Manish S. Patankar

Immunoaffinity-free chromatographic purification of ovarian cancer biomarker CA125 (MUC16) from blood serum enables mass spectrometry characterization

The enrichment of trace proteins from human fluid samples is of great importance in diverse clinical and industrial applications.

A Revised Molecular Model of Ovarian Cancer Biomarker CA125 (MUC16) Enabled by Long-read Sequencing

Abstract The biomarker CA125, a peptide epitope located in several tandem repeats of the mucin MUC16, is the gold standard for monitoring regression and recurrence of high-grade serous ovarian cancer in response to therapy. However, the CA125 epitope along with several structural features of the MUC16 molecule are ill defined. One central aspect still unresolved is the number of tandem repeats in MUC16 and how many of these repeats contain the CA125 epitope. Studies from the early 2000s assembled short DNA reads to estimate that MUC16 contained 63 repeats. Here, we conduct Nanopore long-read sequencing of MUC16 transcripts from three primary ovarian tumors and established cell lines (OVCAR3, OVCAR5, and Kuramochi) for a more exhaustive and accurate estimation and sequencing of the MUC16 tandem repeats. The consensus sequence derived from these six sources was confirmed by proteomics validation and agrees with recent additions to the NCBI database. We propose a model of MUC16 containing 19—not 63—tandem repeats. In addition, we predict the structure of the tandem repeat domain using the deep learning algorithm, AlphaFold. The predicted structure displays an SEA domain and unstructured linker region rich in proline, serine, and threonine residues in all 19 tandem repeats. These studies now pave the way for a detailed characterization of the CA125 epitope. Sequencing and modeling of the MUC16 tandem repeats along with their glycoproteomic characterization, currently underway in our laboratories, will help identify novel epitopes in the MUC16 molecule that improve on the sensitivity and clinical utility of the current CA125 assay. Significance: Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.

Identification of unique clusters of T, dendritic, and innate lymphoid cells in the peritoneal fluid of ovarian cancer patients

AbstractProblemWe hypothesize that activated peritoneal immune cells can be redirected to target ovarian tumors. Here, we obtain fundamental knowledge of the peritoneal immune environment through deep immunophenotyping of T cells, dendritic cells (DC), and innate lymphoid cells (ILC) of ovarian cancer patients.Method of studyT cells, DC, and ILC from ascites of ovarian cancer patients (n = 15) and peripheral blood of post‐menopausal healthy donors (n = 6) were immunophenotyped on a BD Fortessa cytometer using three panels—each composed of 16 antibodies. The data were analyzed manually and by t‐SNE/DensVM. CA125 levels were obtained from patient charts.ResultsWe observed decreased CD3+ T cells and a higher proportion of activated CD4+ and effector memory CD4+/CD8+ T cells, plasmacytoid DC, CD1c+ and CD141+ myeloid DC and CD56Hi NK cells in ascites. t‐SNE/DensVM identified eight T cell, 17 DC, and 17 ILC clusters that were unique in the ascites compared to controls. Hierarchical clustering of cell frequency distinctly segregated the T‐cell and ILC clusters from controls. Increased CA125 levels were associated with decreased CD8+/CD45RA+/CD45RO−/CCR7− T cells.ConclusionThe identified immune clusters serve as the basis for interrogation of the peritoneal immune environment and the development of novel immunologic modalities against ovarian cancer.

Identifying novel ovarian tumor biomarkers through mining of the transcriptome of circulating immune cells: A proof‐of‐concept study

AbstractObjectiveTreatment of high‐grade serous ovarian cancer (HGSOC) will benefit from early detection of cancer. Here, we provide proof‐of‐concept data supporting the hypothesis that circulating immune cells, because of their early recognition of tumors and the tumor microenvironment, can be considered for biomarker discovery.MethodsLongitudinal blood samples from C57BL/6 mice bearing syngeneic ovarian tumors and peripheral blood mononuclear cells (PBMC) from healthy postmenopausal women and newly diagnosed for HGSOC patients were subjected to RNASeq. The results from human immune cells were validated using Affymetrix microarrays. Differentially expressed transcripts in immune cells from tumor‐bearing mice and HGSOC patients were compared to matching controls.ResultsA total of 1282 transcripts (798 and 484, up‐ and downregulated, respectively) were differentially expressed in the tumor‐bearing mice as compared with controls. Top 100 genes showing longitudinal changes in gene expression 2, 4, 7, and 18 days after tumor implantation were identified. Analysis of the PBMC from healthy post‐menopausal women and HGSOC patients identified 4382 differentially expressed genes and 519 of these were validated through Affymetrix microarray analysis. A total of 384 genes, including IL‐1R2, CH3L1, Infitm1, FP42, CXC42, Hdc, Spib, and Sema6b, were differentially expressed in the human and mouse datasets.ConclusionThe PBMC transcriptome shows longitudinal changes in response to the progressing tumor. Several potential biomarker transcripts were identified in HGSOC patients and mouse models. Monitoring their expression in individual PBMC subsets can serve as additional discriminator for the diagnosis of HGSOC.