Early Event In Tumorigenesis Research Articles

The role of imprinting genes' loss of imprints in cancers and their clinical implications.

Genomic imprinting plays an important role in the growth and development of mammals. When the original imprint status of these genes is lost, known as loss of imprinting (LOI), it may affect growth, neurocognitive development, metabolism, and even tumor susceptibility. The LOI of imprint genes has gradually been found not only as an early event in tumorigenesis, but also to be involved in progression. More than 120 imprinted genes had been identified in humans. In this review, we summarized the most studied LOI of two gene clusters and 13 single genes in cancers. We focused on the roles they played, that is, as growth suppressors and anti-apoptosis agents, sustaining proliferative signaling or inducing angiogenesis; the molecular pathways they regulated; and especially their clinical significance. It is notable that 12 combined forms of multi-genes' LOI, 3 of which have already been used as diagnostic models, achieved good sensitivity, specificity, and accuracy. In addition, the methods used for LOI detection in existing research are classified into detection of biallelic expression (BAE), differentially methylated regions (DMRs), methylation, and single-nucleotide polymorphisms (SNPs). These all indicated that the detection of imprinting genes' LOI has potential clinical significance in cancer diagnosis, treatment, and prognosis.

Integrative analysis of DNA methylation and gene expression through machine learning identifies stomach cancer diagnostic and prognostic biomarkers.

DNA methylation is an early event in tumorigenesis. Here, by integrative analysis of DNA methylation and gene expression and utilizing machine learning approaches, we introduced potential diagnostic and prognostic methylation signatures for stomach cancer. Differentially-methylated positions (DMPs) and differentially-expressed genes (DEGs) were identified using The Cancer Genome Atlas (TCGA) stomach adenocarcinoma (STAD) data. A total of 256 DMPs consisting of 140 and 116 hyper- and hypomethylated positions were identified between 443 tumour and 27 nontumour STAD samples. Gene expression analysis revealed a total of 2821 DEGs with 1247 upregulated and 1574 downregulated genes. By analysing the impact of cis and trans regulation of methylation on gene expression, a dominant negative correlation between methylation and expression was observed, while for trans regulation, in hypermethylated and hypomethylated genes, there was mainly a negative and positive correlation with gene expression, respectively. To find diagnostic biomarkers, we used 28 hypermethylated probes locating in the promoter of 27 downregulated genes. By implementing a feature selection approach, eight probes were selected and then used to build a support vector machine diagnostic model, which had an area under the curve of 0.99 and 0.97 in the training and validation (GSE30601 with 203 tumour and 94 nontumour samples) cohorts, respectively. Using 412 TCGA-STAD samples with both methylation and clinical data, we also identified four prognostic probes by implementing univariate and multivariate Cox regression analysis. In summary, our study introduced potential diagnostic and prognostic biomarkers for STAD, which demands further validation.

Clinicopathological features and genomic profiles of a group of secretory breast carcinomas in which progressive cases have more complex genomic features

BackgroundSecretory breast carcinoma (SBC) is a rare malignant breast neoplasm with distinct histological features, including solid, microcystic, tubular, and rarely papillary structures, traditionally characterized by a t (12;15) (p13:q25) translocation, which usually leads to ETV6-NTRK3 fusion, suggesting an early event in tumorigenesis. Due to the rarity of this disease, very few genome sequencing studies have been performed on a series of cases, especially progressive cases.MethodsSeven lesions from 5 patients diagnosed at the Third Affiliated Hospital of Soochow University from 2007 to 2021 were included. Clinicopathological features and prognosis/survival data were collected. Next-generation DNA sequencing was performed on six of the seven lesions.ResultsIn total, 3/7 (42.9%) lesions demonstrated estrogen receptor (ER) expression, including weak, moderate to strong staining, and no lesion demonstrated progesterone receptor (PR) expression. There were no cases of human epidermal growth factor (HER2) overexpression, and the Ki-67 index was low. S-100 and pan-TRK protein were diffusely positively expressed in all cases. All lesions were characterized by a t(12;15) (p13:q25) translocation, leading to ETV6-NTRK3 fusion confirmed by fluorescence in situ hybridization (FISH). The sequencing results showed that ETV6-NTRK3 fusion was the main driver of early tumorigenesis, while SBC with invasive biological behavior had more complex genomic variation in which TERT promoter mutation was detected.ConclusionsImmunohistochemical staining of a biomarker panel, including ER, PR, HER2, Ki-67, S-100 and pan-TRK, can be used as an auxiliary diagnostic tool, and FISH detection can be used as a diagnostic tool. ETV6-NTRK3 gene fusion involving multiple sites may drive tumorigenesis, while mutations in the TERT promoter region may be a factor driving tumor progression.

Cervical Adenocarcinoma: A Still Under-investigated Malignancy.

The aim of the study was to correlate the expression of mismatch repairs proteins (MMR), programmed-death-ligand1 (PDL-1), and estro-progestinic receptors (ER/PgR) in tissue samples from a series of cervical adenocarcinoma (ADC) patients with their clinicopathological features. Thirty-nine ADC specimens were retrospectively retrieved from the Division of Pathology of the University Hospital of Pisa from 2015 to 2021. Histological subtype, grade (G), Silva pattern, presence of lymph vascular space invasion (LVI), and perineural invasion (PNI) were annotated. On representative samples, immunostaining for ER/PgR, MLH1, PMS2, MSH2, MSH6, and PDL-1(sp142) was performed. Thirty-five ADCs were HPV-associated usual type (24 invasive and 11 in situ), 2 were clear cell type, one was a minimal deviation adenocarcinoma (MDA), and one was an invasive stratified mucin-producing carcinoma (iSMC). ADC associated with LVI were mostly G2-3, whereas those associated also with PNI were G3 with Silva pattern C. No difference in the expression of ER/PgR was observed with a dichotomic age stratification (51 years) of patients. Only 6 ADCs were MMR-deficient, all of them were of the usual type (4 invasive and 2 in situ). The heterodimer MLH-1/PMS2 was the one most frequently altered (5/6), whereas only one case had MSH6 loss. None of ADCs express PDL-1, except iSMC which showed PDL-1 expression >1% in neoplastic cells. Both invasive and in situ usual type ADCs indicate MMR deficiency, highlighting how this could be an early event in tumorigenesis. None of the cases, except for iSMC, express PDL-1.

Single-Cell Transcriptome Analysis Reveals Stem Cell-like Subsets in the Progression of Waldenström's Macroglobulinemia

Waldenström's macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by monoclonal immunoglobulin M protein production and bone marrow infiltration by lymphoplasmacytoid cells. It remains an incurable disease. IgM monoclonal gammopathy of undetermined significance (MGUS) is the premalignant condition and is associated with the risk of developing WM. MYD88L265P is a recurrent mutation found in more than 90% of WM patients, and this genetic event is detectable in at least half of IgM MGUS patients. However, the precise cellular landscape and the mechanisms of progression from IgM MGUS to WM remain unclear. Single-cell RNA sequencing (scRNA-seq) provides us a powerful approach to explore tumor heterogeneity and identify rare malignant cells. Anti-CD38 monoclonal antibodies have been introduced into the therapeutic arsenal for plasma cell diseases. Therefore, we analyzed CD38+ immune microenvironment together with B cells and plasma cells to illustrate the first cellular landscape of IgM MGUS and WM in single cell resolution. We isolated bone marrow CD19+ and CD19-CD38+ cells from WM patients(n=3), IgM MGUS patients(n=3) and healthy donors(n=3) and employed the 10x Genomics platform to perform single-cell transcriptomic sequencing. After quality control, our dataset contained a total of 73024 cells. We used UMAP to visualize the cell superclusters and identified clusters including B cells, plasma cells, CD3+CD20+ (CD19+) cells, T cells and NK cells based on the expression of canonical lineage markers and cluster-specific markers. We found that healthy donors had the highest percentage of B cells (72.7% in healthy donors, 19.0% in IgM MGUS patients, 60.5% in WM patients; p<0.001) and that IgM MGUS and WM patients had more NK and T cells than healthy controls (0.06% and 17.7%, respectively, in healthy donors; 24.1% and 20.5% in IgM MGUS patients; 8.5% and 24.0% in WM patients; p<0.001). We also noticed that CD3+CD20+(CD19+) cells were present in both IgM MGUS and WM patients. First, we investigated the cell clusters correlated with WM pathology including B cells and plasma cells. We compared the B-cell gene expression profiles after removing the early B cells. Compared with those of IgM MGUS patients, mature B cells of WM patients showed upregulation of PIM1, DUSP22, HES1, NEAT1, RGS1, RGS16, and GADD45B. Light chain restriction and higher CNV levels were found in B cells of WM patients. SCENIC analysis showed that FOS, FOSB, EGR1 and JUNB were downregulated in B cells from WM and IgM MGUS patients. REL, a proto-oncogene promoting the survival and proliferation of B lymphocytes, was upregulated in B cells of WM patients, and IRF8 and ELF1 were upregulated in B cells of IgM MGUS patients. Nevertheless, plasma cells of WM patients, IgM MGUS patients and healthy donors were colocalized in one cluster, which might indicate their similar gene expression patterns. Next, we further determined the characteristics of B-cell subpopulation with aberrant T-cell antigen expression. Trajectory analysis revealed that CD3+CD20+(CD19+) cells and pro-B cells were located at the same end of the branched structure and were then directed toward mature B cell and plasma cell fates, respectively (Figure. 1A). This inferred developmental trajectory suggested that CD3+CD20+(CD19+) cells may be tumor stem cell-like subset. Using flow cytometry, we validated that CD3 was expressed in a fraction of CD19+ cells (mean: 3.69%, range: 0.28%-15.10%) of bone marrow samples from 8 WM patients. In addition, targeted gene sequencing was performed on CD3+CD19+ and CD3-CD19+ cells from 6 WM patients. MYD88 were proved to be the most frequently mutated gene in this cell subset. And we inferred that MYD88 mutation might be the early events in tumorigenesis by VAF analysis (Figure. 1B). Additional subclonal hits, such as CXCR4 and MAP2K1 mutations, could be acquired during tumor progression. In conclusion, our study provides comprehensive insights into mechanisms of progression from IgM MGUS to WM. We identified the rare CD19+ cell subpopulation with T-cell antigen expression in WM patients. It will be interesting to explore novel therapeutic strategies targeting rare potential cancer-initiating cells in future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Aberrant gene expression pattern in the glycolysis-cholesterol synthesis axis is linked with immune infiltration and prognosis in prostate cancer: A bioinformatics analysis.

Aberrant lipid metabolism is an early event in tumorigenesis and has been found in a variety of tumor types, especially prostate cancer (PCa). Therefore, We hypothesize that PCa can be stratified into metabolic subgroups based on glycolytic and cholesterogenic related genes, and the different subgroups are closely related to the immune microenvironment. Bioinformatics analysis of genomic, transcriptomic, and clinical data from a comprehensive cohort of PCa patients was performed. Datasets included the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) dataset, GSE70768, our previously published PCa cohort. The unsupervised cluster analysis was employed to stratify PCa samples based on the expression of metabolic-related genes. Four molecular subtypes were identified, named Glycolytic, Cholesterogenic, Mixed, and Quiescent. Each metabolic subtype has specific features. Among the 4 subtypes, the cholesterogenic subtype exhibited better median survival, whereas patients with high expression of glycolytic genes showed the shortest survival. The mitochondrial pyruvate carriers (MPC) 1 exhibited expression difference between PCa metabolic subgroups, but not for MPCs 2. Glycolytic subtypes had lower immune cell scores, while Cholesterogenic subgroups had higher immune cell scores. Our results demonstrated that metabolic classifications based on specific glycolytic and cholesterol-producing pathways provide new biological insights into previously established subtypes and may guide develop personalized therapies for unique tumor metabolism characteristics.

Teenage-Onset Colorectal Cancers in a Digenic Cancer Predisposition Syndrome Provide Clues for the Interaction between Mismatch Repair and Polymerase δ Proofreading Deficiency in Tumorigenesis.

Simple SummaryColorectal cancer (CRC) in adolescents and young adults (AYA) is rare. Genetic causes include autosomal recessive and dominant monogenic disorders due to pathogenic variants (PVs) in genes involved in DNA repair. However, the genetic etiology of the majority of AYA-CRC remains unidentified. In two teenage siblings with CRC, we show to our knowledge for the first time that AYA-CRC cases can be caused by digenic inheritance of each a heterozygous pathogenic variant (PV) in the mismatch-repair (MMR) gene PMS2 and the proofreading polymerase (PP) Pol δ gene POLD1. With the aim to elucidate how the constitutional polymerase proofreading defect and the high propensity to MMR deficiency (MMRd) interact, we performed a comprehensive tumor analysis of the two siblings’ tumors. Results indicate that tumorigenesis is initiated by MMRd and the inherited POLD1 PV contributes to fast tumor progression reflected by an ultra-high tumor mutational burden (TMB) and specific mutational signatures.Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings’ CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient’s CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

Loss of Epigenetic Regulation Disrupts Lineage Integrity, Induces Aberrant Alveogenesis, and Promotes Breast Cancer.

Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.

Abstract 1322: The potential effects of thymidine kinase 1 on breast cancer invasion

Abstract Breast cancer is the most common cancer in women and is largely treatable within the early stages of the disease. However, patient mortality drastically declines as the tumor begins to invade other tissues and metastasize, making aggressive phenotypes especially problematic to treat. Such treatment typically requires an aggressive and decisive multidisciplinary approach. The recent expansion of immunotherapy as a viable treatment option has greatly improved treatment outcomes, especially with aggressive breast cancer phenotypes. Thymidine kinase 1 (TK1) is a DNA salvage pathway enzyme that is highly expressed during S phase and involved in cell cycle repair. Past studies indicated that TK1 is secreted into the serum of cancer patients and that its upregulation is an early event in tumorigenesis, thus suggesting that TK1 upregulation may have potential importance in tumor progression. This study’s objective was to further elucidate TK1’s role in tumor progression and metastasis of breast cancer specifically. We hypothesized that TK1 overexpression in breast cancer may affect invasion. To investigate this, TK1 mRNA transcript levels were analyzed between normal and BRCA patient tissue samples. The wild-type breast cancer cell line HCC 1806 was used, as well as a TK1 knockdown (L133) which was produced using CRISPR-Cas9. Successful TK1 knockdown was verified through western blot and qPCR. Bioinformatics was also performed to analyze the relationship between our breast cancer cell lines and various cell adhesion factors. The cell lines HCC 1806 and L133 were then tested with an invasive scratch assay. Additional western blots were run to further investigate the potential relationship TK1 may have with other cell cycle checkpoint proteins. Results showed that TK1 mRNA transcript levels are higher in BRCA tissues compared to normal controls. Western blot and qPCR indicated that TK1 was successfully knocked down in L133 cells. Bioinformatic differential gene analysis revealed several correlations with cell adhesion factors, with contrasting correlations when compared between the HCC 1806 and L133 cell lines. The invasive scratch assay results indicated that the TK1 knockdown L133 cells were less invasive than HCC 1806 cells. In conclusion, results verified that TK1 is highly expressed in breast cancer and that it may help tumor invasion by influencing cell adhesion factors. Further investigation to understand TK1’s potential interactions with important cell adhesion factors, such as FHL1 or GAS6, may help elucidate other therapeutic targets for preventing disease progression. Citation Format: Eliza E. Bitter, Rachel M. Morris, Toni Mortimer, Kai Barlow, Abigail Schekall, Michelle H. Townsend, Jonathan Skidmore, Brett E. Pickett, Kim L. O'Neill. The potential effects of thymidine kinase 1 on breast cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1322.

Metaplasia‐induced Epithelial Heterogeneity Directs Pancreatic Injury and Tumorigenesis

Background and AimsDespite years of research, mechanisms of pancreatic injury and healing remain poorly understood. Acute pancreatitis is a painful and debilitating condition; chronic pancreatitis may be asymptomatic, but greatly enhances the risk of pancreatic ductal adenocarcinoma (PDAC). Acinar to ductal metaplasia (ADM), or the transdifferentiation of digestive enzyme producing acinar cells to ductal cells, is an early event in both conditions. While ADM is thought to function in healing and regeneration, it also represents a first step in tumorigenesis, demonstrating the duplicitous nature of this inherent plasticity. The goal of these studies was to define the populations arising in ADM, associated transcriptional changes, and their role in disease progression.MethodsAcinar cells were lineage traced to follow their fate upon injury. Transcripts of more than 13,000 EYFP+ cells were determined using single cell RNA sequencing (scRNA‐seq). Developmental trajectories were generated using several computational biology approaches that rely on non‐overlapping assumptions. Data were compared to scRNA‐seq studies of gastric metaplasia, oncogenic KrasG12D‐induced ADM, and human pancreatitis. Results were confirmed using immunostaining and electron microscopy. Tuft and enteroendocrine cell (EEC) populations were quantified throughout tumorigenesis. KrasG12D was expressed in injury‐induced ADM populations using several inducible Cre drivers.ResultsscRNA‐seq of ADM from chronically injured pancreata revealed emergence of a mucin/ductal population that resembles gastric pyloric metaplasia. Developmental trajectories suggest that some pyloric metaplasia cells generate tuft or EEC populations as distinct lineages. Comparison to KrasG12D‐induced ADM reveals populations associated with disease progression. Immunostaining demonstrates that tuft and EEC formation is an early event in tumorigenesis. Activation of KrasG12D in ADM populations results in neoplastic transformation and the formation of MUC5AC+ pit cells. Human pancreatitis samples reflect a pyloric metaplasia phenotype as well as the formation of tuft and EEC populations.ConclusionsADM under conditions of chronic injury results in the formation of a pyloric‐type metaplasia which seeds disparate tuft and EEC lineages. This carefully orchestrated plasticity generates myriad epithelial cell types which likely mitigate injury, providing protection from the formation of pancreatitis and PDAC. KrasG12D expression is sufficient to drive neoplasia when targeted to injury‐induced ADM populations offering an alternative origin for tumorigenesis. This program is conserved in human pancreatitis and provides insight into early events in pancreas diseases.

Enteroendocrine Cell Formation Is an Early Event in Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of only 11%, due, in part, to late diagnosis, making the need to understand early events in tumorigenesis critical. Acinar-to-ductal metaplasia (ADM), when not resolved, is a PDAC precursor. Recently, we showed that ADM is constituted by a heterogenous population of cells, including hormone-producing enteroendocrine cells (EECs: gamma, delta, epsilon, and enterochromaffin cells). In this study, we employed histopathological techniques to identify and quantify the abundance of EEC subtypes throughout pancreatic tumorigenesis in mouse models and human disease. We found that EECs are most abundant in ADM and significantly decrease with lesion progression. Co-immunofluorescence identifies distinct lineages and bihormonal populations. Evaluation of EEC abundance in mice lacking Pou2f3 demonstrates that the tuft cell master regulator transcription factor is not required for EEC formation. We compared these data to human neoplasia and PDAC and observed similar trends. Lastly, we confirm that EECs are a normal cellular compartment within the murine and human pancreatic ductal trees. Altogether, these data identify EECs as a cellular compartment of the normal pancreas, which expands early in tumorigenesis and is largely lost with disease progression.

EPCO-28. SINGLE-CELL RNA SEQUENCING IDENTIFIES INTRATUMORAL HETEROGENEITY AND ACTIVATION OF PRO-INVASIVE PATHWAYS IN NON-FUNCTIONING PITUITARY ADENOMAS

Abstract Pituitary adenomas (PA) are one of the most common primary brain tumors and comprise 15% of brain neoplasms. Most PAs are histologically benign but can cause significant morbidity. The genetic profile of PAs is poorly understood. We used single-cell RNA sequencing using the 10X genomic platform to investigate cellular heterogeneity in twelve non-functioning pituitary adenoma samples from nine patients including site-specific (core vs edge) samples from three patients. Our analysis identified discrete clusters of cells associated with activation of specific functional pathways including lipid metabolism, angiogenic, and antigen presentation and processing pathways regardless of location within the tumor. MALT1, a lncRNA associated with increased proliferation and metastasis was ubiquitously expressed amongst these samples. Analysis of the core vs edge samples showed two specific clusters with activated invasion-promoting pathways including PI3k/AKT signaling, Wnt signaling (Wnt6 and FZD4), and epithelial-mesenchymal transition (TGFB1, SMAD1, ZEB1, and SNAI2) in the edge of the tumors. The activated Wnt signaling cascade drove a proinflammatory tumor microenvironment induced by the expression of IL-1, IL-17, and Toll-like receptors (TLR6 and TLR7/8) resulting in suppression of Tregs. Copy number variation analysis using the CONICS-CNV algorithm highlighted distinct chromosomal alterations within our samples that led to insight into clonal variations within each tumor with loss of chromosome 2 an early event in tumorigenesis and gain/loss of chromosome 19 as late events. Mapping the copy number variation analysis with the somatic variant analysis using the Vartrix algorithm identified novel driver mutations within these tumors. These findings help define the molecular fingerprint of pituitary adenomas and provide insights which could be utilized for better management of these tumors.

In This Issue

In This Issue

Analysis of HOXA9 methylated ctDNA in ovarian cancer using sense-antisense measurement

DNA promoter methylation is an early event in tumorigenesis and holds promise as a valuable marker in ovarian cancer (OC). It can be measured using circulating tumor specific DNA (ctDNA) isolated from the bloodstream. Sensitivity, however, is a limiting factor of its diagnostic feasibility in OC. DNA methylation analyses are based on bisulfite conversion, resulting in two DNA strands that are no longer complementary. The current standard strategy would then target only one of the double stranded DNA strands, but the potential to increase the sensitivity by targeting both DNA strands is available. In this study, we aimed at evaluating the diagnostic potential of methylated HOXA9 ctDNA in OC by targeting both the DNA sense and antisense strand. Methylated HOXA9 was detected in the plasma of 47/79 (59.5%) patients with newly diagnosed OC using sense-antisense droplet digital PCR. Simultaneous sense-antisense measurement increased the sensitivity by 14.6% (51.9% to 59.5%) as compared to antisense only. In patients with FIGO stage I-II disease the sensitivity was increased by 25%. In conclusion, simultaneous measurement targeting both DNA strands can increase the sensitivity and the analytical approach appears valuable in the diagnostic setting of OC.

OTEH-11. Single Cell RNA Sequencing to identify cellular heterogeneity with in Pituitary Adenomas

Pituitary adenomas (PA) are one of the most common primary brain tumors and comprise approximately 15% of brain neoplasms. Most PA are histologically benign, but can cause significant morbidity. Previous studies utilized whole genome and exome sequencing to identify a few somatic variants, but no recurrent mutations were observed. Further studies are warranted to identify driver mutations occurring at low frequencies. We used single-cell RNA sequencing (10X Genomics) to investigate cellular heterogeneity in 12 non-functioning pituitary adenomas. Our analysis identified discrete clusters of cells associated with specific functional pathways. One of these clusters corresponded to cells expressing genes related to metabolic pathways, primarily lipid metabolism. Another cluster consistent amongst the three patients comprised cells involved in antigen presentation and processing. In addition, the copy number variation analysis highlighted distinct chromosomal alterations within our samples. Interestingly, we were able to identify clonal variations within each tumor based on chromosomal aberrations. For example, in our first patient we observed a gain of chromosome 19 and loss of chromosome 2. Our analysis showed three different clonal populations within this tumor. All three populations harbored the loss of chromosome 2, one population exhibited gain of chromosome 19, while a third population exhibited loss of chromosome 19. These early results indicate the loss of chromosome 2 as an early event in tumorigenesis and gain/loss of chromosome 19 as late events. We are currently in a process of identifying somatic variations within these tumors by variant calling. Currently we are expanding our analysis to 20 non-functional PA. Mapping the single-cell gene expression profiles with mutational phylogeny will reveal the differences in clonal evolution within the tumor subtypes. This study will help us define the molecular fingerprint of pituitary adenomas and provide insights which could be utilized in the clinic for better management of these tumors.

Abstract 1902: Elevated thymidine kinase 1 (TK1) in cancer serum may suppress immune function

Abstract In this project, we wanted to investigate the role that TK1 may play in macrophage activation and immune suppression. The relationship between immune response and cancer development has been an enigma that researchers have sought to elucidate for decades. Recent and past studies have suggested that the presence and abundance of nucleotides can affect immune response. Thymidine Kinase 1 (TK1) is a salvage pathway enzyme that converts thymidine to thymidine monophosphate, a precursor to DNA synthesis. It is secreted into serum and as such has been suggested to be a tumor biomarker. TK1 has also shown to be present on the surface of many different cancer cell lines including breast, lung, and colorectal cancer. The reason for this upregulation of surface TK1 is unclear, but it has been suspected that the surface expression of TK1 may be beneficial to tumor growth and survival since high TK1 expression and secretion is an early event in tumorigenesis. Although the immunosuppressive properties of purines have been well established, pyrimidines such as thymidine have not been studied as potential immune suppressants. In our investigation, we first confirmed the increased surface expression levels of TK1 in cancerous cell lines. Flow cytometry showed that non-cancerous cells expressed surface TK1 at minimal levels (0.7%), MDA-MB-231 cells expressed TK1 at higher levels (45%), and PC3 cells expressed TK1 at elevated levels as well (64.6%). Bioinformatic analysis of nucleotide transport in relation to TK1 surface expression showed a strong positive correlation between the nucleotide transporter SLC29A2 and surface TK1 expression in colon adenocarcinoma (correlation value 0.455, p-value &amp;lt;0.001), head and neck squamous cell carcinoma (correlation value 0.416, p-value &amp;lt;0.001), rectum adenocarcinoma (correlation value 0.432, &amp;lt;0.001), stomach adenocarcinoma (correlation value 0.405, p-value &amp;lt;0.001), and uterine corpus endometrial carcinoma (correlation value 0.403, p-value &amp;lt;0.001) which further confirms the elevation of TK1 on the surface of cancer cells and suggests a mechanism whereby serum TK1 levels are elevated. Macrophage engulfment assays were then conducted in order to investigate the possibility that exposure to higher concentrations of TK1 leads to immune suppression. We found that macrophages differentiated from THP-1 monocytes that were exposed to varying concentrations of TK1 for 24 hours showed a decrease in macrophage engulfment (29%-37%) compared to controls that were not exposed to TK1(61.2%). These results suggest that TK1 may have some involvement in immune suppression during cancer development, and we intend to further investigate these findings. Citation Format: Kathryn R. Smith, Rachel M. Morris, Edwin J. Velazquez, David M. Bellini, Kim O'Neill. Elevated thymidine kinase 1 (TK1) in cancer serum may suppress immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1902.

Telomere shortening and expression of TRF1 and TRF2 in uterine leiomyoma.

Uterine leiomyoma is a benign smooth muscle tumor of the uterus that can exhibit histopathological traits that mimic malignancy. Telomere shortening is an early event in tumorigenesis and telomerase activation facilitates tumor progression later in the course of carcinogenesis. Telomeric repeat‑binding factor (TRF)1 and TRF2 protect telomeres, and their gene expression levels are dysregulated in various cancer types. However, the roles of telomeres and telomere protection proteins in uterine leiomyoma remain largely unknown. In this study, telomere length and the mRNA levels of various telomere‑related genes in normal tissues and leiomyoma were determined, and their relationships were evaluated. Uterine leiomyoma and normal myometrium were surgically obtained from 18 and 13patients, respectively. Telomere length and gene expression were determined by Southern blot analysis and reverse transcription‑quantitative PCR, respectively. In matched samples, telomeres were consistently shorter in leiomyoma tissue than in adjacent normal tissue. TRF1, TRF2, PIN2‑interacting telomerase inhibitor1(PINX1), and telomerase RNA component were expressed at comparable levels in both leiomyoma and normal tissues. None of these genes were associated with telomere length in leiomyoma. All tested tissues were negative for telomerase reverse transcriptase, which encodes the catalytic component of telomerase, indicating that cells in uterine leiomyoma were not immortalized. In summary, telomere erosion, which reflects active proliferation during tumor evolution, was evident in uterine leiomyoma. Steady‑state expression of TRF1, TRF2 and PINX1 may be important for maintenance of telomere integrity in leiomyoma, where telomere length is shortened.

EPCO-37. USING SINGLE-CELL RNA SEQUENCING TO IDENTIFY CELLULAR HETEROGENEITY WITHIN NON-FUNCTIONING PITUITARY ADENOMAS

Abstract Pituitary adenomas (PA) are one of the most common primary brain tumors and comprise approximately 15% of brain neoplasms. Most PA are histologically benign but can cause significant morbidity. Previous studies utilized whole-genome and exome sequencing to identify a few somatic variants, but no recurrent mutations were observed. Further studies are warranted to identify driver mutations occurring at low frequencies. We used single-cell RNA sequencing (10X Genomics) to investigate cellular heterogeneity in three non-functioning pituitary adenomas. Our analysis identified discrete clusters of cells associated with specific functional pathways. One of these clusters corresponded to cells expressing genes related to metabolic pathways, primarily lipid metabolism. Another cluster consistent amongst the three patients comprised of cells involved in antigen presentation and processing. In addition, the copy number variation analysis highlighted distinct chromosomal alterations within our samples. Interestingly, we were able to identify clonal variations within each tumor based on chromosomal aberrations. For example, in our first patient, we observed a gain of chromosome 19 and loss of chromosome 2. Our analysis showed three different clonal populations within this tumor. All three populations harbored the loss of chromosome 2, one population exhibited gain of chromosome 19, while a third population exhibited loss of chromosome 19. These early results indicate the loss of chromosome 2 as an early event in tumorigenesis and gain/loss of chromosome 19 as late events. We are currently in a process of identifying somatic variations within these tumors by variant calling. Currently we are expanding our analysis to 20 non-functional PA. Mapping the single-cell gene expression profiles with mutational phylogeny will reveal the differences in clonal evolution within the tumor subtypes. This study will help us define the molecular fingerprint of pituitary adenomas and provide insights which could be utilized in the clinic for better management of these tumors.

Current Targets and Bioconjugation Strategies in Photodynamic Diagnosis and Therapy of Cancer.

Photodynamic diagnosis (PDD) and therapy (PDT) are emerging, non/minimally invasive techniques for cancer diagnosis and treatment. Both techniques require a photosensitizer and light to visualize or destroy cancer cells. However, a limitation of conventional, non-targeted PDT is poor selectivity, causing side effects. The bioconjugation of a photosensitizer to a tumor-targeting molecule, such as an antibody or a ligand peptide, is a way to improve selectivity. The bioconjugation strategy can generate a tumor-targeting photosensitizer conjugate specific for cancer cells, or ideally, for multiple tumor compartments to improve selectivity and efficacy, such as cancer stem cells and tumor neovasculature within the tumor microenvironment. If successful, such targeted photosensitizer conjugates can also be used for specific visualization and detection of cancer cells and/or tumor angiogenesis (an early event in tumorigenesis) with the hope of an early diagnosis of cancer. The purpose of this review is to summarize some current promising target molecules, e.g., tissue factor (also known as CD142), and the currently used bioconjugation strategies in PDT and PDD, with a focus on newly developed protein photosensitizers. These are genetically engineered photosensitizers, with the possibility of generating a fusion protein photosensitizer by recombinant DNA technology for both PDT and PDD without the need of chemical conjugation. We believe that providing an overview of promising targets and bioconjugation strategies will aid in driving research in this field forward towards more effective, less toxic, and non- or minimally invasive treatment and diagnosis options for cancer patients.

HIST1H2BB and MAGI2 Methylation and Somatic Mutations as Precision Medicine Biomarkers for Diagnosis and Prognosis of High-grade Serous Ovarian Cancer.

Molecular alterations that contribute to long-term (LT) and short-term (ST) survival in ovarian high-grade serous carcinoma (HGSC) may be used as precision medicine biomarkers. DNA promoter methylation is an early event in tumorigenesis, which can be detected in blood and urine, making it a feasible companion biomarker to somatic mutations for early detection and targeted treatment workflows. We compared the methylation profile in 12 HGSC tissue samples to 30 fallopian tube epithelium samples, using the Infinium Human Methylation 450K Array. We also used 450K methylation arrays to compare methylation among HGSCs long-term survivors (more than 5 years) and short-term survivors (less than 3 years). We verified the array results using bisulfite sequencing and methylation-specific PCR (qMSP). in another cohort of HGSC patient samples (n = 35). Immunoblot and clonogenic assays after pharmacologic unmasking show that HIST1H2BB and MAGI2 promoter methylation downregulates mRNA expression levels in ovarian cancer cells. We then used qMSP in paired tissue, ascites, plasma/serum, vaginal swabs, and urine from a third cohort of patients with HGSC cancer (n = 85) to test the clinical potential of HIST1H2BB and MAGI2 in precision medicine workflows. We also performed next-generation exome sequencing of 50 frequently mutated in human cancer genes, using the Ion AmpliSeqCancer Hotspot Panel, to show that the somatic mutation profile found in tissue and plasma can be quantified in paired urine samples from patients with HGSC. Our results suggest that HIST1H2BB and MAGI2 have growth-suppressing roles and can be used as HGSC precision medicine biomarkers.