous and metachronous tumours. Inter- and intra-patient mutational heterogeneity was also observed. The most frequently mutated gene was KDM6A, which was observed in 53% of the patient samples. Analysis of the immunological landscape of 24 CIS and papillary tumour lesions showed higher levels of immune cells in stromal compartments compared to carcinoma regions. Furthermore, more programmed cell death protein 1 positive cells were observed in CIS lesions compared with papillary tumours (p=0.03). CONCLUSION Collectively, this study identifies a molecular signature that distinguishes CIS lesions from papillary tumours in terms of gene expression levels, mutational landscape, and proportion of programmed cell death protein 1 positive cells that may contribute to an aggressive feature of progressive disease phenotype of the bladder cancer.

Особенности клонального микроразмножения триплоидной осины с применением ростостимулирующих препаратов

The creation of fast-growing triploid aspen plantations in the area of wood processing enterprises has high importance in terms of increasing demand for deciduous wood associated with the development of board production and the prospects for the introduction of innovative technologies for deep processing of wood. The article presents the results of studies of triploid aspen clones of Kostroma origin at the stages of “introduction to in vitro culture”, “proper micropropagation”, and “rooting of microshoots” with the use of growth-regulating substances. At the “introduction culture to in vitro” stage, the most effective sterilizing agents were silver nitrate with the concentration of 0.2 %, Lysoformin 3000, 5 % applied for 15 min and sulema, 0.2 % used for 10 min. At the “proper micropropagation” stage, the total length of triploid aspen shoots did not differ significantly depending on the composition of the nutrient media studied but was slightly exceeded the options with Woody Plant Medium. An increase by 1.2–2.6 times was observed in the total length of triploid aspen microshoots in vitro, when the concentration of 6-benzylaminopurine in the nutrient medium was increased from 0.5 to 1.0 mg/L. The maximum value of the total length (5.6 cm) of tryploid aspen microshoots in vitro was observed on Woody Plant Medium at the 6-benzylaminopurine concentration of 1.0 mg/L and the presence of Epin-Extra preparation of 0.5 mg/L. At the “rooting of microshoots” stage the number, average, and total length of triploid aspen roots in vitro had no statistically significant differences depending on the nutrient medium composition. Increasing the concentrations of indole-3-butyric acid and indole-3-acetic acid from 1.0 to 2.0 mg/L in the nutrient medium increased the total length of regenerated plant roots of triploid aspen in vitro by 2.3–2.4 times. The maximum total length (5.1 cm) of triploid aspen roots in vitro was observed on Woody Plant Medium with indole-3-butyric acid at a concentration of 1.0 mg/L. Clonal micropropagation appears to be a perspective for accelerated production of elite planting material of triploid aspen for the purposes of laying out timber plantations. For citation: Makarov S.S., Bagaev E.S., Chudetsky A.I., Kuznetsova I.B., Lebedeva O.P., Antonov A.M. Features of Triploid Aspen Clonal Micropropagation Using Modern Growth-Stimulating Preparations. Lesnoy Zhurnal = Russian Forestry Journal, 2023, no. 2, pp. 183–194. (In Russ.). https://doi.org/10.37482/0536-1036-2023-2-183-194

Synchronus Oral Squamous Cell Carcinoma in A Female: A Rare Case Report

Worldwide, the number of new cases of lip and oral cavity cancer in both sexes, all age groups in 2020 are around 377713 and the number of deaths in both sexes, all age groups are around 177757. In India, lip and oral cavity cancer ranks at 2nd position with incidence around 10.3%, mortality 8.8% and 5- year prevalence around21.77%. Multiple theories have been put forth describing the multifocal origin of squamous cell carcinoma (SCC) of the oral and aerodigestive tract. “Field Cancerization “is the first theory described in oral squamous cell carcinoma (SCC) which explains the multifocal or synchronous origin of the SCC. Synchronous carcinomas are defined as second neoplasms at the same time or within 6 months period of primary lesions. After that, they are described as metachronous neoplasms. The criteria to describe synchronous tumours were elaborated by Warren and Gates and Moertel et al. “Field Cancerization “theory was explained by widespread epithelial exposure to carcinogens; however, the alternative theory of common clonal origin is now supported by the recent molecular studies. Second Primary Tumours (Synchronous or Metachronous) are usually more aggressive, more resistant, metastasizes early and they require a more aggressive treatment strategy. Hence, their early detection is the crucial step in planning the best possible management. We are reporting an unusual case of synchronous oral squamous cell carcinoma in 58-year-old female who underwent surgery followed by adjuvant treatment after diagnosis.

Abstract 2289: Predicting tumor somatic versus clonal hematopoiesis origin for short variants in liquid assay

Abstract Introduction A common challenge presented by emerging liquid biopsy technology is to distinguish somatic variants originated in CH from those in tumor. While sequencing of genomic DNA from matched peripheral blood mononuclear cells (PBMC) can effectively identify CH variants, its wide application still remains prohibitively expensive. Computational-only solutions are challenging due to shared bioinformatic presentations in both CH and somatic variants such as low allele frequency. Development of such algorithm with clinically meaningful accuracy will have extensive utility in cancer care. Method We sequenced paired plasma-buffy coat samples from 754 pan-cancer patients using the FoundationOne® Liquid CDx (F1LCDx) assay. The samples were randomized into a training and a test set. Ground truth (tumor somatic vs. CH) for SV, including base substitutions and short indels, was determined by comparing variant calls in plasma vs in PBMC. A deep learning prediction model was developed in the training set. The model incorporates genomic and clinical features as input and returns a CH prediction score, which was dichotomized by a learned threshold. After the threshold and model parameters were finalized, we applied the model to the test set to evaluate performance. Results In the test set of 222 paired samples (523 CH and 904 tumor somatic variants), the model achieved 91% sensitivity (probability of predicting true CH variants as CH) and 88% specificity (probability of predicting true tumor somatic variants as tumor somatic). The model accurately identified CH variants in challenging genes such as TP53 (sensitivity = 94%, specificity = 90%, n = 144) and ATM (sensitivity = 93%, specificity = 95%, n = 49), which that are known to harbor both tumor somatic and CH variants. Performance was further evaluated by cancer types and variant allele frequency. CH variants were identified with 90% sensitivity and 89% specificity in non-small cell lung carcinoma (n = 384), and with 87% sensitivity and 100% specificity in pancreas cancer (n = 53). CH variants were identified with 92% sensitivity and 87% specificity in 1%-5% variant allele frequency (n = 645), and with 94% sensitivity and 94% specificity in 5%-10% variant allele frequency (n = 189). Of note, a well-known oncogenic mutation in ATM was predicted as CH in a prostate cancer patient, which would prevent ineffective administration of PARP inhibitors. Multiple plasma specimens also gained more accurate bTMB and tumor fraction estimation by filtering out CH variants. Conclusion For F1LCDx, we developed a computational method that distinguishes tumor somatic from CH variants from plasma cfDNA sequencing alone. The classifier exhibited high sensitivity and specificity in a pan-cancer cohort. Additional analytical validations are required in an external dataset. Clinical utility of this classifier will likely be further demonstrated in CH-corrected bTMB and tumor fraction. Citation Format: Daokun Sun, Zheng Kuang, Alexander D. Fine, Eliana Polisecki, Holley Gettler, Hawra Al-Rekabi, Dean Pavlick, Yanmei Huang, Lee A. Albacker, Brennan Decker, Chang Xu. Predicting tumor somatic versus clonal hematopoiesis origin for short variants in liquid assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2289.

Abstract 1511: Origin 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 18 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 18 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 14 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 38.9 months (7 - 85 months). In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. 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=17), 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, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamigushi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Seishi Ogawa, Hiroko Tanaka, Satoru Miyano. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1511.

Abstract 6346: Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy

Abstract OX40, tumor necrosis factor receptor superfamily member 4, is an immune co-stimulatory receptor, together with its ligand OX40L, forming OX40-OX40L trimer-trimer complex between the surface of T-cells (OX40) and APCs/NKs (OX40L). OX40 expresses constitutively on Treg as well as on conventional Teff, particularly within TME. OX40/OX40L interactions directly triggers conventional T cell activation via NF-kB1 pathway and inhibits bcl-x and survivin to prevent apoptosis; and it also downregulates FoxP3/CTLA4, thus Treg function. OX40 agonistic antibodies have also been shown to activate tumor immunity and increase anti-tumor activity in preclinical models. Here we report that we identified/constructed a novel OX40-agonistic humanized IgG1 monoclonal antibody (mAb), HX011, with high affinity through hybridoma technology. HX011 was subsequently confirmed of the similar binding properties and functions as the original mouse mAb clone, 9C12: 1) binding to human OX40 (EC50 ~0.012 nM; 2) similar function binding in the reporter assay (EC50 ~6.6 nM); but 3) has little blockade of the OX40L binding to OX40. In another word, HX011 is similar to BMS-986178 for the first two assays, but contrastingly different for the third assay. Non-competing with OX40L may actually maximize T-cell activation, a potential advantage over a competing antibody. In vivo pharmacology modeling of HX011 in the humanized syngeneic CRC model, MC38, where syngeneic C57BL/6 mouse mice were knocked in with huOX40 (“HuGEMM-OX40”), demonstrated strong anti-tumor activity (TGI ~100% at 1 mg/kg, twice weekly). HX011 is being further investigated as a potential new immunotherapy for the treatment of cancers. Citation Format: Hang Ke, Faming Zhang, Jialin Li, Feiyu Peng, Cen Chen, Henry Qixiang Li. Discovery of HX011, a novel OX40 mAb, for potential cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6346.

Abstract 6625: Mathematical modeling of single-cell data reveals the key role of stochasticity - not fitness - in determining the clonal origins of relapse in childhood leukemia

Abstract Acute lymphoblastic leukemia (cALL) is the most common pediatric cancer, and since up to 20% of children relapse after initially responding to chemotherapy treatment, the largest cause of cancer-related childhood deaths in the western world. With the idea that dissecting the evolutionary population dynamics leading to relapse would help explain treatment failure from a mechanistic standpoint, several studies have performed comparisons of genetic heterogeneity at diagnosis and relapse. These showed that as many as 70% of relapses are dominated by a single diagnostic subclone or one of its evolutionary progeny; leading to the prevailing view that treatment selection in cALL primarily operates at the genotype level. However, a crucial caveat of these studies is their reliance, due primarily to technical and economic constraints, on very limited data timepoints. While the dynamics linking said timepoints are often interpreted based on simple intuition. To overcome this sparse data challenge and reconstruct the entire spatial and temporal evolutionary trajectories of individual cALL cells from diagnosis to relapse, we have integrated experimental data with an in silico model. Single-cell WGS and RNAseq data were obtained from primary and xenografted leukemic cells before and after chemotherapy. These were then used to parameterize an agent-based mathematical model allowing tracking of the genotype and evolving phenotype of individual cells over time. When short treatment courses akin to those we delivered in vivo were used, the in silico model closely recapitulated the experimental results. Since cALL treatment lasts several years, it cannot be entirely modelled in vivo. Due to the rarity of post-treatment residual cells and the lack of universal cell surface markers to isolate them, direct analysis of clinical samples is also challenging. However, based on the initial treatment data, our calibrated model provided a unique opportunity to simulate longer remission-relapse dynamics of several leukemias. Mechanistic analysis of the collected data showed that, strikingly, in most cases, clonal dominance at relapse results from stochastic sweeps rather than deterministic differences in clonal fitness. Indeed, independent treatment of the same leukemia with an identical drug regimen more frequently ended with a considerably different clonal landscape (either polyclonal disease or a different dominant clone) than with the same outcome initially observed. Rather than genotype differences, the size of residual disease and a founder effect linked to the stochastic exit, post-treatment, of individual cells from quiescence were the key determinants of the relapse genetic landscape. Altogether, our data highlight the misinterpretation risk associated with snapshot analysis and might inform promising alternative treatment schedules. Citation Format: Virginia Turati, Javier Herrero, Mark Robertson-Tessi, Tariq Enver, Andriy Marusyk, Alexander RA Anderson. Mathematical modeling of single-cell data reveals the key role of stochasticity - not fitness - in determining the clonal origins of relapse in childhood leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6625.