Functional and Genetic Studies of Isolated Cells from Parathyroid Tumors Reveal the Complex Pathogenesis of Parathyroid Neoplasia

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Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia

Yuhong Shia, Joyce Hogueb, Darshana Dixitb, James Kohb,1, and John A. Olson, Jr.a,1,2

aDivision of General and Oncologic Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201; and bDivision of Surgical Sciences, Department of Surgery, Duke University Medical Center, Durham, NC 27710

Edited* by Robert C. Gallo, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, and approved January 3, 2014 (received for review November 4, 2013) Parathyroid adenomas (PAs) causing primary hyperparathyroidism thyroid cells leads to abnormal secretion of PTH initially, followed (PHPT) are histologically heterogeneous yet have been historically by proliferation of parathyroid cells in response to chronic de- viewed as largely monotypic entities arising from clonal expansion mand for increased PTH (12, 13). An abnormal calcium–PTH set of a single transformed progenitor. Using flow cytometric analysis point has been well-described in aggregate dispersed cells from of resected adenomatous parathyroid glands, we have isolated parathyroid adenomas, and most reports attribute the impaired and characterized chief cells, oxyphil cells, and tumor-infiltrating set point in these cells to decreased expression of the calcium- lymphocytes. The parathyroid chief and oxyphil cells produce sensing receptor (CASR) (14, 15) or more recently to altered parathyroid hormone (PTH), express the calcium-sensing receptor expression of downstream molecules linked to CASR signaling, (CASR), and mobilize intracellular calcium in response to CASR including RhoGEF and RGS5 (16, 17). In contrast to clonal ex- activation. Parathyroid tumor infiltrating lymphocytes are T cells pansion following genomic tumor-initiating events, attenuated by immunophenotyping. Under normocalcemic conditions, oxyphil cells produce ∼50% more PTH than do chief cells, yet display sig- calcium responsiveness could be expected to drive polyclonal nificantly greater PTH suppression and calcium flux response to proliferation in the parathyroid gland. elevated calcium. In contrast, CASR expression and localization To investigate the composition of parathyroid tumors, we MEDICAL SCIENCES are equivalent in the respective parathyroid cell populations. Anal- sought to characterize a series of parathyroid adenomas at the ysis of tumor clonality using X-linked inactivation assays in a cellular and functional level. In this study, we report the isolation patient-matched series of intact tumors, preparatively isolated and characterization of chief cells, oxyphil cells, and lymphocytes oxyphil and chief cells, and laser-captured microdissected PA speci- present in parathyroid adenomas and histologically normal mens demonstrate polyclonality in 5 of 14 cases. These data dem- parathyroid glands from patients with PHPT. Our results show onstrate the presence of functionally distinct oxyphil and chief that parathyroid adenomas removed from patients with PHPT cells within parathyroid primary adenomas and provide evidence are composed of functionally and genetically distinct oxyphil and that primary PA can arise by both clonal and polyclonal mecha- chief cells and have varying amounts of infiltrating lymphocytes. nisms. The clonal differences, biochemical activity, and relative The chief and oxyphil cells within parathyroid adenomas have abundance of these parathyroid adenoma subpopulations likely differing ability to respond to changes in ambient calcium and reflect distinct mechanisms of disease in PHPT. produce PTH although CASR expression is comparable between these parathyroid cell subtypes. Further, we show that a signifi- tumor heterogeneity | endocrine neoplasia cant proportion of PHPT patients have polyclonal tumors. The relative abundance, functional behavior, and clonal origin of he parathyroid glands maintain serum calcium concentration Twithin a narrow physiological range through regulated syn- Significance thesis and secretion of parathyroid hormone (PTH) (1). Para- thyroid neoplasia results in inappropriate secretion of PTH by one or more glands, leading to hypercalcemia and the disease Parathyroid adenomas, the main cause of primary hyperpara- primary hyperparathyroidism (PHPT) (2). To date, research in thyroidism (PHPT), are thought to result from clonal expansion of tumor cells and to be insensitive to normal calcium feedback the field has focused on histologic and molecular profiling of due to the loss of the calcium-sensing receptor (CASR). Utilizing parathyroid tumors and the investigation of calcium sensing in flow cytometric analysis to isolate and individually study dispersed cells from parathyroid tumors and normal bovine oxyphil cells, chief cells, and lymphocytes from resected para- parathyroid glands. Few studies have characterized the individual thyroid tumors and glands, we now report previously un- cellular constituents of human parathyroid tumors, and no pub- recognized heterogeneity in these tissues with respect to lications have reported live-cell functional evaluation of the dif- calcium responsiveness, CASR expression, and clonal origin of ferent cellular subtypes observed in parathyroid tumors. parathyroid tumors. Such heterogeneity of parathyroid ade- Parathyroid adenomas, the most common cause of PHPT, are nomas likely reflects the complex etiopathogenesis and clinical considered clonal proliferations of a transformed parathyroid heterogeneity of PHPT. cell that has acquired proliferative or survival advantage due to one of several genetic abnormalities including the PRAD1 Author contributions: Y.S., J.K., and J.A.O. designed research; Y.S. and J.H. performed translocation or mutations in the genes encoding menin, P53, research; Y.S., J.H., D.D., J.K., and J.A.O. contributed new reagents/analytic tools; Y.S., J.K., and P27 (3-7). Regardless of type, most authors consider para- and J.A.O. analyzed data; and Y.S., J.K., and J.A.O. wrote the paper. thyroid tumors clonal (8, 9) although intratumoral heterogeneity The authors declare no conflict of interest. has been observed and polyclonality of microdissected para- *This Direct Submission article had a prearranged editor. thyroid adenomas has been reported (10, 11). 1J.K. and J.A.O. contributed equally to this work. Although most data support mutation-driven clonal expansion 2To whom correspondence should be addressed. E-mail: [email protected]. of parathyroid tumors, an alternative model for the origin of This article contains supporting information online at www.pnas.org/lookup/suppl/doi: parathyroid tumors is that abnormal calcium sensing by para- 10.1073/pnas.1319742111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1319742111 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 distinct parathyroid-cell subpopulations in parathyroid tumors ples tested ranged from 2% to 31.9%. The relative abundance likely reflect multiple alternative etiologies of PHPT. of chief cells ranged from 9.4% to 57.8% of the total viable population. The relative abundance of oxyphil cells ranged Results from 0.9% to 59.8%. The distribution of these cells did not Flow cytometric analysis of dispersed parathyroid adenoma cells correlate with any feature of the disease including sex, age, revealed discrete subpopulations of cells resolved on the basis preoperative calcium or PTH, gland weight, or histology. of visible-light diffraction detected by the in-line axis [forward To compare the cellular composition of histologically normal scatter (FSC-A)] and orthogonal axis [side scatter (SSC-A)] and neoplastic parathyroid tissue, we examined a series of five sensors. After excluding nonviable and doublet cells, three dis- paired tumor and histologically normal (but physiologically sup- tinct populations, initially designated P3, P4, and P5, were clearly pressed) parathyroid gland biopsy specimens using flow cytometry and reproducibly resolved as separate peaks in a bivariate SSC- to assay for the presence of the three cell populations. The relative A/FSC-A contour plot (Fig. 1A, Upper Left). Ultrastructural distribution of oxyphil cells, chief cells, and lymphocytes was re- analysis by transmission electron microscopy of preparatively markably consistent between normal tissues. In contrast, adenomas isolated P3, P4, and P5 cells showed clear morphological dif- from different patients have highly variable distribution patterns ferences between the three populations (Fig. 1A). Based upon (Fig. 2 A and B). These data indicate that oxyphil cells, chief cells, size and morphology, P3 cells were provisionally identified and lymphocytes are present in both normal tissue and adeno- as peripheral blood lymphocytes. P4 cells displayed highly matous tissue, but the relative abundance of these subpopulations interdigitated plasma membranes, sparse mitochondria, and can be markedly variable between different adenomas. prominent intracellular secretory granules, all features of To confirm the designation of P3 cells as lymphocytes, we parathyroid chief cells (18). P5 cells were large and contained probed P3 cells with the leukocyte common antigen CD45 an- round nuclei, moderate peripheral chromatin condensation, tibody using standard flow-cytometry methods. P3 cells are uni- and numerous mitochondria densely packed throughout the formly CD45-positive and are indistinguishable in this assay from cytoplasm. These characteristics are consistent with the patient-matched isolated peripheral blood lymphocytes. Sub- sequent cell-surface marker studies revealed that the majority of parathyroid oxyphil cell type (19). We used the same sorting + + − − + strategy to examine the distribution of these cells types among P3 cells are CD45 /CD3 /CD19 /CD24 /CD44 , an immuno- cells dispersed from single adenomas from 20 patients with type consistent with peripheral T cells (Fig. 3A). These results PHPT (Fig. 1B). In these samples, the proportion of lym- unambiguously confirm that P3 cells are lymphocytes. CD3 phocytes present in the dispersed parathyroid adenoma sam- staining on parathyroid adenoma tissue sections showed three predominant patterns: widely separated single cells (Fig. 3B, Pt1), clustered cells extravasated into the parenchyma of the gland but adjacent to vascular structures (Fig. 3B, Pt2), and fo- A 104 P3 cal, densely packed infiltrates (Fig. 3B, Pt3). To verify that the P3 103 cells are tumor-infiltrating lymphocytes as opposed to contami- P5 nating peripheral blood mononuclear cells (PBMCs) adventi- 102 P4 tiously carried over from the tumor vasculature, we examined the + + SSC-A CD4 /CD8 ratio in the P3 population. In contrast to the 2:1 1 + + + 10 P3 ratio of CD4 to CD8 cells found in the bloodstream, CD8 effector cells are highly overrepresented in the P3 lymphocytes 100 0 200 400 600 800 1000 (Fig. 3C). Taken together with the physical distribution of lym- FSC-A phocytes in the parathyroid parenchyma observed by immunohis- P4 P5 tochemistry (IHC), this result confirms the identity of parathyroid adenoma P3 cells as tumor-infiltrating T cells as opposed to contaminating peripheral blood lymphocytes. We then sought to functionally characterize isolated oxyphil and chief cells by evaluating basal PTH production and cellular response (PTH suppression and intracellular calcium flux) to exogenous calcium. Comparison of PTH production under normocalcemic conditions (1.25 mM) in 11 patient-matched pairs of oxyphil and chief cells showed significantly higher baseline PTH production in the oxyphil cells relative to the chief cells (P = B Oxyphil cell 0.026 by paired t test) (Fig. 4A). The paired samples showed significant correlation to each other (r = 0.9017), indicating that adenoma PTH output is strongly influenced by patient origin as Chief cell well as cell type (chief vs. oxyphil). In vitro PTH production by isolated cells correlated well with unsorted cells. We next compared PTH production over a 2-h period by Lymphocyte preparatively isolated oxyphil and chief cells under varied calcium concentrations. Both oxyphil and chief cells secrete PTH when 0 20 40 60 80 100 cultured in 0.5 mM, 1.25 mM, and 3.0 mM calcium-containing Fraction of sorted viable cells (%) media. Regardless of calcium concentration, oxyphil cells secreted more PTH per cell than chief cells over the assay interval. At Fig. 1. Cellular heterogeneity in parathyroid adenomas. (A, Upper Left) Con- elevated calcium concentrations, PTH production was inhibited tour diagram showing the distribution of parathyroid cells (P3, P4, and P5) from incompletely in both oxyphil and chief cells. Normalized PTH dispersed parathyroid adenoma samples based upon the forward and side scatter channel outputs. Nonviable cells and aggregated cells were excluded by secretion profiles of oxyphil and chief cells reveal that both gating for the absence of propidium iodide uptake. In panels labeled P3, P4, and populations show similar PTH suppression at a 3-mM ambient P5 is shown electron microscopy of isolated cells (P3, P4, and P5) from dispersed calcium concentration (Fig. 4B). However, the wide SD clearly parathyroid adenomas. (Scale bar: 500 nm.) (B) Box and whisker plots of lym- reflects heterogeneous responsiveness among the adenomas. The phocytes, chief cells, and oxyphil cells isolated from a total of 20 cases. suppressability of PTH secretion by calcium in vitro did not

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1319742111 Shi et al. Downloaded by guest on September 25, 2021 transcriptase polymerase chain reaction (qRT-PCR). Both cell types were found to contain equivalent amounts of the CASR mRNA (Fig. 5C). Because oxyphil and chief cells differ greatly in their calcium responsiveness as evaluated in the flux assay, the presence of comparable CASR expression in both populations indicates that the degree of calcium responsiveness in parathyroid cells is not solely determined by CASR abundance. The functional and morphological heterogeneity we observed within primary parathyroid adenomas and their cell isolates raised the question of whether the originating tumors were uniformly clonal as is widely believed (8, 9). To test this concept, we probed for allele-specific X-inactivation in parathyroid adenoma cells derived from female patients at two highly polymorphic imprinted loci, the human androgen receptor (HUMARA) (20) and the phosphate glycerate kinase PGK genes (21). Five female HUMARA locus informative cases were subsequently assessed for clonality. Two of the five cases were found to be monoclonal using both whole adenoma tissue and laser-capture microdissected frozen-section specimens as input (Fig. 6A). In contrast, the remaining three cases showed a polyclonal pattern both in whole adenoma tissue and

Fig. 2. Relative abundance and distribution of lymphocytes, chief cells, and oxyphil cells from parathyroid adenomas and matched normal parathyroid gland biopsies from patients with PHPT. (A) Representative contour plots of side scatter and forward scatter parameters from flow-cytometry analysis MEDICAL SCIENCES of parathyroid adenoma and matched normal tissue. (B) Box and whisker plots of lymphocytes, chief cells, and oxyphil cells isolated from a total of five cases.

correlate with severity of clinical disease (i.e., calcium and PTH levels in the blood). We also used flow cytometry-based kinetic analysis to evaluate the relative intracellular responsiveness of the parathyroid cells to changes in extracellular calcium concentration. Stimulation with 3 mM extracellular calcium elicited a rapid and robust intracellular calcium-flux response in the dispersed parathyroid cells as detected by increased Fluo-4 AM mean fluorescent intensity. Clear differences in calcium-flux responses were revealed when we compared the relative activity of the oxyphil cells, chief cells, and lymphocytes (Fig. 4C). Oxyphil cells are the most responsive and account for the great majority of the overall calcium- flux activity in the dispersed parathyroid-cell parental population. Chief cells display a much lower response whereas lymphocytes cells are completely unresponsive. These data indicate that readily identifiable subpopulations of cells within a given parathyroid adenoma display categorically distinct quantitative responses to extracellular-calcium stimulation. To better understand the heterogeneity of parathyroid adenomas and to explore the question of why parathyroid adenomas do not sense calcium properly and fail to regulate PTH secretion, we examined CASR expression in parathyroid adenomas from a series of patients with PHPT. First, we used immunofluorescence detection to evaluate CASR protein expression in isolated oxyphil and chief cells from a panel of primary parathyroid adenoma specimens. As shown in Fig. 5A, CASR immunoreactivity is detected on the surface of both oxyphil and chief cells. For quantitation of the relative abundance of CASR-positive cells Fig. 3. Immunophenotypic characterization of cells isolated from para- within each population, at least 100 cells per field were counted thyroid adenomas. (A) Flow-cytometric analysis of cell-surface markers CD45, in each of three different fields, and the proportion of CASR- CD3, CD19, CD24, and CD44 on tumor-infiltrating lymphocytes (TILs) (Lower) positive cells was calculated as a percentage. Both cell pop- and patient-matched PBLs (Upper). (B) Tissue sections from three independent parathyroid adenomas were probed with an anti-CD3 antibody, and ulations were found to contain similar proportions of CASR- ± ± reactivity was visualized by DAB staining and with hematoxylin/eosin coun- positive cells (39.5 6.3% for chief cells and 41.0 6.2% for terstaining. (C) CD4 and CD8 cell-surface expression in P3 cells derived from + oxyphil cells) (Fig. 5B). Second, we examined CASR transcript three parathyroid adenomas was determined by FACS. The ratio of CD4 to + abundance in chief and oxyphil cells by quantitative reverse- CD8 cells is based upon quadrant gating using standard conditions.

Shi et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 proportions of chief cells, oxyphil cells, and infiltrating T lymphocytes and that the two parathyroid cell types manifest distinct functional properties. Parathyroid chief cells and oxyphil cells express similar amounts of CASR, and both exhibit attenuated PTH secretory responses to changes in ambient calcium. However, the respective cell types are markedly different in their baseline PTH production and intracellular flux response to extracellular calcium challenge. As the basis for this differential calcium responsiveness is not attributable solely to CASR relative abundance, additional mechanisms, including enhanced biochemical opposition to CASR signaling, altered CASR protein trafficking, or CASR- independent calcium sensing, may prove to be important functional determinants in these cells. Further, we show that a significant proportion of parathyroid adenomas causing PHPT are polyclonal rather than monoclonal. These results indicate that parathyroid tumors causing primary hyperparathyroidism are heterogeneous at the cellular and functional level and further show that some patients’ tumors are monoclonal whereas other patients have polyclonal tumors. Expanded studies of a large number of patients Fig. 4. Parathyroid hormone (PTH) secretion and intracellular calcium-flux with PHPT to assess frequency of these different tumor types and response to 3.0 mM extracellular calcium in dispersed primary cells isolated from parathyroid adenomas. (A) Basal PTH secretion over 2 h by tumor- the clinical phenotypes associated with them will be needed to matched chief and oxyphil cells from 11 PHPT patients under normocalcemic fully understand the meaning of these findings. It will be important (1.25 mM) conditions. (B) PTH secretion over 2 h under varied calcium con- in future studies to relate the variable biochemical behavior and centration by tumor-matched chief and oxyphil cells (n = 6). (C) Intracellular clonal status of PHPT tumors to the presence of driver mutations flux response among isolated lymphocytes, chief cells, and oxyphil cells. One in known parathyroid tumor suppressor genes and oncogenes such representative experiment out of seven is shown. as MEN1 and CCND1 (22). Earlier investigations using histologic methods have revealed that parathyroid adenomas comprise mainly chief cells, transi- microdissected frozen-section specimens (Fig. 6A). Patient-matched tional oxyphil cells, and oxyphil cells. These observational studies peripheral blood lymphocyte genomic DNA was used in as an allelic were limited to analysis of fixed parathyroid tissue sections. Until control to demonstrate the polyclonal status of the HUMARA lo- the current work, the successful preparative isolation of living cus in all five of the cases tested. We next probed for the clonal status of isolated oxyphil and chief cells and found that these cells oxyphil and chief cells from human parathyroid adenoma tissue reflected the same clonality pattern observed in the originating has not been reported. Using a flow cytometry-based approach, patient-matched whole adenoma tissue (Fig. 6A). In the mono- we analyzed 20 adenomas resected from patients with PHPT and clonal cases, the oxyphil and chief cells both harbored the same determined the proportional representation of chief cells, oxy- maternally imprinted HUMARA allele, indicating that these phil cells, and infiltrating T lymphocytes in the primary tumor. populations arose from a common progenitor. Conversely, in Wide variations among adenomas were observed. In contrast, the polyclonal specimens, both the oxyphil and chief populations the distribution of these cell types proved remarkably consistent were found to contain equal representations of each imprinted HUMARA allele, indicating the nonclonal provenance of both cell types. To confirm the HUMARA findings, we also performed methylation-sensitive PCR of the imprinted PGK gene (21). Of 8 HUMARA locus informative cases, 4 were monoclonal and 4 were polyclonal; of 9 PGK locus informative cases, 7 cases were monoclonal and 2 were polyclonal (Fig. 6B). Three cases were informative at both HUMARA and PGK loci, and all 3 yielded concordant results (2/3 monoclonal, 1/3 polyclonal). Altogether, 14 informative cases assayed by either method or both methods showed polyclonal origin in 5 and monoclonal origin in 9 parathyroid adenomas. A single tumor from a patient with renal failure-induced secondary HPT was investigated as control and showed polyclonal status in both HUMARA and PGK assays. Discussion Parathyroid neoplasias result in abnormal PTH secretion from an expanded population of parathyroid cells. With few excep- tions, a significant body of prior work has shown that parathyroid tumors in PHPT are clonal and that aggregate dispersed parathyroid tumor cells secrete PTH in culture but can be poorly responsive to negative feedback by calcium. In our work to identify differential gene expression and mechanisms of abnormal Fig. 5. Expression of CASR in isolated parathyroid cells. (A) Immunofluo- calcium sensing in parathyroid adenomas, we noticed substantial rescence (IF) for CASR protein in chief and oxyphil cells using anti-CASR monoclonal antibody (20x). (Inset) CASR expression-magnified (63x, oil). (B) heterogeneity in these tumors. This finding, coupled with the Quantitation of CASR IF-positive cells in chief and oxyphil cells from A.A observed histologic heterogeneity of parathyroid adenomas, led total of more than 100 cells per field were counted in each of three different us to design the current study to isolate and functionally char- fields. Data are from three patients. (C) Pairwise comparison of CASR mRNA acterize individual populations of cells from parathyroid ade- expression measured by qRT-PCR in chief and oxyphil cells from parathyroid nomas. We report that parathyroid adenomas comprise variable adenomas from seven patients, normalized to an internal GAPDH control.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1319742111 Shi et al. Downloaded by guest on September 25, 2021 secretory properties of parathyroid tissue. Although the numbers of patients studied are too small to form clear conclusions, the behavior of preparatively isolated chief and oxyphil cells appears similar to the activity of each of these cell types when analyzed as unsorted cell suspensions. Further, in vitro basal and calcium stimulated PTH production by isolated cells did not correlate with severity of patient disease, underscoring the complex physiology of calcium homeostasis in patients. CASR down-regulation is generally believed to be the principal mechanism for the abnormal calcium-PTH set-point relationship in patients with parathyroid tumors. If this assumption is true, then CASR expression should be significantly reduced in all parathyroid tumors, and calcium responsiveness should be de- monstrably dependent upon CASR expression. The data in our study challenge this prevailing model. We show that chief and oxyphil cells display comparable CASR expression at both the protein and transcript levels, yet oxyphil cells are clearly more responsive to calcium and exhibit consistently higher basal PTH secretion. These data indicate that CASR expression is not the sole determinant of PTH production and calcium responsiveness in the parathyroid gland and suggest that the elevated PTH associated with PHPT could be reflective of not only increased tumor-cell number but also the higher basal PTH secretion profile of oxyphil cells compared with chief cells. Our study indicates that the presence of lymphocytic infiltration is a common feature in parathyroid adenomas. Using flow cytometry, we identified the parathyroid adenoma-derived P3 population as T lymphocytes expressing the leukocyte common antigen CD45 and T-cell receptor unit CD3. Additional MEDICAL SCIENCES + + marker studies revealed the T lymphocytes to be CD45 /CD3 / − − + Fig. 6. Clonality of parathyroid adenomas. (A) Clonality of parathyroid cell CD19 /CD24 /CD44 . The role of immune cells in the patho- populations isolated from five parathyroid adenomas. HUMARA alleles were genesis of human parathyroid adenoma is less clear. Immune amplified using methylation-sensitive PCR in HhaI undigested (−) and digested (+) genomic DNA from peripheral blood lymphocytes (PBL), whole tumor tissue cells can release inflammatory mediators with proangiogenic and (Adenoma), preparatively sorted chief and oxyphil cells, or microdissected tumor prometastatic effects. Tumor cells can express antigens and be- tissue (LCM). The presence of a single band reveals a single methylated allele come targets for a T cell-mediated adaptive immune response. from a monoclonal tumor (Pt3 and -14) whereas two bands reflects random The presence of high numbers of tumor-infiltrating lymphocytes, methylation of alleles in a polyclonal tumor (Pt15, -16, and -17). (B) Clonality of 14 particularly T cells, has been found to be a major predictor of parathyroid adenomas identified by HUMARA and PGK methods. favorable clinical outcomes in several solid cancers (24–26). These findings support the hypothesis that the adaptive immune response influences the behavior of human tumors. However, in a series of five histologically normal parathyroid glands ipsilateral lymphocytic infiltration in parathyroid adenoma is rarely repor- to adenomatous tissue. This result suggests that the variable com- ted (27), which suggests that our current findings may represent position of parathyroid adenomas relative to normal tissue could a newly recognized histologic feature. Because we found lym- reveal a greater degree of functional heterogeneity among primary phocytes in both normal and adenomatous parathyroid glands, parathyroid adenomas than is currently appreciated. the role of these T cells is still unclear. As the ratio of effector + + To address this question, we isolated live oxyphil and chief (CD8 ) to helper (CD4 ) T cells in P3 lymphocytes recovered cells from parathyroid adenomas and compared the behavior of from parathyroid adenoma tissue is consistently distinct from the respective cell types in functional assays measuring calcium the 1:2 ratio maintained in the bloodstream, it is clear that the responsiveness and PTH secretion, two definitive performance P3 population represents tumor infiltrating lymphocytes (TILs) metrics of parathyroid gland function. Our data indicate that the rather than adventitious contaminating cells carried over from subpopulations of chief and oxyphil cells within a given para- vascular elements. It is possible that the TILs may be homing to thyroid adenoma display clearly different quantitative responses antibodies targeting CASR, PTH, or other parathyroid antigens to extracellular calcium stimulation. In all cases tested, oxyphil and that antibody-mediated inhibition of CASR or clearance of cells responded much more strongly than chief cells to increased CASR-expressing or PTH-producing cells could induce compen- ambient calcium concentration. Baseline PTH production under satory hyperplasia in the gland leading to adenoma development. normocalcemic conditions (1.25 mM) in individual patient- Alternatively, the P3 TILs could be responding to inflamma- matched pairs of chief and oxyphil cells was significantly higher tory mediators, tumor-specific antigens, or senescence-associated in oxyphil cells relative to the chief cells. The enhanced calcium proteins released upon oncogene-induced senescence and may sensitivity and increased PTH production we observed in oxyphil be participating in tumor-clearance activities. Further study will cells relative to chief cells is consistent with a recent report that be necessary to verify the functions of these immune cells and to used immunohistochemistry to compare oxyphil and chief cell discover any potential relationships to parathyroid disease. gene expression in hyperplastic parathyroid tissue from patients Our results are at odds with the assumption of clonal expan- with hyperparathyroidism secondary to chronic kidney disease sion as the sole tumorigenic mechanism in parathyroid adeno- (23). In their study, Ritter et al. (23) demonstrated elevated mas. The detection of 5 polyclonal adenomas out of 14 tested expression of parathyroid-relevant genes including PTH and certainly suggests that monoclonal expansion and multi- or poly- PTHrP in oxyphil cells relative to chief cells. In conjunction with clonal proliferation may coexist as alternative mechanisms in the this finding, our data support the notion that oxyphil cells are origin of primary parathyroid adenomas. Seminal work (8, 9) important functional participants in the calcium-sensing and established that monoclonal proliferation is commonly found in

Shi et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 primary hyperparathyroidism, yet subsequent studies using whole demonstration of polyclonality within primary adenomas raises tissue (10, 11) have shown that polyclonal origin can occur in a the possibility that such expansions may occur during an un- significant subset of primary parathyroid adenomas. These recognized prodrome phase analogous to the abnormal physio- studies were limited by the potential for contamination in samples logical contexts that drive the development of secondary not specifically enriched for individual cell populations. In our parathyroid hyperplasia. Further work will be necessary to test study, we used multiple independent methods of tissue sampling these concepts in expanded patient cohorts to define the re- and two independent methods of clonality determination to es- lationship of these findings to the clinical presentation and tablish definitively that both polyclonal and monoclonal mecha- disease course of PHPT and to improve our understanding of nisms can give rise to primary parathyroid adenomas. Differences the causes of primary and secondary parathyroid neoplasia. in reported frequencies of polyclonal versus monoclonal origin may be attributable to the lack of definitive pathological criteria Materials and Methods for distinguishing adenoma from hyperplasia, sample size limi- Patients and Samples. Informed consent was sought from patients scheduled tations in the number of parathyroid glands tested, as well as the to undergo parathyroidectomy for PHPT. Consenting patients donated application of different clonality assays with variations in meth- resected parathyroid tissue for study (Protocol Number 00007056, approved odology and the ability to exclude contaminating nonadenoma by the Institutional Review Board of the Duke University Medical Center). All elements (11). Recently, a new model has been described in which of the patients were diagnosed with sporadic, nonfamilial PHPT on the basis tumor heterogeneity arises through recruitment of proximate of standard clinical and biochemical parameters, and all had a single para- untransformed but hyperplastic cells into aggregation chimeras thyroid tumor removed at curative surgery. Viable dispersed parathyroid cells collected from the surgical specimens were preparatively sorted by induced by a monoclonal population of tumor-initiating cells (28). flow cytometry prior to further analysis. Cellular morphology was eval- It is possible that a similar mechanism may contribute to the uated by electron and light microscopy. Marker gene expression was origin of the polyclonal adenomas observed in our study. Future detected by immunofluorescence, immunohistochemistry, quantitative studies will be required to analyze the genomic composition reverse-transcriptase PCR, and analytical flow cytometry. Calcium re- of parathyroid adenomas at single-cell resolution to determine sponsiveness was detected via kinetic analysis of Fluo-4 AM fluorescence whether polyclonal tumors are constitutively transformed or whether intensity. Clonality status was determined by PCR assays detecting X-chromosome a monoclonal initiating component has induced hyperplastic expan- inactivation at the highly polymorphic HUMARA and PGK loci. A full de- sion of neighboring untransformed parathyroid cells. scription of the experimental procedures may be found in the SI Materials The current study demonstrates that flow cytometry can be and Methods. used to identify, analyze, and recover functionally distinct sub- ACKNOWLEDGMENTS. We thank Dr. Sara Miller and Mr. Phillip Christopher populations of live cells from human parathyroidectomy speci- (Duke Electron Microscopy Core Facility) for excellent technical assistance in mens. Using this approach, we show that parathyroid oxyphil and the preparation and analysis of the EM data, Dr. Michael Cook and Ms. Lynn chief cells derived from parathyroid adenomas display clear Martinek (Duke Flow Cytometry Shared Resource, Duke Cancer Institute) for differences in calcium responsiveness and PTH secretion. The advice and assistance, Ms. Cynthia Webb for assistance with parathyroid tissue procurement and banking, and Dr. Yasheng Gao (Duke Light Micros- differential activity and relative abundance of these distinct cell copy Core Facility) for assistance with the live-cell imaging studies. This work types could contribute to heterogeneity in clinical presentation, was supported by National Institutes of Health Grant R01 DK088188-03 (to therapeutic response, and outcome in PHPT. Importantly, the J.A.O. and J.K.).

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