Parathyroid Tumor Clonal Status

To define the frequency of monoclonal-X and polyclonal-X tumors in PHPT patients having PTX and to define the relationship between parathyroid tumor clonal status and MGN, we will compare surgical and pathologic outcomes to tumor clonal status in a multicenter cohort of patients having bilateral neck exploration (BNE) and PTX.

Study participants will be recruited from four high-volume centers at Washington University in St. Louis (WU. Eligible patients will receive standard of care treatment (parathyroidectomy) and de-identified FFPE tumor samples (1 slide H\&E stained, 6 x 10 um sections unstained from each abnormal gland) will be sent to WU for study. DNA will be extracted from FFPE samples and the HUMARA assay will be performed according to our established protocol. (3) Our two published studies show >90% concordance between replicate HUMARA assays of the same tumor. For additional rigor, two regions of each tumor will be assayed independently to ensure concordance of clonal status. Tumors where the clonality call from the two within-tumor samples are discordant will be recorded as such, and we will perform sensitivity analyses, for aims where this is relevant, of assigning one or the other clonal state to these samples.

Further, we will employ a secondary assay (Cytoscan HD array, ThermoFisher) to assess DNA copy number variation (CNV) in a random set of samples from 58 polyclonal-X cases and 49 monoclonal-X cases (estimated 107 total assays). Published and unpublished data have shown that CNV occurs with considerable frequency in parathyroid tumors, including adenomas. CNV assessment can provide independent verification of an X-inactivation-based finding of polyclonality by identifying heterogeneous CNV within a tumor sample indicating polyclonality, or more uniform CNV reflecting monoclonality. Cases with discordant results (estimated <10%) from HUMARA and CNV assays will be comprehensively studied in the exploratory objectives.

De-identified pathologic data including the number and weight of abnormal glands removed from patients will be recorded and entered in a REDCap database maintained at WU. We then will review operative and pathologic reports for correlation of tumor clonality and the presence of SGN or MGN. In cases of MGN we will perform ms-PCR of HUMARA alleles on all resected tumors to assess for concordance of clonality. We will also determine the impact of two common surgical approaches on outcomes in tumors of different clonal status. Participating surgeons in the trial estimate that between 40% and 50% of their cases have unilateral neck exploration (UNE), and all use intraoperative PTH (ioPTH) during UNE. The frequency of MGN stratified by tumor clonality will be examined in patients who undergo UNE with ioPTH monitoring and compared BNE. Operative and pathology reports will be reviewed as well as ioPTH levels drawn before and both 5 and 10 minutes (PTH T1/2 = 5 min.) after tumor removal. Underlying tumor clonality will be determined as described above and will be compared to pathologic results (MGN versus SGN), as well as ioPTH kinetics (% decline from pre-op PTH levels at 5 and 10 minutes after final tumor removal).

To define the relationship between parathyroid tumor clonal status and biochemical outcomes following PTX for PHPT, we will compare baseline clinical features, surgical/pathologic findings and postoperative biochemical outcomes following PTX to tumor clonal status in a large, multicenter cohort of patients having PTX. A total of 645 patients with known tumor clonal status will have standard clinical and biochemical data (serum calcium, albumin, intact PTH, 25(OH)D, and creatinine) recorded at baseline (before PTX), and at 2 weeks, 3 months, and 6 months post-PTX (not all labs are recorded at each follow-up time point). We will compare the frequency of elevated PTH (ePTH) at each time point in patients with monoclonal-X and polyclonal-X tumors. We have previously shown that vitamin D status impacts ePTH following PTX. To investigate abnormal vitamin D metabolism, the most common mechanism of secondary hyperparathyroidism as a cause of polyclonal-X disease, we also will perform a comprehensive analysis of vitamin D status in a subset of 111 WUSM patients with monoclonal-X and polyclonal-X tumors. Our analysis will include biochemical indices of vitamin D metabolism (25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and vitamin D binding protein levels).

Genomic approaches, e.g. whole genome sequencing plus RNA sequencing, will be performed at Wash U, using fresh frozen tissue collected following PTX or from FFPE tissue.