Recurrent novel HMGA2-NCOR2 fusions characterize a subset of keratin-positive giant cell-rich soft tissue tumors.

Giant cell tumors of soft tissue (GCT-ST) are rare low-grade neoplasms that were at one time thought to represent the soft tissue counterparts of GCT of bone (GCT-B) but are now known to lack the H3F3 mutations characteristic of osseous GCT. We present six distinctive giant cell-rich soft tissue neoplasms that expressed keratins and carried a recurrent HMGA2-NCOR2 gene fusion. Patients were five females and one male aged 14-60 years (median, 29). All presented with superficial (subcutaneous) masses that were removed by conservative marginal (3) or wide (2) local excision. The tumors originated in the upper extremity (2), lower extremity (2), head/neck (1), and trunk (1). Five patients with follow-up (median, 21 months; range, 14-168) remained disease-free. Grossly, all tumors were well-demarcated but not encapsulated with variable lobulation. Histologically, they were composed of bland plump epithelioid or ovoid to spindled mononuclear cells admixed with evenly distributed multinucleated osteoclast-type giant cells. Foci of stromal hemorrhage and hemosiderin were seen in all cases. The mitotic activity ranged from 2 to 14/10 high power fields (median: 10). Foci of necrosis and vascular invasion were seen in one case each. The mononuclear cells were immunoreactive with the AE1/AE3 keratin cocktail and less frequently/less diffusely for K7 and K19 but lacked expression of other lineage-associated markers. RNA-based next-generation sequencing revealed an HMGA2-NCOR2 fusion in all tumors. None of the keratin-negative conventional GCT-ST showed the HMGA2-NCOR2 fusion (0/7). Metaplastic bone (4/9) and SATB2 expression (3/4) were frequent in keratin-negative conventional GCT-ST but were lacking in keratin-positive HMGA2-NCOR2 fusion-positive tumors. The distinctive immunophenotype and genotype of these tumors strongly suggest that they represent a discrete entity, differing from conventional GCT-ST and other osteoclast-rich morphologic mimics. Their natural history appears favorable, although a study of additional cases and longer follow-up are warranted.

Making sense of giant cell lesions of the jaws (GCLJ): lessons learned from next-generation sequencing.

Next-generation sequencing has revealed mutations in several bone-related lesions and was recently used to uncover the genetic basis of giant cell lesions of the jaws (GCLJ). Consistent with their benign nature, GCLJ show a low tumor mutation burden. They also harbor somatic, heterozygous, mutually exclusive mutations in TRPV4, KRAS, or FGFR1. These signature mutations occur only in a subset of lesional cells, suggesting the existence of a 'landscaping effect', with mutant cells inducing abnormal accumulation of non-mutant cells that form the tumor mass. Osteoclast-rich lesions with histological similarities to GCLJ can occur in the jaws sporadically or in association with genetically inherited syndromes. Based on recent results, the pathogenesis of a subgroup of sporadic GCLJ seems closely related to non-ossifying fibroma of long bones, with both lesions sharing MAPK pathway-activating mutations. In this review, we extrapolate from these recent findings to contextualize GCLJ genetics and we highlight the therapeutic implications of this new information. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prognostic role of PD-L1 and immune-related gene expression profiles in giant cell tumors of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastatic tumor, with a relatively unpredictable clinical course. A retrospective series of 46 GCTB and a control group of 24 aneurysmal bone cysts (ABC) were selected with the aim of investigating the PD-L1 expression levels and immune-related gene expression profile, in correlation with clinicopathological features. PD-L1 and Ki67 were immunohistochemically tested in each case. Furthermore, comprehensive molecular analyses were carried out using NanoString technology and nCounter PanCancer Immune Profiling Panel, and the gene expression results were correlated with clinicopathological characteristics. PD-L1 expression was observed in 13/46 (28.3%) GCTB (and in 1/24, 4.2%, control ABC, only) and associated with a shorter disease free interval according to univariate analysis. Moreover, in PD-L1-positive lesions, three genes (CD27, CD6 and IL10) were significantly upregulated (p < 0.01), while two were downregulated (LCK and TLR8, showing borderline significance, p = 0.06). Interestingly, these genes can be related to maturation and immune tolerance of bone tissue microenvironment, suggesting a more immature/anergic phenotype of giant cell tumors. Our findings suggest that PD-L1 immunoreactivity may help to select GCTB patients with a higher risk of recurrence who could potentially benefit from immune checkpoint blockade.

ZNF687 Mutations in Severe Paget Disease of Bone Associated with Giant Cell Tumor.

Paget disease of bone (PDB) is a skeletal disorder characterized by focal abnormalities of bone remodeling, which result in enlarged and deformed bones in one or more regions of the skeleton. In some cases, the pagetic tissue undergoes neoplastic transformation, resulting in osteosarcoma and, less frequently, in giant cell tumor of bone (GCT). We performed whole-exome sequencing in a large family with 14 PDB-affected members, four of whom developed GCT at multiple pagetic skeletal sites, and we identified the c.2810C>G (p.Pro937Arg) missense mutation in the zinc finger protein 687 gene (ZNF687). The mutation precisely co-segregated with the clinical phenotype in all affected family members. The sequencing of seven unrelated individuals with GCT associated with PDB (GCT/PDB) identified the same mutation in all individuals, unravelling a founder effect. ZNF687 is highly expressed during osteoclastogenesis and osteoblastogenesis and is dramatically upregulated in the tumor tissue of individuals with GCT/PDB. Interestingly, our preliminary findings showed that ZNF687, indicated as a target gene of the NFkB transcription factor by ChIP-seq analysis, is also upregulated in the peripheral blood of PDB-affected individuals with (n = 5) or without (n = 6) mutations in SQSTM1, encouraging additional studies to investigate its potential role as a biomarker of PDB risk.

ZNF687 mutations are frequently found in pagetic patients from South Italy: implication in the pathogenesis of Paget's disease of bone.

Paget's disease of bone (PDB) is a skeletal disorder whose molecular basis is not fully elucidated. However, 10% of patients show a familial PDB and 35% of them carry mutations in the SQSTM1 gene. We recently reported a founder mutation (p.Pro937Arg) in the ZNF687 gene, underlying PDB complicated by giant cell tumor (GCT/PDB) and rarely occurring in PDB patients without neoplastic degeneration. Since 80% of Italian GCT/PDB patients derive from Avellino, we hypothesized that ZNF687 mutation rate was higher in this region than elsewhere. Interestingly, our molecular analysis on 30 PDB patients showed that 33% hosted ZNF687 mutations, with the p.Pro937Arg identified in 8 familial cases. Two novel ZNF687 mutations (p.Pro665Leu and p.Gln784Glu) were detected in 2 sporadic patients. Only 2 subjects were positive for the p.Pro392Leu mutation in SQSTM1. ZNF687-mutated patients showed a severe PDB, with a remarkable number of affected sites. in vitro studies revealed that the ZNF687-mutant osteoclasts appeared as giant sized with up to 150 nuclei, never described in PDB. Finally, we also confirmed the causality of the p.Pro937Arg mutation in 4 additional GCT/PDB cases deriving from the same geographic area, indicating that PDB and GCT/PDB represent 2 sides of the same coin.

Giant cell tumor of soft tissue is genetically distinct from its bone counterpart.

Giant cell tumors of bone are locally aggressive bone neoplasms with a predilection for young adults. Histologically, they are composed of histiocytoid to spindled mononuclear cells, admixed with numerous large osteoclastic giant cells. Giant cell tumors of soft tissue are rare tumors that bear striking histological resemblance to giant cell tumors of bone and might be regarded as a soft tissue analog thereof. Point mutations of the H3F3A gene (coding for a histone H3.3 protein) at the Gly34 codon, mostly G34W resulting from a GGG>TGG nucleotide change, have recently been identified in a vast majority of giant cell tumors of bone. To delineate the possible pathogenic linkage between both tumor types, we analyzed the H3F3A genotypes in a series of 15 giant cell tumors of soft tissue by Sanger sequencing and found no mutation in any case. We then sequenced cognate histone H3 genes with an identical nucleotide sequence ('GGG') at the codon Gly34, including the H3F3B, H3F3C, HIST2H3A, HIST2H3C, and HIST2H3D genes, and no somatic mutation was detected. These results reveal that giant cell tumors of soft tissue are probably genetically distinct from their bone counterparts and suggest that they might be pathogenically unrelated. Given the prominence of non-neoplastic cells in these tumors and the limitations of the current study, however, analyses using more sensitive techniques might be required to solve the issue.

Phenotypic and molecular differences between giant-cell tumour of soft tissue and its bone counterpart.

AIMS: Giant-cell tumour (GCT) of soft tissue (GCT-ST) is a primary soft tissue neoplasm that is histologically similar to GCT of bone (GCT-B). Recently, it has been reported that >90% of GCT-Bs have a driver mutation in the H3F3A gene. As the relationship between GCT-ST and GCT-B is unclear, the aim of this study was to compare a series of GCT-STs and GCT-Bs with regard to the presence of H3F3A mutations and several immunophenotypic markers. METHODS AND RESULTS: Eight GCT-STs were retrieved from our institutional archives. Fifteen GCT-Bs served as controls. Direct sequencing for H3F3A mutations in coding regions between codons 1 and 42, including the hotspot codons (28, 35, and 37), was performed on DNA extracted from formalin-fixed paraffin-embedded tissue. Tumours were studied immunohistochemically for the expression of CD14, CD33, RANKL, RANK, p63, and the osteoblastic markers SATB2 and RUNX2. None of the seven GCT-STs that could be analysed showed H3F3A mutations, whereas 14 GCT-Bs (93.3%) were mutated. All eight GCT-STs were positive for RANK and RUNX2, whereas RANKL and SATB2 were detected in only two cases (25%). CD14 was detected only in mononuclear elements, whereas multinucleated giant cells and a proportion of the mononuclear population expressed CD33. Few mononuclear cells of GCT-STs expressed p63. In comparison, GCT-Bs showed higher expression of p63 (14 of 15 cases with >50% of positive mononuclear cells), RANKL, and SATB2, whereas CD14, CD33, RANK and RUNX2 were similarly expressed. CONCLUSIONS: Although GCT-ST and GCT-B are similar in histological appearance, our results indicate that they are immunophenotypically and genetically distinct.

Xanthogranulomatous Epithelial Tumors and Keratin-Positive Giant Cell Rich Tumors of Soft Tissue and Bone: Two Sides of the Same Coin.

Xanthogranulomatous epithelial tumor is a recently described soft tissue tumor characterized by subcutaneous location, partial encapsulation, a xanthogranulomatous inflammatory cell infiltrate, and keratin-positive mononuclear cells. It shares some morphologic features with keratin-positive, giant cell-rich soft tissue tumors. Both have recently been shown to harbor HMGA2::NCOR2 fusions. The relationship between these tumors and their differential diagnosis with other osteoclast-containing soft tissue tumors is discussed.

Subependymal nodules and giant cell tumours in tuberous sclerosis complex patients: prevalence on MRI in relation to gene mutation.

PURPOSE: The purpose of this study was to estimate the association among the presence of subependymal nodules (SENs), subependymal giant cell tumours (SGCTs) and gene mutation in tuberous sclerosis complex (TSC) patients. METHODS: Clinical records and images of 81 TSC patients were retrospectively reviewed by two neuroradiologists in consensus. All patients were assessed for gene mutations and were categorized as TSC1 or TSC2 mutation carriers, or no-mutations-identified (NMI) patients. They underwent a brain magnetic resonance imaging (MRI) using 0.1 mmol/kg of gadobutrol. Any enhancing SEN ≥ 1 cm and placed near the foramen of Monro was considered SGCT. Two MRI follow-up exams for each patient with SGCT were evaluated to assess tumour growth using Wilcoxon and chi-squared tests. RESULTS: Of 81 patients, 44 (54%) were TSC2 mutation carriers, 20 (25%) TSC1 and 17 (21%) NMI. Nine (11%) had a unilateral and three (4%) a bilateral SGCT. Fifty of 81 patients (62%) showed at least one SEN. None of the 31 patients without SEN showed SGCTs, whilst 12 (24%) of the 50 patients with at least one SEN showed SGCTs (p = 0.003). The association between the presence of SGCT or SEN and gene mutation was not significant (p = 0.251 and p = 0.187, respectively). At follow-up, the median SGCT diameter increased from 14 to 15 mm (p = 0.017), whilst the median SGCT volume increased from 589 to 791 mm(3) (p = 0.006). CONCLUSIONS: TSC patients with SENs are more likely to present with SGCT than those without SENs, in particular for TSC2 mutation carriers. The SGCT growth rate may be missed if based on the diameter instead of on the volume.

Recurrent Fusion of the Genes for High-mobility Group AT-hook 2 (HMGA2) and Nuclear Receptor Co-repressor 2 (NCOR2) in Osteoclastic Giant Cell-rich Tumors of Bone.

BACKGROUND/AIM: Chimeras involving the high-mobility group AT-hook 2 gene (HMGA2 in 12q14.3) have been found in lipomas and other benign mesenchymal tumors. We report here a fusion of HMGA2 with the nuclear receptor co-repressor 2 gene (NCOR2 in 12q24.31) repeatedly found in tumors of bone and the first cytogenetic investigation of this fusion. MATERIALS AND METHODS: Six osteoclastic giant cell-rich tumors were investigated using G-banding, RNA sequencing, reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization. RESULTS: Four tumors had structural chromosomal aberrations of 12q. The pathogenic variant c.103_104GG>AT (p.Gly35Met) in the H3.3 histone A gene was found in a tumor without 12q aberration. In-frame HMGA2-NCOR2 fusion transcripts were found in all tumors. In two cases, the presence of an HMGA2-NCOR2 fusion gene was confirmed by FISH on metaphase spreads. CONCLUSION: Our results demonstrate that a subset of osteoclastic giant cell-rich tumors of bone are characterized by an HMGA2-NCOR2 fusion gene.

Intramuscular Tenosynovial Giant Cell Tumor Harboring a Novel CSF1-CD96 Fusion Transcript.

Tenosynovial giant cell tumors typically arise in the synovium of joints, bursae, or tendon sheaths. They may occur in an intra- or extra-articular location and can be divided into localized and diffuse types. The neoplastic nature of the lesion has been supported by a recurrent CSF1 gene rearrangement in a small subset of lesional cells, of which the most common fusion partner is COL6A3. Herein, we report a case of intramuscular localized tenosynovial giant cell tumor harboring a novel CSF1-CD96 fusion transcript, thus expanding the molecular profile of this tumor.

Silencing of the UCHL1 gene in giant cell tumors of bone.

Giant cell tumors are heterogeneous tumors consisting of multinucleated giant cells, fibroblast-like stromal cells and mononuclear histiocytes. The stromal cells have been identified as the neoplastic cell population, which promotes the recruitment of histiocytes and the formation of giant cells. Strong evidence exists that these cells develop from mesenchymal stem cells (MSCs) but little is known about the molecular mechanisms involved in GCT tumorigenesis. The aim of our study was the identification of cancer-related genes differentially expressed in GCTs compared to MSCs in order to identify possible targets for aberrant promoter methylation, which may contribute to MSC transformation and GCT development. Gene expression of 440 cancer-related genes was analyzed by DNA microarrays in GCT stromal cells and bone marrow-derived MSCs (BMSCs) isolated from the same patient (n = 3) to avoid interindividual variations. Differential expression was identified for 14 genes, which could be confirmed by quantitative PCR in further 21 GCT and 10 BMSC samples. The most pronounced difference in gene expression was detected for UCHL1, an important regulator of the ubiquitin proteasome pathway. Methylation-specific PCR and bisulfite sequencing revealed a strong methylation of the CpG island covering the UCHL1 promoter in GCT stromal cells, whereas methylation was completely absent in BMSCs. UCHL1 expression in stromal cells could be restored by the methylation inhibitor 5-aza-dC. These data demonstrate that the UCHL1 gene is inactivated in GCTs but not in MSCs, suggesting a possible role of UCHL1 in MSC transformation and GCT development.

Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors.

Podoplanin is known as a lymphatic marker because its expression is detected in lymphatic but not vascular endothelium. Podoplanin is also expressed in several normal tissues including osteocytes or osteoblasts. A systematic examination of the podoplanin expression was conducted in normal skeletal tissues and some bone tumor cell lines, and the diagnostic value determined in primary bone tumors. Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7). Therefore, podoplanin may be a sensitive immunohistochemical marker of osteogenic and chondrogenic bone tumors.

What is new about the molecular genetics in matrix-producing soft tissue tumors? -The contributions to pathogenetic understanding and diagnostic classification.

Soft tissue tumors encompass a wide variety of mesenchymal neoplasms exhibiting diverse clinical, pathologic, and molecular features. Among these, osteoid and/or chondroid matrix deposition in some soft tissue tumors represents a noticeable characteristic. Unlike matrices present in bone tumors where they likely reveal the respective cells of origin (i.e., osteoblastic or chondroblastic precursors), those existing in soft tissue tumors more often denote a metaplastic phenomenon and reflect the diversity of differentiation these tumors can display. While many soft tissue tumor types can occasionally harbor metaplastic bone or cartilage as an incidental component or heterologous differentiation, in some other tumor types, the production of these matrices is a frequent and distinctive, if not diagnostic, feature. This review focuses on the latter tumor types where emerging immunohistochemical and molecular evidence has significantly improved our understanding of their respective pathogenesis and histopathological spectra. These tumor types include ossifying fibromyxoid tumor, phosphaturic mesenchymal tumor, synovial chondromatosis, soft tissue chondroma, calcifying aponeurotic fibroma, giant cell tumor of soft tissue, myositis ossificans and related diseases, mesenchymal chondrosarcoma, and extraskeletal osteosarcoma.

Immunohistochemical and biogenetic features of diffuse-type tenosynovial giant cell tumors: the potential roles of cyclin A, P53, and deletion of 15q in sarcomatous transformation.

PURPOSE: Diffuse-type tenosynovial giant cell tumor (D-TSGCT) is an aggressive proliferation of synovial-like mononuclear cells with inflammatory infiltrates. Despite the COL6A3-CSF1 gene fusion discovered in benign lesions, molecular aberrations of malignant D-TSGCTs remain unidentified. EXPERIMENTAL DESIGN: We used fluorescent in situ hybridization and in situ hybridization to evaluate CSF1 translocation and mRNA expression in six malignant D-TSGCTs, which were further immunohistochemically compared with 24 benign cases for cell cycle regulators involving G(1) phase and G(1)-S transition. Comparative genomic hybridization, real-time reverse transcription-PCR, and a combination of laser microdissection and sequencing were adopted to assess chromosomal imbalances, cyclin A expression, and TP53 gene, respectively. RESULTS: Five of six malignant D-TSGCTs displayed CSF1 mRNA expression by in situ hybridization, despite only one having CSF1 translocation. Cyclin A (P = 0.008) and P53 (P < 0.001) could distinguish malignant from benign lesions without overlaps in labeling indices. Cyclin A transcripts were more abundant in malignant D-TSGCTs (P < 0.001). All malignant cases revealed a wild-type TP53 gene, which was validated by an antibody specifically against wild-type P53 protein. Chromosomal imbalances were only detected in malignant D-TSGCTs, with DNA losses predominating over gains. Notably, -15q was recurrently identified in five malignant D-TSGCTs, four of which showed a minimal overlapping deletion at 15q22-24. CONCLUSIONS: Deregulated CFS1 overexpression is frequent in malignant D-TSGCTs. The sarcomatous transformation involves aberrations of cyclin A, P53, and chromosome arm 15q. Cyclin A mRNA is up-regulated in malignant D-TSGCTs. Non-random losses at 15q22-24 suggest candidate tumor suppressor gene(s) in this region. However, P53 overexpression is likely caused by alternative mechanisms rather than mutations in hotspot exons.

Genomic gains of COL1A1-PDFGB occur in the histologic evolution of giant cell fibroblastoma into dermatofibrosarcoma protuberans.

Giant cell fibroblastoma (GCF) is a subcutaneous mesenchymal neoplasm characterized by the chromosomal t(17;22), which results in the formation of the fusion gene COL1A1-PDGFB. This same fusion gene is also seen in the supernumerary ring chromosome of dermatofibrosarcoma protuberans (DFSP). Several studies have addressed the molecular genetics of DFSP but molecular cytogenetic characterization of individual areas and cell components in pure GCF and GCF/DFSP hybrids have not been performed. Herein, we studied the frequency and genomic copy number of COL1A1-PDGFB in pure GCF and GCF/DFSP hybrids, and identified the molecular cytogenetic signatures in individual cells in each component. Four pure GCF and nine GCF/DFSP hybrids were studied. All tumors exhibited classical histological features and CD34 expression. COL1A1 and PDGFB rearrangements were evaluated by fluorescence in situ hybridization (FISH) using probes for COL1A1 and PDGFB on paraffin-embedded thin tissue sections. All GCF and GCF/DFSP hybrids showed unbalanced rearrangements of COL1A1-PDGFB at the molecular cytogenetic level. Genomic gains of COL1A1-PDGFB were found predominantly in the DFSP component of GCF/DFSP hybrids but in none of the pure GCF, suggesting that these gains are associated with the histologic evolution of GCF into DFSP. The molecular cytogenetic abnormalities were found not only in the spindle/stellated cells but also in individual nuclei of the multinucleated giant cells, suggesting that these cells may result from the fusion of individual neoplastic cells.

Cytogenetic Single-Institution Analysis: 101 Consecutive Cases of Soft-Tissue Tumors of the Extremities.

We summarize the results and clinical usefulness of cytogenetic analysis that is routinely performed for musculoskeletal tumors. We performed cytogenetic analysis and traditional histologic evaluation on 101 (51 malignant/ 50 benign) consecutive tumors that were surgically excised. The successful culture rate for cytogenetic analysis was 86% (87/101). Fifty-four percent (25/46) of clearly malignant tumors that were successfully cultured demonstrated significant clonal abnormalities. Fifty-one percent (21/41) of benign tumors that were cultured had significant cytogenetic clonal aberrations. Increased cellular ploidy (> 50 chromosomes/cell) was demonstrated in 14/46 malignant and 1/41 benign tumors that were successfully cultured. Hyperploidy was highly correlated with malignancy (p < 0.001); the only "benign" tumor was a multiply recurrent and giant cell, demonstrating histologic changes consistent with early sarcomatous transformation. As expected, cytogenetic abnormalities frequently occurred in malignant tumors. Surprisingly, almost half of the benign tumors had significant clonal cytogenetic aberrations. Consistent findings of extra chromosomes 5 and 7 in samples of pigmented villonodular synovitis strongly favored a neoplastic origin for this condition. Although the presence or absence of cytogenetic aberrations cannot be used to determine malignant potential, increased cellular ploidy is highly indicative of malignancy. Modern molecular genetics have become more popular, but cytogenetic analysis can be useful for classifying the malignant potential of recurrent and difficult to diagnose tumors of the musculoskeletal system.

Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction.

Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system.