Positive Pregnancy Tests in a Nongravid, Premenopausal Woman Due to hcg β-chain Production by Multiple Myeloma

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1 Anatomic Pathology / HCG β-chain PRODUCTION BY MULTIPLE MYELOMA Positive Pregnancy Tests in a Nongravid, Premenopausal Woman Due to hcg β-chain Production by Multiple Myeloma Stephen P. Slone, MD, 1 Zakaria Ahmed, PhD, 1 Laurence A. Cole, PhD, 2 Ronald J. Elin, MD, PhD, 1 Alvin W. Martin, MD, 1 Roger H. Herzig, MD, 3 Geoffrey P. Herzig, MD, 3 and James J. Miller, PhD 1 Key Words: Immunohistochemistry; Multiple myeloma; hcg; Pregnancy; Heterophil antibody; Paraprotein; HAMA; Interference; Immunoassay DOI: /7BRDM5A17XN3QFKM Abstract Positive pregnancy test results occurred in a nongravid, premenopausal woman while she was receiving chemotherapy for multiple myeloma. We tested 2 hypotheses to account for this finding: (1) Heterophil antibodies caused positive interference in the immunoassays. (2) Genuine human chorionic gonadotropin (hcg) originated from a nonsyncytiotrophoblastic source. Paraprotein was eliminated as a source of positive interference because 3 different instruments with unique capture and signal antibodies gave similar results (83, 90, and 97 miu/ml [83, 90, and 97 IU/L]). Human antimouse antibodies (HAMAs) were unlikely to cause positive interference because immunoreactivity was maintained after serum was treated to neutralize heterophil antibodies. Immunoassays performed after gel filtration of serum indicated that immunoreactivity was due to genuine hcg. The high-molecular-weight fraction (heterophil antibody) had 6 miu/ml (6 IU/L) of hcg. The lowmolecular-weight fraction (hcg) had 86 miu/ml (86 IU/L) of hcg. Immunohistochemical stains revealed that myeloma cells expressed immunoreactive hcg. Hence, multiple myeloma caused positive pregnancy test results in a nongravid woman. Human chorionic gonadotropin (hcg) is a glycoprotein produced by syncytiotrophoblastic cells of the placental villi. hcg maintains the function of the corpus luteum during early pregnancy until the luteoplacental shift of progesterone production occurs. Other physiologic functions of hcg are to promote steroidogenesis in the fetoplacental unit and to promote gonadal development in the fetus. 1 hcg is a dimer composed of nonidentical α and β subunits joined noncovalently by charge interactions. 2 hcg belongs to a family of dimeric glycoproteins including human luteinizing, follicle-stimulating, and thyroid-stimulating hormones. 3 Members of the family are related by α subunits with identical amino acid sequences. 4 The β subunits are unique and confer biologic and immunologic specificity to each hormone. 3,4 hcg is an unusual glycoprotein because a large proportion of its molecular weight (approximately 30%) is due to sugar side chains. 5 There are 4 N-linked carbohydrate side chains, 2 on the α subunit and 2 on the β subunit, and 4 O-linked carbohydrate side chains, all on the β subunit. 2,5 A predictable consequence of combining 2 dissimilar subunits with 8 total sugar side chains is structural heterogeneity. In addition to intact hcg (the predominant isoform of hcg in serum during normal pregnancy), there are 5 key variants: (1) hyperglycosylated hcg, (2) nicked hcg, (3) hcg missing the β-subunit C-terminal peptide, (4) free β subunit, and (5) nicked free β subunit. 2 Unlike placental trophoblasts, which produce intact hcg, 1 a host of tumors produce variant hcg. 3,6-12 Trophoblastic neoplasms and nonseminomatous testicular tumors are associated with high serum levels of hcg-related molecules (>1 ng/ml). 3 Low serum levels of variant hcg (<1 ng/ml) occasionally occur in patients with nontrophoblastic tumors 3 ; sites of origin include the stomach, liver, 108 Am J Clin Pathol 2005;124: DOI: /7BRDM5A17XN3QFKM

2 Anatomic Pathology / CASE REPORT lung, urinary bladder, endocrine and exocrine pancreas, colon, cervix, and endometrium. 3,8-12 Multiple myeloma joins the list of malignant tumors that make variant hcg. 3,6-12 Positive pregnancy test results in a nongravid, premenopausal woman with multiple myeloma prompted clinical investigation that led to an unexpected finding: the hcg originated from myeloma cells. The following case report documents this unusual phenomenon and, to the best of our knowledge, is the first such account to appear in the literature. Case Report A 38-year-old African American woman sought care in October 2000 with a 2-month history of worsening low back pain. Clinical evaluation established a diagnosis of multiple myeloma (IgG κ) and a compression fracture of the L2 vertebral body. She was treated with 5 cycles of VAD (vincristine, doxorubicin [Adriamycin], dexamethasone) ending in April Initial response to chemotherapy was good, with decline of the serum M protein from 5 g/dl to 1.6 g/dl. In July 2001, the patient was treated with high-dose melphalan with autologous stem cell support. Thalidomide was given after hematopoietic recovery. She remained progression-free until September 2002 when she was treated with involved-field radiation for bone pain. In January 2003, she was enrolled in a randomized phase 3 study using dexamethasone with or without oblimersen sodium (Genasense), a bcl-2 antisense oligonucleotide (G3139) for patients with relapsed or refractory multiple myeloma. She went off-study in April 2003 because of progressive disease and was treated with high-dose pulse dexamethasone and thalidomide. Although resolution of hypercalcemia occurred initially, destruction of bone was relentless, and the serum concentration of M protein continued to rise. Monthly pregnancy tests were done because the patient was premenopausal and being treated with thalidomide. In April 2003, qualitative test results for serum and urine β-hcg were positive. The initial quantitative immunoassay for β- hcg revealed a serum concentration of 90 miu/ml (90 IU/L). Twenty-four hours later, the serum concentration had decreased to 84 miu/ml (84 IU/L), a finding inconsistent with pregnancy. Obstetric consultation confirmed that the patient was not pregnant. A neoplasm typically associated with β-hcg production, such as a germ cell tumor, could not be detected. After clinical laboratory testing established that the β-hcg was genuine and not a false-positive result, attention turned to the source of hormone production. Evaluation of immunohistochemically stained sections of the bone marrow biopsy specimen revealed that myeloma cells expressed the β- chain of hcg. Hence, β-hcg produced by myeloma cells caused positive pregnancy test results in a nongravid woman. The patient died of disease 3 years after diagnosis. Materials and Methods Formalin-fixed, paraffin-embedded bone marrow biopsy specimens (before treatment and after disease progression) were retrieved from the pathology files of the University of Louisville Hospital and Norton Hospital, Louisville, KY. Light Microscopy Sections of tissue (5 µm thick) were stained with H&E for histomorphologic evaluation. Immunohistochemical Analysis Sections of tissue (5 µm thick) were stained immunohistochemically with antibodies against hcg α and β chains. Heatinduced epitope retrieval was performed by immersing sections of tissue in citrate buffer (ph 6.0) and steam heating for 15 minutes in an electric pressure cooker. Clone M (hcg α; Fitzgerald Industries International, Concord, MA) was diluted 1:2,000. A polyclonal antibody to hcg β (DAKO, Carpinteria, CA) was diluted 1:1,000. Immunohistochemical stains were performed on a DAKO Autostainer. By using appropriate positive and negative control samples, sections of tissue were stained immunohistochemically via the labeled streptavidin-biotin method. A 5-minute incubation with 3% hydrogen peroxide was followed by a 25-minute incubation with primary antibody, a 20-minute incubation with biotinylated secondary antibody, and a 20-minute incubation with labeled streptavidin-biotin. Antibody localization was accomplished by a 10-minute incubation in diaminobenzidine. Measurement of Serum hcg Instruments used to measure serum hcg included the Elecsys (Roche, Indianapolis, IN), the Access (Beckman Coulter, Fullerton, CA), and the Centaur (Dade Behring, Deerfield, IL). Free β-subunit and hyperglycosylated hcg were quantified using the DPC (Diagnostics Products, Los Angeles, CA) Immulite platform and the Nichols Institute Diagnostics (San Clemente, CA) invasive trophoblast antigen assay, respectively. Blockade of Antibodies That Cause Interference The Heterophile Blocking Tube (Scantibodies, Santee, CA) and addition of mouse serum to patient serum were used to block antibodies that could cause interference in the immunoassays. Gel Filtration of Serum Gel filtration of serum was performed with the Bio-Spin (Bio-Rad Laboratories, Hercules, CA) apparatus with a 40-kd molecular weight cutoff. Am J Clin Pathol 2005;124: DOI: /7BRDM5A17XN3QFKM 109

3 Slone et al / HCG β-chain PRODUCTION BY MULTIPLE MYELOMA Results Light Microscopy Neoplastic plasma cells accounted for 80% of the marrow cellularity in the bone marrow biopsy specimen obtained to establish the diagnosis of multiple myeloma, ie, before chemotherapy Image 1. The plasma cells did not have anaplastic cytomorphologic features. There was no amyloid. Neoplastic plasma cells accounted for 90% of the marrow cellularity in the bone marrow biopsy specimen procured 3 years after diagnosis, ie, after disease progression Image 2. About 20% to 30% of the plasma cells had anaplastic cytomorphologic features. Amyloid was absent. Immunohistochemical Analysis Before treatment, about 30% to 40% of myeloma cells stained positively for the hcg β chain; immunoreactivity localized to the cell membrane and Golgi zone, and staining intensity varied from weak to moderate to strong Image 3. After disease progression, virtually 100% of myeloma cells stained positively for the hcg β chain; immunoreactivity localized to the cell membrane and Golgi zone, and staining intensity varied from moderate to strong Image 4. Neoplastic plasma cells in the bone marrow biopsy specimens obtained before treatment and after disease progression were devoid of hcg α-chain staining. Serum hcg Levels Concentrations of serum hcg measured on the Elecsys (90 miu/ml [90 IU/L]), Access (97 miu/ml [97 IU/L]), and Centaur (83 miu/ml [83 IU/L]) were similar. Blockade of Antibodies That Interfere With Immunoassays Immunoreactivity for hcg persisted after use of the Heterophile Blocking Tube and addition of mouse serum to the patient serum sample. Gel Filtration of Serum Immunoassays performed after gel filtration of the patient s serum sample revealed an hcg concentration of 6 miu/ml (6 IU/L) in the high-molecular-weight fraction (>40 kd) and an hcg concentration of 86 miu/ml (86 IU/L) in the low-molecular-weight fraction (<40 kd). Fractionation of Total hcg There was 86% free β subunit; regular hcg and hyperglycosylated hcg constituted the remainder. Discussion Clinical investigation of the patient s elevated hcg level was methodical. Likely reasons for hcg elevation were eliminated before esoteric causes were considered. Although pregnancy was an obvious consideration in a premenopausal woman, the patient maintained that pregnancy was impossible because bone pain precluded sexual intercourse. An obstetric consultation confirmed the patient s assertion that she was not pregnant. Although multiple primary malignant tumors occur in a subset of patients with cancer, 13,14 subsequent clinical studies failed to detect a second neoplasm that produced hcg, such as an ovarian germ cell tumor. Image 1 Plasma cells accounted for 80% of the cellularity in the pretreatment bone marrow (H&E, 400). Image 2 After three years of disease progression, plasma cells accounted for 90% of the bone marrow cellularity. Some of the myeloma cells had anaplastic features (arrows) (H&E, 400). 110 Am J Clin Pathol 2005;124: DOI: /7BRDM5A17XN3QFKM

4 Anatomic Pathology / CASE REPORT After probable causes for hcg elevation were excluded, the possibility of a false-positive result was studied. Paraproteins (monoclonal immunoglobulins) are known to cause interference (positive or negative) in various laboratory assays One report indicated that an IgG κ paraprotein acted like a heterophil antibody and produced negative interference in a 2-site (sandwich) immunoassay for thyrotropin. 20 However, positive interference by the patient s paraprotein was highly unlikely because 3 different instruments used to measure serum hcg gave essentially the same result. It would be virtually impossible for a paraprotein to produce the same magnitude of positive interference in each instrument because the capture and signal antibodies are different in each automated immunometric assay. Human antimouse antibodies (HAMAs) were excluded as a source of positive interference because of failure to quench immunoreactivity with a heterophilic antibody blocking agent (Heterophile Blocking Tube) or addition of mouse serum to the patient s serum sample. HAMAs are present in the plasma of more than 10% of patients and occur idiopathically or after treatment with mouse monoclonal antibodies. 21 The interference caused by heterophilic antibodies can be positive or negative. 21 Immunoassays performed after gel filtration of serum provided direct evidence that immunoreactivity was due to real hcg and not to positive interference by paraprotein or HAMAs. The high-molecular-weight fraction (>40 kd), where the paraprotein and HAMAs would be expected, had 6 miu/ml (6 IU/L) of hcg, and the low-molecularweight fraction (<40 kd), where hcg (molecular weight, 34 kd) would be expected, had 86 miu/ml (86 IU/L) of hcg. Once a false-positive result was ruled out, attention turned to the source of hcg production. The possibility of hcg production by multiple myeloma was considered because the patient was not pregnant and the clinical workup failed to detect a second malignant neoplasm. Immunohistochemical stains provided incontrovertible evidence that the hcg originated from the patient s plasma cell dyscrasia. The myeloma cells displayed intense membranous immunoreactivity for the β chain of hcg but not the α chain. The immunohistochemical findings correlated with the results from the immunoassays, which revealed that 86% of the serum hcg was due to free β-subunit and not intact hcg. To the best of our knowledge, this is the first reported case of hcg production by multiple myeloma. There was at least a relative increase, if not an absolute increase, in the number of hcg β-chain positive myeloma cells in the bone marrow after 3 years of disease progression. This finding suggests that the clone of myeloma cells positive for expression of the hcg β chain was more resistant to chemotherapy than the negative clone. Although hcg β-chain positive myeloma cells were present in the pretreatment bone marrow, the amount of variant hcg secreted into the serum at that time was insufficient to cause positive pregnancy test results. With disease progression, 2 possible mechanisms could account for the positive pregnancy test results: (1) An alteration in cellular physiology caused an increased rate of hcg β-chain secretion by myeloma cells. (2) There was an absolute increase in the number of myeloma cells that secreted variant hcg. Reports in the literature indicate that a variety of gonadal and nongonadal neoplasms produce hcg-related molecules. 3,6-12 Unlike placental trophoblasts, which produce intact hcg, 1 the neoplastic trophoblasts in choriocarcinomas and invasive moles make hyperglycosylated hcg, 2,5 whereas nontrophoblastic cancers make free β subunit Multiple myeloma Image 3 About 30% to 40% of myeloma cells stained positively for the β chain of human chorionic gonadotropin before chemotherapy (immunoperoxidase, 400). Image 4 After disease progression, virtually 100% of myeloma cells stained intensely positive for the β chain of human chorionic gonadotropin (immunoperoxidase, 400). Am J Clin Pathol 2005;124: DOI: /7BRDM5A17XN3QFKM 111

5 Slone et al / HCG β-chain PRODUCTION BY MULTIPLE MYELOMA joins the list of malignant neoplasms that produce hcg free β subunit. However, the hcg concentration in the patient s serum (6 ng/ml [6 IU/L]) was much higher than the level that occurs in patients with cancer, which is typically much less than 1 ng/ml. 3 Although a variety of tumors produce hcg, the hormone serves as a useful marker for cancer screening only when titers are high, such as in nonseminomatous testicular tumors. 3 Despite the high concentration in the patient s serum, hcg was unsuitable for cancer screening because a high titer of free β subunit was absent in the pretreatment serum samples and hcg was heretofore unrecognized as a tumor marker for myeloma. Further study will be required to determine what proportion of patients with myeloma have elevated hcg titers at the time of diagnosis. However, we maintain that hcg production by multiple myeloma occurs infrequently because, until now, there have been no reports of this phenomenon in the literature. Serendipity accounted for discovery of the hcg-producing multiple myeloma described in this case report. Periodic pregnancy tests were done because the patient was a premenopausal woman who was being treated with chemotherapy, including thalidomide, an antiangiogenic agent notorious for its teratogenic side effects. Positive pregnancy test results in a nongravid woman prompted an exhaustive clinical evaluation to determine the source of hcg production. After the unexpected discovery of hcg production by multiple myeloma, the unusually high serum titers of the hormone allowed oncologists to monitor disease progression with a marker other than M protein. The variety of hcg-related molecules produced by tumors, including this case of multiple myeloma, emphasizes the importance of selecting an immunoassay that detects not only intact hcg, but also the variant forms of the molecule. This will decrease false-negative results when screening for cancer and will ensure accuracy when hcg is used to monitor the disease course. From the 1 Department of Pathology and Laboratory Medicine and 3 Department of Medicine, Division of Blood and Marrow Transplantation, School of Medicine, University of Louisville, Louisville, KY; and 2 Department of Obstetrics and Gynecology, University of New Mexico Health Science Center, Albuquerque. Address reprint requests to Dr Slone: Dept of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY 40292; spslon01@gwise.louisville.edu. Acknowledgments: We are grateful to Mary Williams for manuscript preparation and to Kevin Radford for computer assistance. 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