Aggressive Tumor Biology Research Articles

Abstract 2117: Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers

Abstract Genomic instability is a hallmark of cancer. Unlike normal diploid cells, cancer cells exhibit abnormal centrosome numbers and chromosomal instability. Mitosis with supernumerary centrosomes yields progeny with aberrant chromosome segregation and aneuploidy. While most progeny cells with abnormal chromosomes are eliminated through apoptosis, some have distinct fates with a growth advantage. These cancer cells can acquire resistance to antineoplastic agents. This confers an aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) regulates cell cycle progression and the centrosome cycle. We previously reported that CDK2 inhibition prevents supernumerary centrosome clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. This was associated with residual transplanted tumors in mice. To elucidate cell fates of progeny with multipolar mitosis after CDK2/9 inhibition with CYC065 (0.2 µM) treatment, a panel of murine and human lung cancer cells underwent time-lapse fluorescent microscopy. This elucidated multipolar mitotic events within the progeny. Intriguingly, distinct outcomes occurred after mitosis including formation of multipolar and multinucleated cells. Cell death of progeny is the predominant fate, but some progeny survive despite continuous CDK2 inhibition. Surprisingly, some of these cells fuse together. Those multinucleated cells can undergo multiple cell cycles without successful cytokinesis. To confirm these effects were through CDK2 inhibition, the selective CDK2 inhibitor Tagtociclib (PF-07104091, at the 2 µM dosage) was used. This treatment statistically-significantly increased multipolar lung cancer cells and increased apoptotic death during time-lapse fluorescent microscopy. Findings were independently validated by CDK2 shRNA knockdown. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent staining revealed the ultrastructure of multinucleated cells. Insights into tumor biology came from intravital imaging of transplanted lung cancer cells in mice. Multipolar and multinuclear cells followed CDK2 antagonism. Incucyte® Live-Cell Analysis System and Artificial Intelligence-based imaging determined distinct growth responses in aneuploid as compared to non-aneuploid lung cancer cells after CDK2 inhibitor treatments. In summary, these findings are translationally relevant. CDK2 inhibition of aneuploid lung cancers yields distinct cell fates. This is linked to resistance to CDK2 antagonism and to formation of residual in vivo tumors. Citation Format: Liliya Tyutyunyk, Zibo Chen, Xiuxia Liu, Yeap Ng, Aayush Bhatawadekar, Kedar Narayan, Roberto Weigert, Xi Liu, Ethan Dmitrovsky. Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2117.

Abstract 5156: Is the formation of Stroma AReactive Invasion Front Areas in colorectal cancer driven by characteristic extracellular matrix reorganization processes based on the plasmin/plasminogen system

Abstract Background: As patient stratification still needs to be refined in colorectal cancer (CRC), we established SARIFA (Stroma AReactive Invasion Front Areas) as a novel H&E-based prognostic biomarker that is defined by a direct tumor-adipocyte interaction with a lack of desmoplastic reaction. Even though we could prove the high prognostic relevance of SARIFA now in multiple cohorts in gastric, colorectal, and pancreatic cancer, as well as could link an upregulation of lipid metabolism and immune alterations to this specific aggressive tumor biology, it remains an open question how SARIFAs are formed mechanistically. Materials/Methods: Based on the results of our recently established SARIFA-scores in the TCGA-CRC (TCGA-COAD & TCGA-READ cohorts; SARIFA-positive n=69, SARIFA-negative n=138) and the corresponding differential gene expression analysis, which are part of a previous study, protein levels (in ng/mL) measured by commercially available uPA and PAI1 enzyme-linked immunosorbent assays (ELISAs) were compared between SARIFA-positive and SARIFA-negative CRC patients (SARIFA-positive n=35, SARIFA-negative n=62). Additionally, immunohistochemistry for FABP4 as a key player in lipid metabolism was performed. Moreover, spatial transcriptomics (digital spatial profiling, DSP) was performed with tumor and stroma section segmentation on a subset of cases. Results: In TCGA-CRC, SERPINE1 (PAI1), as well as PLAU (uPA), are both significantly upregulated on mRNA level (LFC: 1.14 and 0.74, respectively; both adjusted p-values ≤0.001). Logically, SERPINE1 and PLAU are highly expressed in mesenchymal CMS4 CRCs, which overlap with SARIFA-positivity. This is reflected by an upregulation of matrix metalloproteinases in predicted functional protein networks (STRING analysis). In our local cohort, we could now validate this upregulation of uPA in SARIFA-positive CRCs on protein level (p=0.0027). For PAI1, a trend towards higher protein levels in SARIFA-positive CRCs could be observed (p=0.209). Moreover, SARIFA-positive CRCs show a higher number of tumor buds (p=0.013) as well as increased FABP4 expression at the invasion front (p<0.001) in our local cohort. In DSP analysis, the stroma fraction of SARIFA-positive CRCs showed significant changes compared to SARIFA-negative cases. Conclusion: Our current data shows that the formation of SARIFAs and the consecutive direct tumor-adipocyte interaction with a tumor-promoting increase in lipid metabolism may rely on alterations in the plasmin/plasminogen system, which could be a novel potential drug target in the aggressive subset of SARIFA-positive CRCs. Citation Format: Bianca Grosser, Nic G. Reitsam, Florian Sommer, Jochen Hardt, Bruno Märkl. Is the formation of Stroma AReactive Invasion Front Areas in colorectal cancer driven by characteristic extracellular matrix reorganization processes based on the plasmin/plasminogen system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5156.

Abstract 6108: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. Within cancerous cells, striking intra- and inter-tumoral heterogeneity was observed by genetic ancestry even within molecular subtype groups. Current efforts focus on in-depth molecular characterization of ancestry- and risk factor-related differences in the tumor epithelium and microenvironment and distinct signatures present in lethal disease. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6108.

Critical appraisal of surgical margins according to KRAS status in liver resection for colorectal liver metastases: Should surgical strategy be influenced by tumor biology?

BackgroundKRAS mutation is a negative prognostic factor for colorectal liver metastases. Several studies have investigated the resection margins according to KRAS status, with conflicting results. The aim of the study was to assess the oncologic outcomes of R0 and R1 resections for colorectal liver metastases according to KRAS status. MethodsAll patients who underwent resection for colorectal liver metastases between 2010 and 2015 with available KRAS status were enrolled in this multicentric international cohort study. Logistic regression models were used to investigate the outcomes of R0 and R1 colorectal liver metastases resections according to KRAS status: wild type versus mutated. The primary outcomes were overall survival and disease-free survival. ResultsThe analysis included 593 patients. KRAS mutation was associated with shorter overall survival (40 vs 60 months; P = .0012) and disease-free survival (15 vs 21 months; P = .003). In KRAS-mutated tumors, the resection margin did not influence oncologic outcomes. In multivariable analysis, the only predictor of disease-free survival and overall survival was primary tumor location (P = .03 and P = .03, respectively). In KRAS wild-type tumors, R0 resection was associated with prolonged overall survival (74 vs 45 months, P < .001) and disease-free survival (30 vs 17 months, P < .001). The multivariable model confirmed that R0 resection margin was associated with prolonged overall survival (hazard ratio = 1.43, 95% confidence interval: 1.01–2.03) and disease-free survival (hazard ratio = 1.42; 95% confidence interval: 1.06–1.91). ConclusionsKRAS-mutated colorectal liver metastases showed more aggressive tumor biology with inferior overall survival and disease-free survival after liver resection. Although R0 resection was not associated with improved oncologic outcomes in the KRAS-mutated tumors group, it seems to be of paramount importance for achieving prolonged long-term survival in KRAS wild-type tumors.

Molecular Classification and Pathogenesis of Pancreatic Adenocarcinoma and Targeted Therapies: A Review.

Pancreatic adenocarcinoma (PDAC) is disease with a 5-year survival of only 12%. Many patients with PDAC present with late-stage disease and even early-stage disease can often be characterized by an aggressive tumor biology. Standard therapy for metastatic PDAC consists mainly of chemotherapy regimens like FOLFIRINOX, FOLFOX, or gemcitabine and nab-paclitaxel. Research has focused on sequencing PDAC tumors to understand better the mutational landscape and transcriptomics of PDAC with the goal to develop targeted therapies. Targeted therapies may potentially minimize the toxic risks of chemotherapy and provide a long-term survival benefit. We herein review the underlying molecular pathogenesis of PDAC, as well as the classification schema created from current sequencing data, and recent updates related to targeted therapy for PDAC.

Investigating the Impact of Tumor Biology and Social Determinants on Time to Diagnosis and Stage at Presentation of Wilms Tumor.

Delays in diagnosis and time to diagnosis generally are used interchangeably in cancer disparity research, but these terms may have important differences. Although these terms are related, we hypothesize that time to diagnosis is determined by the aggressiveness of the tumor based on intrinsic factors such as tumor biology, whereas delays in diagnosis are caused by extrinsic factors such as socioeconomic status, leading to presentation at higher stage of disease due to barriers of care. We conducted a retrospective study of 306 patients diagnosed with Wilms tumor at Children's Hospital Colorado between 1971 and 2016 identifying patient barriers as extrinsic markers and using unfavorable histology and loss of heterozygosity as markers of aggressive tumor biology. Multivariable logistic regression was performed. Patients with Medicaid were more likely to present greater than 4 days after initial symptoms compared to those with private insurance, and those with housing concerns were more likely to be diagnosed greater than 9 days from initial symptoms. Tumor biology was noted to be associated with higher stage at diagnosis, but patient barriers were not. These findings suggest the interplay between tumor biology, patient barriers, diagnostic timing, and stage at diagnosis is more complex, multifactorial, and in need of further study.

Diminution of Primary Cilia in the Stromal Cells at the Tumor-stromal Interface Correlates With an Aggressive Tumor Biology in the Urothelial Carcinoma of the Urinary Bladder.

Primary cilia (PC) are cellular organelles that regulate the cellular homeostasis. They are the seats of many oncogenic pathways and indirectly regulate the epithelial-mesenchymal transition (EMT) and extracellular matrix, both critical for the tumor microenvironment (TME). Though there are a few studies highlighting the alteration of PC in the tumor cells of various malignancies, none depict the PC in the stromal cells in the urothelial carcinoma of the urinary bladder (UC), the stromal cells being an essential component of TME. Therefore, we intend to evaluate the PC in the stromal cells at the tumor-stromal interface in UC. Immunohistochemistry for acetylated-α-tubulin (for PC), Ki67, E-cadherin, and SNAI1 was performed in 141 cases of UC and 5 normal controls, and primary cilium: nucleus (C:N) ratio was counted in the stromal cells at the tumor-stromal interface. The C:N ratio was correlated with various clinical and histopathological parameters. The C:N ratio showed significant diminution from normal control (mean=0.75) to low-grade UC (mean=0.24) ( P =0.001) to high-grade UC (mean value=0.17) ( P =0.001). There was a significant diminution of the C:N ratio from the noninvasive to invasive UC ( P =0.025). The C:N ratio did not show any correlation with EMT although negatively correlated with the Ki67 index ( r =-0.32; P =0.001), and a higher ratio showed a trend with a higher recurrence-free survival ( P =0.07). The diminution of the PC in the stromal cells at the tumor-stromal interface is an early event and correlates with an aggressive tumor biology of UC.

Emerging need of molecular profiling in hepatobiliary cancer

: Gallbladder cancer is a rare malignancy but represents almost 50% of all biliary tract cancer. Biliary cancers are highly fatal malignancies with a 5-year survival rate of approximately 20%. The prognosis of gallbladder cancer is poor due to the aggressive tumor biology, late presentation, complicated anatomic position, and advanced stage at diagnosis. Locally advanced and metastatic disease treatment is with palliative chemotherapy. Alarming sign of gallbladder cancer is overall decreased in incidence in older patients but increased in the younger population. So many mutations have been reported for the gall bladder cancer till date. : A prospective observational study was conducted over a period of 1 year at Asian Institute of Medical Sciences Faridabad which includes hepatobiliary carcinoma patients who are at stage III and stage IV of cancer. After getting the consent formalin fixed paraffin embedded biopsy samples, and 5 ml serum sample was collected in serum separator tube (SST). A whole genome sequencing was performed using Illumina HiSEQ, Illumina (NGS) technology, allows for high-throughput sequencing of DNA and RNA. Illumina's NGS is based on "sequencing by Synthesis" to detect the mutations.: Most common mutation found was in the P53 gene. TP53 (p.Arg175His), TP53 (p.Arg306Ter), TP53 (p.Cys238Tyr), TP53 (p.Leu43Ter), TP53 (p.Glu339Ter), TP53 (p.Pro190Leu). Mutations in the TP53 gene are a common feature of carcinoma of the gallbladder, and are associated with a more aggressive tumor phenotype, resistance to chemotherapy, and poorer overall survival.

Latissimus Dorsi Flap Revisited: Coverage of Large Chest Wall Defects Following Mastectomy for Locally Advanced Breast Carcinoma and Angiosarcoma.

The latissimus dorsi myocutaneous flap (LDMF) remains a significant tool in the armamentarium of the oncoplastic breast surgeon. Despite declining popularity for reconstruction, owing to the increasing use of implants and free flaps, it still has an important role in certain salvage situations and as a flap to cover large defects. We report our experience with the use of LDMF for immediate coverage of large mastectomy defects when options are limited. Retrospective series of prospectively collected patient records. Patient and tumour characteristics, length of stay, and post-operative and oncologic outcomes are reported. Patients with angiosarcoma were discussed at tertiary sarcoma centres as per national guidelines. Operations were carried out by oncoplastic breast surgeons. The case series was approved by the institutional information governance department in line with institutional requirements for patient data sharing. All patients provided written consent for photography. Descriptive statistics were used to report findings.Median (IQR) was used for continuous variables. Six women were included, with a median age of 62.5 years, from December 2019 to October 2022. Follow-up ranged from 15 to 49 months. Median tumour size was 72.5 (16.25) mm. Four patients had locally advanced breast carcinoma (LABC), and two had breast angiosarcoma. The donor site and chest wall defects were closed primarily in all cases. Median length of stay was three nights. All mastectomy wounds healed without issues and any delay to their adjuvant treatment. One patient had a minor latissimus dorsi (LD) donor site wound breakdown managed conservatively. Three patients had adjuvant radiotherapy after surgery. Four patients, one after high-grade angiosarcoma and three after aggressive breast carcinoma, had a locoregional recurrence or distant metastases and succumbed within 20 months of surgery. The LDMF can be a reliable option for the primary closure of large post-mastectomy wounds. Its use can lead to timely wound healing, allowing patients to undergo adjuvant treatment without delay. However, the overall oncologic outcomes in patients with LABC and angiosarcoma are poor due to the underlying aggressive tumour biology. Long-term outcomes are to be interpreted with caution due to the small number of patients with diverse pathologic features.

Epithelioid angiosarcoma of the pericardium: Case report of the challenges in the management of a rare malignancy with poor prognosis.

A primary epithelioid angiosarcoma of the pericardium is a rare soft tissue malignancy. This report describes a young adult woman who presented with progressive dyspnea and pericardial effusion. She was found to have pericardial mass on imaging along with extensive myocardial infiltration. The biopsy of the mass revealed epithelioid angiosarcoma, which was CD34 and CD31-immuno-positive. Due to unresectable disease, she was given a trial of immunotherapy followed by conventional chemotherapy. She showed partial response on interim assessment, but progressed soon after the completion of six cycles and succumbed to her rapidly progressive illness within nine months of diagnosis. This report discusses the diagnostic and therapeutic challenges faced while managing this disease of aggressive tumor biology.

Perioperative and palliative systemic treatments for biliary tract cancer.

Due to the fact biliary tract cancer (BTC) is often diagnosed at an advanced stage, thus, not eligible for resection, and due to the aggressive tumor biology, it is considered as one of the cancer types with the worst prognosis. Advances in diagnosis, surgical techniques, and molecular characterization have led to an improvement of the prognosis of BTC patients, recently. Although neoadjuvant therapy is expected to improve surgical outcomes by reducing tumor size, its routine is not well established. The application of neoadjuvant therapy in locally advanced disease may be indicated, the routine use of systemic therapy prior to surgery for cholangiocarcinoma patients with an upfront resectable disease is less well established, but discussed and performed in selected cases. In advanced disease, only combination chemotherapy regimens have been demonstrated to achieve disease control in untreated patients. Molecular profiling of the tumor has demonstrated that many BTC might bear actionable targets, which might be addressed by biological treatments, thus improving the prognosis of the patients. Furthermore, the addition of the immunotherapy to standard chemotherapy might improve the prognosis in a subset of patients. This review seeks to give a comprehensive overview about the role of neoadjuvant as well as palliative systemic treatment approaches and an outlook about novel systemic treatment concept in BTC.

Long-Term Survival of Patients with Stage T1N0M1 Renal Cell Carcinoma.

Metastatic renal cell carcinoma (RCC) is among the most lethal urological malignancies. However, small, localized RCCs (≤7 cm, stage T1) have an excellent prognosis. There is a rare patient subgroup diagnosed with synchronous distant metastasis (T1N0M1), of which very little is known in terms of survival outcomes and underlying disease biology. Herein, we examined the long-term survival of 27 patients with clear cell RCC (ccRCC) stage T1N0M1 in comparison to 18 patients without metastases (T1N0M0). Tumor tissue was stained by immunohistochemistry for CD8+ tumor infiltrating lymphocytes (TILs). As expected, patients with stage T1N0M1 showed a significantly worse median cancer specific survival (CSS; 2.8 years) than patients with stage T1N0M0 (17.7 years; HR 0.077; 95% CI, 0.022-0.262). However, eight patients (29.6%) with ccRCC stage T1N0M1 survived over five years, and three of those patients (11.1%) survived over a decade. Some of these patients benefitted from an intensified, multimodal treatment including metastasis-directed therapy. The number of CD8+ TILs was substantially higher in stage T1N0M1 ccRCCs than in stage T1N0M0 ccRCCs, suggesting a more aggressive tumor biology. In conclusion, long-term survival is possible in patients with ccRCC stage T1N0M1, with some patients benefitting from an intensified, multimodal treatment approach.

Abstract LB_C03: Protein translation inhibition enforces histone deacetylase inhibitor activity resulting in synergistic pancreatic cancer cell death

Abstract Management of pancreatic cancer presents significant challenges due to late-stage diagnosis, aggressive tumor biology, and limited efficacy of existing treatment options. Gemcitabine and FOLFIRINOX (folinic acid, 5-FU, irinotecan hydrochloride, oxaliplatin) are standard treatments for advanced pancreatic cancer. Despite these therapies the overall 5-year survival rate for pancreatic cancer patients is at 12%, largely due to drug resistance. Thus, there is a critical need to explore new therapeutic strategies. Our study explores a small molecule combination that induces epigenetic reprogramming as a potential novel therapeutic strategy for pancreatic cancer. We have identified marked sensitivity of pancreatic cancer cells to the combination of a novel inhibitor of RNA helicase eIF4A, DMPatA (MZ735), and a histone deacetylase (HDAC) inhibitor, romidepsin. A short-term, low dose romidepsin treatment, when combined with MZ735, triggers persistent global histone acetylation, transcriptional upregulation, replication inhibition, and augmented DNA damage. This combined effect induces a synergistic anti-proliferative response in pancreatic cancer cells. We have identified a tolerable protocol for this combination regimen in animals, demonstrating its efficacy against tumor growth in a MIA PaCa-2 xenograft mouse model of pancreatic cancer. Treatment with the combination resulted in inhibition and then complete suppression of tumor growth in animals for 39 days. Our in vitro studies aimed at understanding the mechanism of this combination treatment showed that at low doses of romidepsin monotherapy, histone acetylation rapidly faded, while robust acetylation persisted to 18 and 24 hours with the combination. Additionally, R-loop accumulation was observed using confocal microscopy and DRIP-qPCR, along with a reduction in replication fork speed, which might account for the identified DNA damage and genome instability. The combination treatment significantly reduces acetyl-CoA and c-MYC protein levels and disrupts glycolysis and oxidative phosphorylation. We show that a structurally different HDAC inhibitor exhibits the same synergistic effects when co-administered with MZ735, confirming a unique method of eliciting synthetic lethality in PDAC cells. In conclusion, this combination shows significant antitumor activity in both in vitro and in vivo models of pancreatic cancer and warrant translation to the clinic. Citation Format: Maryam Safari, Luigi Scotto, Agnes Basseville, Thomas Litman, Haoran Xue, Luba Petrukhin, Ping Zhou, Christopher Damoci, Minhzhao Zhu, Kenneth Hull, Kenneth P Olive, Antonio T Fojo, Daniel Romo, Susan E Bates. Protein translation inhibition enforces histone deacetylase inhibitor activity resulting in synergistic pancreatic cancer cell death [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C03.

Abstract C044: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=32), Kenyan (n=18), and European American (EA, n=20) women in relation to ancestry and risk factor exposure. In an initial pilot in 10 Kenyan women, we successfully isolated intact, high-quality single nuclei from frozen breast tumor tissue through an optimized combination of enzymatic digestion and an automated tissue homogenizer. We performed multiome sequencing on a total of 36525 nuclei across these 10 pilot tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Seurat-v4 for snRNA; Harmony for snATAC), we characterized tumor, stromal, and immune cell populations (totaling 10 distinct cell types). We observed striking intra- and inter-tumoral heterogeneity across Kenyan women, with increased abundance of immune subpopulations (myeloid, T-cells, B-cells) in triple-negative subtypes. After demonstrating technical feasibility and success in this sample subset, we are now performing integration, cell type annotation, and downstream analyses in our larger cohort to characterize differences in both tumor biology and the tumor microenvironment in European American, African American, and Kenyan women at the single cell level. To date, 70 tumors spanning breast cancer subtypes have been sequenced. We are currently performing downstream analyses in this cohort to characterize ancestry- and risk factor-related differences in the tumor epithelium and microenvironment at the single-cell level. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C044.

Novel biomarker SARIFA in colorectal cancer: highly prognostic, not genetically driven and histologic indicator of a distinct tumor biology

SARIFA (Stroma AReactive Invasion Front Areas) has recently emerged as a promising histopathological biomarker for colon and gastric cancer. To elucidate the underlying tumor biology, we assessed SARIFA-status in tissue specimens from The-Cancer-Genome-Atlas (TCGA) cohorts COAD (colonic adenocarcinoma) and READ (rectal adenocarcinoma). For the final analysis, 207 CRC patients could be included, consisting of 69 SARIFA-positive and 138 SARIFA-negative cases. In this external validation cohort, H&E-based SARIFA-positivity was strongly correlated with unfavorable overall, disease-specific, and progression-free survival, partly outperforming conventional prognostic factors. SARIFA-positivity was not associated with known high-risk genetic profiles, such as BRAF V600E mutations or microsatellite-stable status. Transcriptionally, SARIFA-positive CRCs exhibited an overlap with CRC consensus molecular subtypes CMS1 and CMS4, along with distinct differential gene expression patterns, linked to lipid metabolism and increased stromal cell infiltration scores (SIIS). Gene-expression-based drug sensitivity prediction revealed a differential treatment response in SARIFA-positive CRCs. In conclusion, SARIFA represents the H&E-based counterpart of an aggressive tumor biology, demonstrating a partial overlap with CMS1/4 and also adding a further biological layer related to lipid metabolism. Our findings underscore SARIFA-status as an ideal biomarker for refined patient stratification and novel drug developments, particularly given its cost-effective assessment based on routinely available H&E slides.

Inhibition of autophagy initiation: A novel strategy for oral squamous cell carcinomas

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancer and is known to have poor prognostic outcomes. Autophagy is known to be associated with aggressive tumor biology of OSCC. Hence, this study aimed to develop a novel therapeutic strategy against OSCC by targeting the autophagic pathway. MethodsImmunoblotting, and confocal microscopy were used to examine the effect of tumor microenvironmental stressors on the autophagy activity. Cellular proliferation and migration assays were performed to assess the anti-cancer activity of standard chemotherapy and autophagy initiation inhibitors, either alone or in combination. High resolution mass-spectrometry based proteomic analysis was utilized to understand the mechanisms behind chemoresistance in OSCC models. Finally, immunohistochemistry was performed to determine associations between autophagy markers and clinicopathological characteristics. ResultsTumor microenvironmental stressors were shown to induce autophagy activity in OSCC cell lines. Novel combinations of chemotherapy and autophagy inhibitors as well as different classes of autophagy inhibitors were identified. Combination of MRT68921 and SAR405 demonstrated marked synergy in their anti-proliferative activity and also showed synergy with chemotherapy in chemoresistant OSCC cell models. Autophagy was identified as one of the key pathways involved in mediating chemoresistance in OSCC. Furthermore, TGM2 was identified as a key upstream regulator of chemoresistance in OSCC models. Finally, positive staining for autophagosome marker LC3 was shown to be associated with low histological grade OSCC. ConclusionIn conclusion, this study identified a combination of novel autophagy inhibitors which can potently inhibit proliferation of both chemosensitive as well as chemoresistant OSCC cells and could be developed as a novel therapy against advanced OSCC tumors.

DNA Liquid Biopsies for Cancer-Associated Venous Thromboembolism Prediction

Introduction Cancer-associated venous thromboembolism (VTE) is a major source of oncologic cost, morbidity and mortality. Identifying an optimal population for prophylactic anticoagulation is challenging and adds to clinician overhead. Circulating tumor (ct)DNA sequencing assays (“liquid biopsies”) are increasingly deployed in clinic, with multiple US FDA approvals for matching to molecularly targeted therapy. Preliminary data suggest that cell-free (cf)DNA, which is also measured as part of liquid biopsies and may consist of tumor or wild-type DNA, is thrombogenic due to its association with neutrophil extracellular traps. However, clinical validation of cfDNA for VTE prediction is lacking. ctDNA detection is associated with aggressive tumor biology and worse survival; however, whether it is associated with VTE is unknown. To address these gaps, we studied the clinical utility of ctDNA and cfDNA from liquid biopsies for predicting VTE. Methods We conducted an observational study in two cohorts: 1) Patients with any cancer type at Memorial Sloan Kettering Cancer Center (MSK, New York USA), an academic medical center, with plasma ctDNA sequencing using MSK-ACCESS, a NY State-approved, 129-gene assay (N=4,141). 2) A nonoverlapping cohort of patients at MSK or GenesisCare (Sydney, Australia), a community oncology setting, with stage IV or recurrent non-small cell lung cancer and ctDNA sequencing using a separate commercial assay (Agilent ctDx Lung, N=463). We studied the independent contribution of ctDNA, cfDNA and related variables for predicting VTE using Cox proportional hazards. We studied the potential utility of DNA liquid biopsies for predicting VTE within six months using machine learning models, comparing their dynamic area under the receiver operating curve, precision, and recall to those of the Khorana score, the most widely implemented risk metric. We tested the impact of ongoing anticoagulation for non-VTE indications on future VTE in patients with and without detectable ctDNA using nonrandomized, real-world evidence. Results ctDNA detection was associated with an increased risk of VTE in a dose-dependent manner (Figure 1). This association was observed across assays and treatment sites and regardless of genomic subtype. ctDNA was associated with VTE risk in non-small cell lung, breast, pancreatic, prostate, and less represented cancers as well as melanoma but not in bladder, hepatobiliary, or colorectal cancer. By contrast, cfDNA concentration was associated with VTE risk in all cancer types. In multivariate analysis, ctDNA detection, cfDNA concentration, Khorana score, chemotherapy treatment within 30 days, and number of organ sites of metastasis were all independently associated with VTE. In a cohort of patients with treatment naive lung adenocarcinoma with segmented positron emission tomography scans, ctDNA was an independent predictor of VTE after controlling for metabolic tumor volume. A random survival forest machine learning model to predict VTE within six months of plasma draw using liquid biopsy data (i.e. cfDNA and ctDNA levels as well as genomic content), cancer type, and chemotherapy receipt (“LB+”) outperformed the Khorana score in 5-fold cross-validation and was similarly superior in the external lung cohort (Table 1). The superiority of the model was observed in patients treated or not treated with chemotherapy. Additional clinical and radiologic data, including demographics, metastatic sites of disease, and Khorana score components (“All”), did not improve the ctDNA assay-based LB+ model. Patients with anticoagulation had lower VTE rates if ctDNA was detectable (adjusted HR 0.54, 95%CI 0.33-0.87) but not if ctDNA was undetectable. Conclusion In patients with cancer, ctDNA and cfDNA are independent biomarkers for VTE. Risk models incorporating liquid biopsy data are feasible in academic or community settings and, if prospectively validated, would provide a means of VTE risk stratification at scale.

External Validation of PRISM Score in Multiple Myeloma Patients in a Community Setting: A Retrospective Cohort Study

Background: Newly diagnosed Multiple Myeloma (MM) patients have a 10 to 20 times higher risk of developing venous thromboembolism (VTE) than the general population. The pathogenesis may involve several factors such as activation of procoagulant factors, acquired activated protein C resistance, and inflammation. In addition to the general risk factors for venous thromboembolism such as older age, immobility, and surgery, some MM treatment-related factors such as the use of immunomodulatory drug therapy and abnormal cytogenetics contribute to the increased risk. The PRISM score (Prior VTE, Race, IMiD, Surgery, Metaphase Cytogenetics) is a risk prediction tool for VTE in MM and was published in 2022. The score ranges from 0 to 18 and patients were stratified into three groups: low (0), intermediate (1-6), and high risk (≥ 7). Our primary objective was to assess the external validity of the PRISM score in our community hospital setting. Methods: All consecutive newly diagnosed MM treated at Saint Vincent Hospital from 1/1/2008 to 12/22/2022 were included in our analysis. Patients receiving therapeutic anticoagulation for other indications or a diagnosis of VTE within 6 months preceding the MM diagnosis were excluded. Variables were collected through a retrospective chart review during MM treatment initiation. Model discrimination was assessed by calculating the area under the receiver operating characteristic curve (AUROC). Logistic regression was used to calculate the odds ratio (OR) of VTE occurrence in the risk prediction model. Results: A total of 214 patients with newly diagnosed MM and available data on VTE occurrence were included. The median age of our cohort at treatment initiation was 74 years (range 31- 96). The median BMI was 27.8. 56% were male, and 44% were female. The mean PRISM score was 2.9, and 73% of MM were IgG/IgA subtype. IMiD-based induction regimen was used in 63 % of patients. A total of 24 patients (11.2%) were diagnosed with VTE after treatment initiation, of which 8 (3.7%) patients had a previous history of VTE. Prophylactic aspirin was used in 53.8% and abnormal metaphase cytogenetics was identified in 13% of MM cases. The median time to VTE from induction therapy was 4.8 months. The PRISM score was low in 116 patients, intermediate in 89, and high in 9 patients. The AUROC of the model was 0.759 (95% CI, 0.644-0.874) (1). The cumulative incidence of VTE at 6 months, 12 months, and more than 1 year was 6%, 7%, and 11.2% of the patients respectively. Each 1-point increase in the score was associated with a significantly higher chance of VTE occurrence in the model [ HR= 1.53, 95% CI (1.27-1.85), P&amp;lt;0.001]. Among the parameters, patients with a history of VTE [ HR= 1.55, 95% CI (1.26-1.91), P&amp;lt;0.001] and with abnormal metaphase cytogenetics [HR= 2.35, 95% CI (1.37-4.05), P&amp;lt;0.001] had a higher strength of association with the development of VTE after induction (2). Furthermore, among 9 patients with a high-risk PRISM score (≥ 7), 7 (77%) developed VTE within the first 6 months after treatment initiation. Conclusions: Our findings suggest that PRISM [AUROC=0.75, 95% CI (0.644-0.874), P&amp;lt;0.001] score could statistically predict VTE outcomes in our patient population. Abnormal metaphase cytogenetics was a significant predictor of VTE in the validation cohort. Abnormal cytogenetics has been well established to be an indicator of aggressive tumor biology and proliferation in stroma-independent MM. It has been established to be a predictor of VTE in acute myeloid leukemia as well. Also, high-risk scores were predictive of earlier development of VTE after treatment induction. Our study strengthens the evidence of using PRISM as a predictive tool. Further studies are needed to enhance the sensitivity of our existing tool.

An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies

Background and AimsThe highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology but contradicts the current concept of one size fits all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and the therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the Nuclear Factor of Activated T-cells (SMAD4-/-/NFATc1High). MethodsTranscriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of the SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. ResultsOur findings determine the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition (MEKi) normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. ConclusionOur results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a MEKi/gemcitabine combination therapy.

The Role of Immune Checkpoint Inhibitors in Metastatic Pancreatic Cancer: Current State and Outlook.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, characterized by its aggressive tumor biology and poor prognosis. While immune checkpoint inhibitors (ICIs) play a major part in the treatment algorithm of various solid tumors, there is still no evidence of clinical benefit from ICI in patients with metastatic PDAC (mPDAC). This might be due to several reasons, such as the inherent low immunogenicity of pancreatic cancer, the dense stroma-rich tumor microenvironment that precludes an efficient migration of antitumoral effector T cells to the cancer cells, and the increased proportion of immunosuppressive immune cells, such as regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), and myeloid-derived suppressor cells (MDSCs), facilitating tumor growth and invasion. In this review, we provide an overview of the current state of ICIs in mPDAC, report on the biological rationale to implement ICIs into the treatment strategy of pancreatic cancer, and discuss preclinical studies and clinical trials in this field. Additionally, we shed light on the challenges of implementing ICIs into the treatment strategy of PDAC and discuss potential future directions.