Abstract Background Ductal carcinoma in-situ (DCIS) is a pre-invasive form of invasive breast cancer (IBC). Due to improved breast cancer screening, it now accounts for ~ 25% of all breast cancers. While the treatment success rates are over 90%, this comes at the cost of considerable morbidity, considering that the majority of DCIS never become invasive and our understanding of the molecular changes occurring in DCIS that predispose to invasive disease is limited. The aim of this study is to characterize molecular changes that occur in DCIS, with the goal of improving DCIS risk stratification. Methods We identified and obtained a total of 197 breast tissue samples from 5 institutions (93 DCIS progressors, 93 DCIS non-progressors, and 11 adjacent normal breast tissues) that had at least 10-year follow-up. We isolated DNA and RNA from archival tissue blocks and characterized genome-wide mRNA expression, DNA methylation, DNA copy number variation, and RNA splicing variation. Results We obtained all four genomic data sets in 122 of the 197 samples. Our intrinsic expression subtype-stratified analyses identified multiple molecular differences both between DCIS subtypes and between DCIS and IBC. While there was heterogeneity in molecular signatures and outcomes within intrinsic subtypes, several gene sets that differed significantly between progressing and non-progressing DCIS were identified by Gene Set Enrichment Analysis. Conclusion DCIS is a molecularly highly heterogenous disease with variable outcomes, and the molecular events determining DCIS disease progression remain poorly defined. Our genome-wide multi-omic survey documents DCIS-associated alterations and reveals molecular heterogeneity within the intrinsic DCIS subtypes. Further studies investigating intrinsic subtype-stratified characteristics and molecular signatures are needed to determine if these may be exploitable for risk assessment and mitigation of DCIS progression. The highly significant associations of specific gene sets with IBC progression revealed by our Gene Set Enrichment Analysis may lend themselves to the development of a prognostic molecular score, to be validated on independent DCIS cohorts.

Introduction: Our current understanding of the molecular changes predisposing Ductal carcinoma in situ (DCIS) to progress to invasive breast cancer (IBC) is limited. The aim of this study is to characterize the molecular changes of DCIS to improve DCIS risk assessment. Methods: We obtained paraffin-embedded tissue of 210 DCIS samples with no concurrent or antecedent IBC from 5 cancer institutions, and extracted DNA and RNA. Transcriptome analysis: Illumina’s TruSeq RNA Exome kit was used for library construction, followed by sequencing. Methylome analysis: Bisulfite treated genomic DNA was restored and arrayed using the Illumina 450K methylation chips. DNA Copy number (CNV) analysis: CNV was estimated from the methylation data set using the EpiCopy R package. Results: Samples passing Q/C metrics: Transcriptome: 59 cases, 63 controls. Methylome & CNV: 93 cases, 98 controls. We classified samples into their intrinsic PAM50 subsets. Cases showed an increase in the Her2 subtype, and the controls were enriched in LuminalA (LumA) samples, while Basal and LuminalB (LumB) subtypes were evenly distributed. Compared to the TCGA IBC dataset, proportions of Her2 and LumB subtypes in DCIS were increased while the LumA cohort was decreased. Unsupervised clustering of the transcriptome data resulted in 3 clusters with key differences between PAM50 subtypes: one cluster predominated in Basal and Her2 subtypes, and a second was enriched in hormone-positive samples (LumA and LumB). For the methylome data, the optimal number of clusters was 6. Again, several clusters showed correlation with PAM50 subtype, e.g., one was enriched for hormone-negative subtypes (Basal and Her2), while another was enriched for hormone-positive subtypes (LumA and LumB). For the CNV data, the optimal number of clusters was 4. Here, one CNV cluster appeared predominantly in the Basal subtype, and there were multiple regions showing significant subtype-specific differences with the TCGA IBC data set. While the well-known heterogeneity of DCIS prevents the above broad molecular categories from strongly stratifying DCIS by outcome, we are continuing to analyze our data sets for predictive molecular signatures. Since the long-term outcome of all DCIS in this cohort is known, we are in the unique position to pursue additional questions such as PAM50 subtype differences in times-to-events or lineage fidelity (i.e., whether the subtype of the subsequent IBC matches the preceding DCIS), and it appears that the time to IBC diagnosis is shortest in Basal DCIS, while at the same time, lineage fidelity is lowest in Basal DCIS, as has been previously reported. Conclusions: Our subtype-stratified analyses identified multiple molecular differences both between intrinsic subtypes as well as between DCIS and IBC that suggest subtype-specific characteristics that may be exploitable for risk stratification of DCIS. Citation Format: Marija Debeljak, Soonweng Cho, Bradley Downs, Michael Considine, Brittany Avin-McKelvey, Yongchun Wang, William Grizzle, Katherine Hoadley, Charles Lynch, Brenda Hernandez, Paul van Diest, Wendy Cozen, Ann Hamilton, Debra Hawes, Edward Gabrielson, Ashley Cimino-Mathews, Liliana Florea, Leslie Cope, Christopher B. Umbricht. Multimodal genome-wide survey of progressing and non-progressing DCIS [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 3126.

Previous studies, on mostly Non-Hispanic White (NHW), have identified genetic and epigenetic markers for pediatric acute lymphoblastic leukemia (ALL) relapse. Higher incidence rates and worse outcomes in Hispanic/Latinx (HSP) patients suggest that the genesis and progression of disease may vary by population. We compared methylation signatures and mutation spectrum between HSP and NHW ALL patients at diagnosis as well as by relapse status, defined as relapse within three years of diagnosis (early relapse) versus remission (>=4 years after diagnosis). Study participants included pediatric (1-19 years) patients in the Therapeutically Applicable Research to Generate Effective Treatments program. We included 26 HSP and 51 NHW patients with bone marrow samples at diagnosis including 11 HSP and 32 NHW patients with both diagnosis and relapse samples. The mean age at diagnosis for HSP was 8.8 years (standard deviation, sd=5.6) and NHW was 6.4, sd=4.9. The proportion of males (56%) was higher than females (44%). At diagnosis, the top methylated regions for the combined sample harbored the following genes: AB13, GNGT2, MAPK10, ARHGAP24, TEX264 and IQCF6 (all p<0.000001, all q<0.1) (all p<0.000005, all q<0.1). However, the top methylated regions for HSP did not overlap with those for NHW. For HSP, the top methylated regions harbored the following genes: ITPA, COX16, ADAM21, GALR1, MBP, DCLRE1A, PPP4R12 and IGSF11 (all p<0.000005, all q<0.1); while for NHW, the top methylated regions harbored the following genes: ARMC10, FBXL13, TEX264 and IQCF6 (all p<0.000002, all q<0.1). Similar results were observed for differentially methylated regions between diagnosis and relapse samples. The top regions for the overall sample harbored the following genes: RINT1, RPS16, PDE12, TDRKH, PLEKHG2, TPO, PXDN, DTNBP1, YTHDC1. For HSP, the top regions differentially methylated between diagnosis and relapse harbored SPOCK3, B3GALT6, KIAA1804, FAM201A, HEMK1 and CLRN1OS; while for NHW, the top regions differentially methylated between diagnosis and relapse harbored SRPK2, RINT1, TMPRSS11E, YTHDC1, GSTT1, SFTPB, GNLY, JARID2, DTNBP1, ZFP36 and RPS16. A comparison of mutation spectrum of frequently mutated genes in pediatric ALL revealed higher mutations in NRAS (12.5% vs 0%) and FLT3 (6.25% vs 0%), but not KRAS (12.5% vs 14.3%), for NHW (n=16) than HSP (n=14). Few of the candidate genes observed in HSP (ITPA, MBP, PPP4R12, and SPOCK3) have been previously implicated in leukemia. The findings suggest that the methylation signatures and mutation spectrum for HSP and NHW pediatric acute ALL patients might differ and further studies among HSP, a group with the highest prevalence of acute pediatric ALL, could potentially identify new genetic and epigenetic markers for acute pediatric ALL. Citation Format: Alexander Miller, Michael Considine, Leslie Cope, Ernest K. Amankwah. DNA methylation and mutation spectrum among pediatric acute lymphoblastic leukemia patients by Hispanic/Latinx ethnicity [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 1969.

Hepatocellular carcinoma (HCC) is a major global cause of mortality. The epithelial-mesenchymal transition (EMT) transcription factor TWIST1 has been implicated in the invasion and metastasis of HCC, but the mechanism is unclear. Integrated transcriptomic and proteomic analyses on primary liver tumors and lung metastases from a spontaneous Twist1-dependent metastasis mouse model of MYC-induced HCC identified the hexosamine biosynthetic pathway (HBP) as a cancer cell autonomous mechanism for EMT-mediated invasion and metastasis, which was further validated in vitro and ex vivo. RNA-sequencing and mass spectrometry-based proteomics were carried out on primary liver tumors, lung metastases, normal livers and normal lungs from a Twist1-dependent metastasis mouse model of MYC-induced HCC (LMT); a metastatic MYC-induced HCC mouse model dependent on Twist1 phosphorylation mimetic mutant (LMT-DQD); a non-metastatic mouse model of MYC-induced HCC (LM); a non-metastatic MYC-induced HCC mouse model with the Twist box mutant (LMT-F191G); and wildtype mice. Data was analyzed in R. Organoids were derived from LM liver tumors, embedded into collagen and imaged using time-lapse DIC microscopy. Cell lines were derived from LM and LMT liver tumors, and along with human HCC cell lines Huh7, Hep3B and HepG2, were utilized for in vitro migration and invasion assays. 91 genes were differentially expressed between lung metastases versus primary liver tumors; primary liver tumors versus normal liver; and normal lung versus normal liver with p<0.05. Gene Ontology and KEGG Pathway Enrichment analyses revealed an enrichment of metabolic processes. Gfpt2, the gene encoding for the rate-limiting enzyme of the hexosamine biosynthetic pathway, was overexpressed >2 fold in metastases compared to primary tumor (p = 0.007). Kaplan-Meier estimates from cBioPortal show that HCC patients with genetic alterations in GFPT2 and/or its isoform GFPT1 have significantly worse overall median survival compared to patients without these alterations (27.5 months vs 83.24 months, p = 0.006). Functional validation studies demonstrated that increased O-GlcNAcylation by pharmacologic treatment with TMG or genetic manipulation by GFPT2 overexpression was sufficient to increase migration and invasion in vitro in a panel of human and murine HCC cell lines. Decreased HBP-O-GlcNAcylation by pharmacologic treatment with DON or genetic manipulation by GFPT2 shRNA knockdown inhibited migration and invasion in vitro. Similarly, using a novel organoid invasion assay, TMG treatment increased invasion of organoids derived from LM tumors ex vivo 2-fold (p<0.03) while DON treatment inhibited invasion 2-fold (p<0.03). The hexosamine biosynthetic pathway is required and sufficient for invasion and metastasis in vitro in HCC cell lines and ex vivo in HCC tumor-derived organoids. The HBP may be a potential therapeutic target for advanced HCC patients.

A 30-UTR KRAS-variant is associated with cisplatin resistance in patients with recurrent and/or metastatic head and neck squamous cell carcinoma C. H. Chung1,2*, J. W. Lee3, R. J. Slebos4, J. D. Howard1, J. Perez1, H. Kang1, E. J. Fertig1, M. Considine1, J. Gilbert5, B. A. Murphy5, S. Nallur6, T. Paranjape6, R. C. Jordan9, J. Garcia10, B. Burtness7, A. A. Forastiere1 & J. B. Weidhaas6,8 Departments of Oncology; Otolaryngology-Head and Neck Surgery, Sidney Kimmel Cancer Center, Johns Hopkins University, Baltimore; Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston; Department of Cancer Biology; Division of Hematology/Oncology, Department of Medicine, Vanderbilt University, Nashville; Department of Therapeutic Radiology; Section of Medical Oncology, Department of Internal Medicine, Yale University School of Medicine, New Haven; Department of Radiation Oncology, University of California, Los Angeles; Department of Pathology, University of California, San Francisco; Mayo Clinic, Rochester, USA

A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) which disrupts microRNA regulation has previously been associated with altered patient outcome and drug sensitivity in various cancers. Our study suggests this KRAS-variant is a potential predictive biomarker for poor platinum response in recurrent/metastatic head and neck squamous cell carcinoma patients.A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings.KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04).The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients.NCT00503997, NCT00425750, NCT00003809.

MicroRNAs (miRNAs) play a critical role in cell cycle and pro-survival signal regulation. Consequently, their deregulation can enhance tumorigenesis and cancer progression. In the current investigation, we determined whether cancer- or human papillomavirus (HPV)-specific miRNA deregulation could further elucidate signal transduction events unique to head and neck squamous cell carcinoma (HNSCC). Twenty-nine newly diagnosed HNSCC tumors (HPV-positive: 14, HPV-negative: 15) and four normal mucosa samples were analyzed for global miRNA expression. Differential miRNA expression analysis concluded HNSCC is characterized by a general upregulation of miRNAs compared to normal mucosa. Additionally, miR-449a and miR-129-3p were statistically significant miRNAs differentially expressed between HPVpositive and HPV-negative HNSCC. The upregulation of miR-449a was also validated within an independent dataset obtained from TCGA containing 279 HNSCCs and 39 normal adjacent mucosa samples. To gain a better understanding of miRNA-mediated cell cycle deregulation in HNSCC, we functionally evaluated miR-205, a transcript upregulated in our cancer-specific analysis and a putative regulator of E2F1. Modulation of miR-205 with a miRNA mimic and inhibitor revealed miR-205 is capable of regulating E2F1 expression in HNSCC and overexpression of this transcript enhances proliferation. This study demonstrates miRNA expression is highly deregulated in HNSCC and functional evaluations of these miRNAs may reveal novel HPV context dependent mechanisms in this disease.