Humoral Hypercalcemia of Malignancy: The Case for Parathyroid Hormone-Related Protein, with Caveats
Hypercalcemia due to various pathologies is associated with high morbidity and mortality. Both elevated parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) cause hypercalcemia resulting in symptoms of stones (kidney stones), bone loss (bone resorption), and psychotic overtones (depression, anxiety, cognitive dysfunction, insomnia, coma). In cancer patients, elevated PTHrP levels lead to increased osteoclastic bone resorption and hypercalcemia, a condition known as humoral hypercalcemia of malignancy (HHM). Ravinder Singh, Ph.D., Director of the Mayo Clinic Endocrine Laboratory, authored an article in Clinical Laboratory News discussing humoral hypercalcemia of malignancy.
Various malignancies, such as leukemia, lymphoma, and cancers of the lung, breast, head, neck, bladder, and ovaries, secrete PTHrP, which exists in several isoforms, ranging in size from 60 to 173 amino acids. The N terminus and secondary structure of multiple isoforms of PTHrP resemble PTH, allowing PTHrP to bind to the same receptor as PTH. This calcium mobilization is complex, involving G coupled protein receptor and activation of the cyclic adenosine monophosphate pathway (cAMP). This syndrome, called HHM, produces effects similar to that of primary hyperparathyroidism. However, lab test results are crucial for making the differential diagnosis between the two.
In hyperparathyroidism, one would expect an elevated Ca, PTH, and serum 1,25 dihydroxyvitamin D3, as well as hypercholeremic acidosis with a decreased pH confirmed by bicarbonate and chloride lab measurements. In HHM, typical lab values include elevated Ca and PTHrP, decreased PTH, and suppressed serum 1,25 dihydroxyvitamin D3 levels. Given their similar names, physicians might mistakenly order PTHrP when intending to order PTH or vice versa. This confusion, if not recognized, could lead to misdiagnosis, making it incumbent on laboratorians to confirm that the correct test has been ordered if they have any concerns in this regard. Studies have suggested that the most accurate diagnosis of hypercalcemia should involve ruling out primary hyperparathyroidism.
PTH assays are reliable and readily available from various commercial diagnostic manufacturers. The quality and characterization of these tests have evolved over the past few decades. Historically, they were either research use only (RUO) or laboratory-developed test devices and were targeted to PTH fragments, such as N-terminal, C-terminal, mid molecule, or intact molecules. With improved technology, PTH assays are now reliable and standardized. The consensus is that full length PTH/intact PTH assays are more relevant clinically and have reference ranges that maximize sensitivity and specificity to confirm primary hyperparathyroidism.
In contrast, PTHrP testing is in its early stages and requires more research and development to achieve a similar level of clinical utility and standardization. Compared to PTH, PTHrP testing is less common and is performed by only a few reference laboratories. Most PTHrP assays are based on its fragments and results are not comparable among various assays. The Food and Drug Administration has yet to approve a PTHrP assay. Preanalytics also are crucial in PTHrP testing because PTHrP is unstable in plasma, and samples should be collected in tubes containing protease inhibitors and EDTA and separated within 30 minutes.
More on PTHrP Assays
PTHrP is cleaved into N-terminal (1-86) and C-terminal (109-141) peptides, and various attempts have been made to develop assays that target the N-terminal or C-terminal PTHrP peptide. The Nichols Institute of Diagnostics in 1993 first described a sandwich immunoradiometric assay for PTHrP, with a reference range of <0.7 – 2.6 pmol/L. This assay used two antibodies targeting different PTHrP peptides and was shown to be specific for PTHrP, but now has been discontinued. Quest currently offers a C-terminal PTHrP assay with a reference range of 14–27 pg/mL. While C-terminal fragments are more stable and easier to develop an assay for, they also are impacted by renal function, producing higher values and more false-positive results.
In contrast, N-terminal fragments are less influenced by renal function, thereby producing fewer false positive results. For this reason, detection of the N-terminal fragment is considered to be a more useful indicator of HHM.
Mayo Medical Laboratories developed an assay using a sandwich approach to capture the N-terminal and mid region of PTHrP. A similar DSL-8100 PTHrP IRMA (RUO) assay from Beckman is available commercially and also targets the PTHrP sequence 1-86. Esoterix (LabCorp) has improved the sensitivity and precision of this assay by converting to chemiluminescent technology. ARUP Laboratories recently developed a liquid chromatography-mass spectrometry (MS) assay for the C-terminal (103-110) peptide of PTHrP, but it had very poor correlation with immunoassay. Competitive immunoassays have lower specificity, precision, and analytical sensitivity compared to sandwich or MS assays.
At this time, nephrogenous cAMP remains the gold standard as a bio-assay until the PTHrP or other protein assays are able to achieve maximum clinical sensitivity and specificity. Existing PTHrP assays lack clinical sensitivity and specificity for HHM. Without optimal standardized assays, but with potential for PTHrP to be a biomarker for HHM, this is an excellent opportunity for clinical investigators, laboratories, and manufacturers to work together to create a reliable PTHrP test.
Co-Author: Parmpreet Kaur, medical student at the University of Missouri–Kansas City