Title: MicroRNA biomarkers for Determining Response in colorectal cancer
Grant: R01; NIH/NCI
Colorectal cancer (CRC) is a common malignancy and remains the second leading cause of cancer-related deaths in the United States. The present guidelines for CRC treatment recommend that patients with stage III and IV colon cancers (with lymph node or distant metastasis) should be treated with adjuvant or palliative chemotherapy using cytotoxic drugs such as 5-fluorouracil (5-FU) and oxaliplatin after surgical resection of primary cancer. Despite our best intentions, a significant proportion of patients treated with chemotherapy derive no clinical benefit, though all are exposed to toxic and expensive therapeutic regimens. Therefore, there is a clear need to develop biomarkers to help predict which subset of patients will or will not benefit from these treatment regimens. Currently, no biomarkers are available for determining response for oxaliplatin-based chemotherapy (FOLFOX), which serves as standard-of-care for treating patients with advanced CRC. The significance of the current proposal stems from the huge unmet clinical need for the lack of availability of a single established predictive/prognostic biomarker for stage III or IV CRC patients treated with oxaliplatin-based chemotherapy, the standard-of-care treatment for such patients. This knowledge gap encouraged us to undertake systematic and comprehensive microRNA (miRNA) biomarker discovery using next-generation sequencing approaches, rigorous biomarker prioritization and validation of candidate markers in multiple, independent patient cohorts, and potential translation of some of these aberrantly methylated genes/loci as blood-based, noninvasive biomarkers.
To achieve these goals, we will pursue the following three specific aims:
- Aim-1: MiRNA biomarkers will be "discovered" using miRNA-Seq in matched tumor and normal mucosa tissues from advanced CRC patients with and without response to FOLFOX chemotherapy.
- Aim-2: Candidate miRNA biomarkers will be "clinically validated" and their "performance evaluated" in primary tumor tissues from independent cohorts of stage III (adjuvant treatment) and stage IV (palliative treatment) CRC patients treated with FOLFOX.
- Aim-3: Determine the feasibility of translating "tissue-based" miRNAs into "plasma-based" predictive biomarkers in metastatic CRC patients treated with FOLFOX.
This project will be undertaken by a team of investigators with longstanding experience and track records in cancer epigenetics and biomarker discovery. Our findings will have an important clinical impact in identifying patients who will benefit from current chemotherapeutic regimens and those likely to experience adverse outcomes without benefit. Identifying such biomarkers will help reduce the overall healthcare cost burden associated with such treatments and provide a significant step forward in the era of personalized or precision medicine.
Title: Exosomal biomarkers for the noninvasive detection of colorectal cancer
Grant: R01; NIH/ NCI
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. However, unlike other cancers, most early-stage CRCs are surgically curable. Therefore, early detection of CRCs and removal of precursor lesions, particularly advanced colorectal adenomas (A-CRA), is considered the best approach to reducing CRC-associated mortality. However, the currently available screening procedures are impractical. Despite the efficacy of colonoscopy as a screening tool, its invasive nature and expense often dissuade individuals from following CRC screening guidelines; the inaccuracy of fecal-based tests remains insufficient as a diagnostic modality. Accumulating evidence indicates that microRNA (miRNA) dysregulation occurs in all human cancers. As biomarkers, miRNAs are more resilient than mRNAs as they are less prone to degradation and are frequently deregulated even in the earliest stages of neoplasia compared to genetic alterations.
Furthermore, the recent discovery that cancers actively excrete small extracellular vesicles called "exosomes" into systemic circulation has brought additional enthusiasm to the field of translational biomarker research. However, even though exosomes are considered promising due to their structural stability and molecular profiles reflecting their cell-of-origin, utilization of exosomes in biomarker research has been hampered due to multiple reasons, including the Lack of standardized protocols for their isolation and purification. Use of cell line-derived, not patient-derived specimens for biomarker discovery; Lack of molecular profiling studies on cancer-derived exosomes from matched tissues and plasma specimens to establish their cancer specificity; Albeit the perception that exosomal-miRNAs (exo-miRNAs) may be superior to circulating cell-free miRNAs (cf-miRNAs), no studies have undertaken an effort to directly compare these two types to support or negate the superiority of either type, as clinically-relevant disease biomarkers. This proposal will address these concerns by undertaking the following specific aims:
- Aim-1: Optimization of patient-derived exosome isolation, followed by RNA-Seq-based discovery of circulating cell-free (cf-miRNAs) and exosomal miRNAs (exo- miRNAs) in matched tissue and plasma specimens collected from patients with A-CRAs, CRCs, and individuals with a normal colon.
- Aim-2: Development of circulating cell-free and exosomal miRNA biomarker panels that distinguish patients with A-CRAs and CRCs from healthy individuals.
- Aim-3: Clinical validation and performance evaluation of optimized cf-miRNAs and exo-miRNAs in large, independent patient cohorts with colorectal neoplasia.
If successful, this proposal will provide a much-needed molecular characterization of cell-free and exosomal miRNA biomarkers as liquid biopsy biomarkers, which may transform early detection of CRC into a robust, noninvasive, and inexpensive clinical assay.
Title: Exosomal biomarkers for the early detection of hepatocellular carcinoma
Grant: R01; NIH/ NCI
Liver cancer is estimated to afflict 42,230 individuals and result in approximately 30,230 deaths in the US in 2021. The incidence rates for liver cancer have more than tripled since 1980, and the death rates for this disease have increased more than 2% per year since 2007, making it the 5th leading cause of cancer-related deaths in the US. Approximately three-fourths of liver cancer cases are hepatocellular carcinoma (HCC). As for all cancers, detecting HCC at an earlier stage is critical to elicit the best chance of a cure. Early detection of HCC has significant potential to reduce mortality rates due to the significant efficacy of local treatments for early-stage disease vs. systemic therapy for advanced-stage cancers. Furthermore, early detection of HCC before basic liver function has been impaired offers the only hope for a cure. Although the surveillance of patients with virus-associated chronic hepatitis (CH) or liver cirrhosis (LC) is widely performed, the population of HCC patients without viral etiologies (e.g., those with alcohol abuse, obesity, type 2 diabetes mellitus, and non-alcoholic steatohepatitis and fatty liver disease) is rapidly increasing for whom the screening modalities remain inadequate. This highlights the important unmet clinical need to identify early detection biomarkers for all HCC patients, particularly those without viral etiologies. At present, alpha-fetoprotein (AFP) is the most widely used blood tumor marker for surveillance and detection of HCC; however, its clinical utility for detecting early-stage HCCs is limited by low sensitivity. Hepatic ultrasound is a low-cost imaging method for surveillance of HCCs but is limited by low specificity for detecting early-stage HCC. These data highlight the imperative need to develop robust, blood-based, noninvasive biomarkers for the early detection of HCC.
Accumulating evidence indicates that dysregulation of microRNAs (miRNAs) occurs in all human cancers, including HCC. As biomarkers, miRNAs are more resilient than mRNAs, as they are less prone to degradation; compared to genetic alterations, they are frequently deregulated even in the earliest stages of neoplasia. Furthermore, the recent discovery that cancers actively excrete small extracellular vesicles, called exosomes, into the systemic circulation has brought additional enthusiasm to the burgeoning field of translational biomarker research. However, although exosomes are considered promising due to their structural stability and molecular profiles that reflect their respective cells-of-origin, use of exosomes in biomarker research has been hampered due to multiple reasons, including 1) lack of standardized protocols for their isolation and purification; 2) use of cell line-derived, but not patient-derived specimens for biomarker discovery; and 3) lack of molecular profiling studies on cancer-derived exosomes from matched tissues and plasma specimens to establish their cancer specificity. Furthermore, despite the perception that exosomal-miRNAs (exo-miRNAs) may be superior to circulating cell-free miRNAs (cf-miRNAs), no studies have undertaken an effort to directly compare these two types to support or negate the superiority of either type as clinically relevant disease biomarkers.
This proposal will first establish rigorous exosome isolation and characterization methodologies, followed by small RNA-Seq-based biomarker discovery for cf- and exo-miRNAs from patients’ plasma and matched tissues. We will use machine-learning-based bioinformatic approaches to discover and develop biomarker panels in HCC patients vs. non-cancerous controls (patients with CH or LC and healthy individuals). Through the following specific aims, we will subsequently validate their diagnostic performance in multiple large, independent patient cohorts using rapid, high-throughput, and reproducible kit-based approaches.
- Aim-1: Discover candidate cf-miRNA and exo-miRNA biomarkers using small RNA-Seq in matched tissue and plasma from patients with early-stage HCC vs. controls;
- Aim-2: Develop a biomarker panel composed of cf-miRNAs and exo-miRNAs for the identification of patients with HCC in an independent cohort.
- Aim-3: Clinically validate the optimized panel of non-invasive plasma miRNA biomarkers in a large prospective cohort of patients with HCC.
Title: Exosome-based microRNA Biomarkers for Non-invasive and Early Detection of Pancreatic Cancer
Grant: U01; NIH/ NCI
Pancreatic cancer and the importance of early detection. Pancreatic cancer is a highly aggressive malignancy that is estimated to become the second leading cause of cancer-related deaths by 2026. Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of all pancreatic cancer cases and has an overall five-year survival rate of ~8%, the lowest among the major cancers. In PDAC, only 15–20% of patients present with localized, resectable, potentially curable tumors at initial diagnosis. PDAC develops through a gradual, multi-step progression from pancreatic precancerous neoplasms (PNs), including pancreatic intraepithelial neoplasms and pancreatic cystic neoplasms (PCNs), to invasive cancer. Thus, early detection and monitoring of precancerous and early-stage cancers is the ideal approach for early diagnosis of PDAC. Unfortunately, other approaches, such as the measurement of serum CA19-9 or current imaging tools, lack adequate diagnostic sensitivity and specificity. Thus, there is an unmet clinical need to develop highly robust diagnostic strategies for the early detection of PDAC.
miRNAs as promising biomarkers for PDAC. MicroRNAs (miRNAs) are small non-coding RNAs that regulate genes implicated in every human cancer, including PDAC, and may thus be ideal biomarkers. Indeed, circulating cell-free miRNAs (cf-miRNAs) have been shown to have diagnostic potential. Furthermore, the recent discovery that cancer cells actively excrete miRNAs in small extracellular vesicles called exosomes (exo miRNAs) has revolutionized the field, as tumor-derived exosomal cargo enables the identification of cancer-specific molecular markers. However, to date, our studies have been among only very few to compare the clinical significance of cf-miRNAs vs. exo-miRNAs directly, and our team is unique in proposing a combination of cf- and exo-miRNAs as a potentially superior biomarker for cancer diagnosis compared to cf- or exo-miRNAs individually.
Importance of exosome-based miRNAs for early detection of PDAC. Early studies achieved limited success in defining effective cf-miRNA biomarkers for various reasons, including patients with advanced disease (stage III/IV) rather than early-stage cancers, use of statistically underpowered patient cohorts, and lack of key non-disease controls. To address the limitations of cf-miRNA biomarkers and to interrogate the potential clinical significance of exo-mRNAs, during the previous cycle of funding (U01-CA214254), we completed a systematic and comprehensive biomarker discovery and validation effort in patients with PDAC and non-disease control participants. Utilizing the power of unbiased and genome-wide sequencing-based miRNA profiling approaches, together with rigorous bioinformatics and machine-learning algorithms, we: 1) identified panels of 5 cf-miRNAs and 8 exo-miRNAs that could robustly identify patients with early-stage PDAC (area under the curve [AUC] for cf-miRNAs = 0.90; AUC for exo-miRNAs = 0.96); 2) combined the cf- and exo-miRNAs into a “transcriptomic signature” that was superior to individual biomarker panels in identifying patients with all stages of PDAC (AUC = 0.98), including patients with early-stage (stage I/II) disease (AUC = 0.93; sensitivity = 80%; specificity = 91%); 3) showed that combining our transcriptomic signature with established serological tumor marker CA19-9 further improved diagnostic performance (AUC = 0.99); and 4) most importantly, showed that our transcriptomic signature accurately identified patients with PDAC who were CA19-9-negative (<37 U/ml; AUC = 0.96; sensitivity = 91%; specificity = 90%). We hold patent-pending intellectual property for our transcriptomic signature with the USPTO. These successes collectively demonstrate our ability to establish a transcriptomic signature for the early detection of PDAC.
In this competing renewal application in response to PAR-21-334, we will build upon our previous success by demonstrating the clinical significance of our transcriptomic signature in real-life clinical settings. We will evaluate its performance as a non-invasive assay in large, racially/ethnically diverse, prospective cohorts of PDAC patients with various risk profiles; decipher its diagnostic potential for detecting PCNs with high-grade dysplasia (HGD) or invasive disease; and use it to determine lead-time for disease development in pre-diagnosis specimens from individuals who later developed PDAC. We will complete four milestone-based specific aims:
- Aim-1: Expand our biorepository via continued prospective enrollment of patients with PDAC and PNs, including those with PCNs and familial risk, with an additional focus on enrollment of and specimen collection from patients of racial/ethnic minority populations to support our project and NCI’s PCDC biobanking efforts.
- Aim-2: Further validate the transcriptomic signature and establish its performance in prospective cohorts of patients with early-stage PDAC for its clinical translation.
- Aim-3: Determine the clinical significance of our transcriptomic signature to detect the presence of HGD and invasive cancer in pre-operative plasma collected from patients clinically diagnosed as PCNs with high-risk stigmata.
- Aim-4: Evaluate the ability of our transcriptomic signature to detect PDAC at its earliest stages in pre-diagnosis plasma specimens and to determine lead time before disease presentation.
Title: A circulating cell-free epigenetic signature for early detection of pancreatic cancer
Grant: AACR-Lustgarten Foundation
Pancreatic cancer and the importance of early detection: Pancreatic cancer is one of the most lethal malignancies, which is estimated to become the second leading cause of cancer-related deaths by 2030. Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of all pancreatic cancer cases and has a 5-year survival rate of ~12%, the lowest among the major cancers. PDAC is rarely curable when treatment options are limited and not highly effective because it is often diagnosed at an advanced stage. As the pancreas is a retroperitoneal organ, PDAC can invade neighboring organs with few symptoms, making curative resection impossible. In addition, its symptoms are frequently absent or non-specific, resulting in delayed diagnosis. There is no question that early detection is the best strategy to improve the prognosis of patients with cancers, including PDAC. Hence, developing accurate biomarkers, particularly non-invasive markers, will have a transformative clinical impact. However, unlike many other cancers, there is no effective strategy for the early detection of PDAC. Screening for PDAC is not a standard practice in the general population because of its low incidence, and even periodic screening in non-familial high-risk groups is controversial and not yet routine. Furthermore, unfortunately, current diagnostic approaches, such as serum CA19-9 or imaging tools, lack adequate diagnostic potential. Thus, there is a strong, unmet clinical need for robust, sensitive, and specific biomarker assays as an alternative to existing approaches to diagnose the earliest stages of PDAC.
Newly advanced, highly sensitive techniques for genomewide epigenetic profiling: DNA methylation at 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the mammalian genome is a key epigenetic event critical for various cellular processes. 5mC methylation is the most abundant and well-known epigenetic alteration that plays an important role in regulating gene expression. 5hmC methylation, an oxidized form of 5mC, is mainly distributed in transcriptionally active regions coincident with open chromatin and permissive histone. As circulating cell-free DNA (cfDNA) contains epigenetic information, including 5mC and 5hmC, which are chemically stable, abundant in the genome, and closely associated with disease states, combined analysis of both 5mC and 5hmC alterations offer an attractive platform for biomarker development. There are several advantages to using genomewide 5mC and 5hmC profiling for biomarker development, including 1) aberrant alterations in DNA methylation occur early in cancer development and thus are appropriate for detecting processes surrounding cancer initiation; 2) the global, genomic nature of epigenetic alterations provide a rich dataset for developing widely applicable diagnostic tests, whereas genetic mutations are frequently unique to specific tumors; and 3) changes in the genomic distributions of 5mC and 5hmC occur in cancer-specific and tissue-specific manners and track with disease burden. Furthermore, the specific locations of 5mC and 5hmC modifications in the genome provide information about genomic regions undergoing transcriptional suppression or activation in disease versus healthy states, thus potentially revealing new information about the loci driving cellular transformation and malignancy. However, due to the lack of effective collection, standardized quality control, and analysis procedures, the clinical utilization of these data cannot be effectively realized. Our proposal uses two newly advanced molecular technologies, the Linear Amplification of Bisulfite Sequencing (LABS-seq) and nano-hmC-Seal, developed to probe 5mC and 5hmC, respectively. These innovative techniques involve analytical sensitivity to robustly determine genomewide distributions of 5mC and 5hmC modifications using minimal amounts of DNA (1 ng or less). This proposal aims to identify diagnostic 5mC and 5hmC biomarkers from cfDNA for developing sensitive, specific, minimally invasive, and clinically feasible blood tests for early PDAC detection.
Improved early diagnosis of PDAC represents the best opportunity to select patients for resection before their cancer progresses to invasive PDAC. To achieve this goal, we propose two Specific Aims, as follows.
- Aim-1: Discovery of 5mC and 5hmC biomarkers to distinguish patients with PDAC from non-cancerous controls in circulating cfDNA through genomewide methylation profiling using LABS-seq and nano-hmC-Seal.
- Aim-2: Prioritization, training, and validation of 5mC and 5hmC biomarker signatures using qPCR-based approaches in cfDNA to facilitate their clinical translation for detecting early-stage PDAC.
Title: Development of a DNA methylation-based, liquid biopsy assay for the early detection of pancreatic cancer
Grant: Susan E. Riley Foundation
Dr. Goel's lab is seeking funding to develop a blood test for the early detection of pancreatic cancer. This noninvasive test would be used for early detection of pancreatic cancer, particularly in high-risk populations, long before patients show symptoms of this deadly disease. The test would use abnormal DNA methylation patterns specific to pancreatic ductal adenocarcinoma (PDAC) tumors. Healthy DNA methylation is a biological process essential for normal cell function; however, this process is altered in cancer cells, leading to abnormal patterns that induce chromosomal instability and other changes that are a hallmark of cancer cells. These patterns have emerged as a significant marker enabling us to target tumor cells because methylated DNA is a highly stable and cancer tissue-specific change that can be measured in bodily fluids such as blood (e.g., plasma or serum), urine, and stool. In the last ten years, such liquid biopsy assays have gained attention in the scientific community, leading to several tests approved by the FDA for the early detection of other cancers. No such test yet exists, however, for pancreatic cancer. Dr. Goel's project aims to produce a test of this kind for pancreatic cancer.
Title: A next-generation blood test for the early detection of ovarian cancer
Grant: City of Hope Circle 1500 Award
Ovarian cancer-associated poor survival and a clinical need for early-detection biomarkers: Ovarian cancer (OC) ranks fifth in cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. A woman’s risk of getting OC during her lifetime is about 1 in 78. Her lifetime chance of dying from ovarian cancer is about 1 in 108. The American Cancer Society estimates that in 2023, about 19,710 new cases of OC will be diagnosed, and 13,270 women will die of this cancer in the United States alone. The survival rates in OC are stage-dependent, with 5-year survival rates for a localized disease (cancer confined to the ovary) decreasing significantly as cancer spreads to regional lymph nodes and other distant organs. These unfavorable survival rates are primarily because more than two-thirds of OC patients are diagnosed in advanced stages of the disease (stage III or IV), which is associated with a 5-year survival of 27% for stage III and 13% for stage IV cancer patients – hence, preventing a definite cure. In contrast, the 5-year survival rate would improve significantly if ovarian cancer was detected in stage I; the 5-year survival in these cases is 90%, and the 10-year survival is 84%.
These sobering statistics highlight the limitations of current OC screening methods: the CA125 serum marker and transvaginal ultrasound (TVUS). These screening modalities lack the sensitivity and specificity to effectively diagnose the disease, minimize false positives, and downstage the disease to improve survival, highlighting the pressing unmet clinical need for identifying and developing early-detection biomarkers for women with OC, especially in women with elevated disease risk. These high-risk patients include those with a genetic predisposition (e.g., carriers of BRCA1, BRCA2, Lynch syndrome genes, BRIP1, RAD51C, and RAD51D), who possess an up to 80% lifetime risk of developing ovarian cancer.
Rationale: By undertaking a systematic and comprehensive research effort over the past several years by performing whole genome sequencing of ovarian cancer tissues, Dr. Goel and colleagues have identified a novel 8-microRNA (miRNA) signature that is over-expressed explicitly in OC, especially in the earliest stages of disease (stages I-II). Enthused by these exciting findings, Goel’s team has taken the next step. It has made a significant advance by translating these tissue-based biomarkers into a blood-based, liquid biopsy non-invasive assay with a sensitivity of 86% and a specificity of 92%, which is remarkably superior to CA125, making it an attractive blood-based test for the early detection of OC. These results were published in a very high-impact peer-reviewed article (Kandimalla et al., Clinical Cancer Research, 2021).
The overarching goal of this proposal is to optimize and analyze the performance of this 8-miRNA panel in a prospective cohort of women and detect the earliest stage of invasive OC without false positives from non-neoplastic lesions. Since the process of carcinogenesis is hypothesized to take decades, our goal would be to develop an effective strategy to detect and intercept this process in its inception or pre-invasive phase before the development of overt cancer. This will be achieved through two specific aims:
- Aim-1: To design and conduct a prospective cross-sectional study to validate the performance of this blood-based miRNA panel in women with a) a genetic predisposition for ovarian cancer, b) a significant family history of ovarian cancer, and c) a new diagnosis of epithelial OC.
- Aim-2: Compare the performance of miRNA-biomarkers with CA125 and TVUS and develop a final assay that offers the highest sensitivity and specificity for non-invasive, early detection of OC.