Association between axillary lymph node status and Ki67 labeling index in triple-negative medullary breast carcinoma.

OBJECTIVE: Medullary breast carcinoma is a rare breast carcinoma with good prognosis. Although it has been established that axillary lymph node metastasis is a poor prognostic factor, little is known about the relationship between axillary lymph node metastasis and clinicopathological characteristics of medullary breast carcinoma patients. The aim of this study was to identify factors that predict occurrence of axillary lymph node metastasis in medullary breast carcinoma patients. METHODS: We performed a retrospective study of axillary lymph node status and the relevant clinicopathological characteristics in 49 triple-negative medullary breast carcinoma patients with axillary lymph node dissection between November 2004 and July 2011. RESULTS: A total of 49 patients were enrolled in the study. Fourteen patients (28.6%) had axillary lymph node metastasis that was confirmed pathologically. Axillary lymph node metastasis was not associated with age, menopausal status, primary tumor size or its location, the degree of inflammation within the tumor or mitotic count. However, we found a statistically significant association between axillary lymph node metastasis and Ki67 labeling index in primary tumors (P < 0.001). CONCLUSIONS: There is a positive association between Ki67 labeling index in the primary tumor and axillary lymph node metastasis in triple-negative medullary breast carcinoma patients.

Localization of medullary thyroid carcinoma after surgery using (11)C-methionine PET/CT: comparison with (18)F-FDG PET/CT.

Tumor localization is difficult in patients with medullary thyroid carcinoma (MTC) that have persistent hypercalcitoninemia after thyroidectomy. In this study, the (11)C-methionine positron emission tomography/computed tomography (PET/CT) was compared with the (18)F-FDG PET/CT for diagnostic sensitivity in detecting residual or metastatic disease. (11)C-methionine PET/CT and (18)F-FDG PET/CT were performed on 16 consecutive patients with MTC that had persistent hypercalcitoninemia after surgery in this prospective, single-center study. Patient- and lesion-based analyses were performed using a composite reference standard which was the sum of the lesions confirmed by all combined modalities, including neck ultrasonography (US) with or without fine needle aspiration cytology, CT, bone scan, magnetic resonance imaging (MRI), and surgery. By patient-based analysis, the sensitivities of (11)C-methionine PET/CT and (18)F-FDG PET/CT were both 63%. By lesion-based analysis, the sensitivity of (11)C-methionine PET/CT was similar to (18)F-FDG PET/CT (73% vs. 80%). Excluding hepatic lesions, which could not be detected because of physiological uptake of methionine by the liver, the sensitivity of (11)C-methionine PET/CT was better than (18)F-FDG PET/CT especially for detecting cervical lymph node lesions; however, it was not superior to US. All patients with serum calcitonin levels ≥370 pg/mL showed uptake by (11)C-methionine PET/CT and (18)F-FDG PET/CT. This preliminary data showed that despite its similar sensitivity to (18)F-FDG PET/CT for detecting residual or metastatic MTC, (11)C-methionine PET/CT provided minimal additional information compared to combined (18)F-FDG PET/CT and neck US.

Medullary thyroid carcinoma with poor differentiation and atypical radiographic pattern of metastasis.

A diagnosis of sporadic medullary thyroid carcinoma (MTC) is complicated. On first diagnosis it may present with distant metastasis. There has been inconsistency regarding metastatic MTC tissue expression of calcitonin, its tumor marker. Adding to the difficulty is the fact that the radiographic pattern of pulmonary metastasis from MTC may vary substantially among patients. Herein is reported the case of a 73-year-old man who presented with two ill-defined pulmonary opacities, clinically resembling primary lung carcinoma. MTC was diagnosed on histopathology of tissue obtained from a total thyroidectomy. The pulmonary biopsy specimens were confirmed to be MTC metastasis on positive immunohistochemical staining of chromogranin-A and synaptophysin, even though only a few cells were stained for calcitonin. To the authors' knowledge this is the first reported case of MTC presenting initially as complex pulmonary metastasis with weakened expression of calcitonin in the metastatic lesion.

How basal are triple-negative breast cancers?

The basal molecular subtype of breast cancer (BC) is defined by the mRNA expression pattern of an intrinsic approximately 500-gene set. It is the most homogeneous subtype in transcriptional terms, and one of the most aggressive in prognostic terms. Clinical trials testing new systemic therapeutic strategies have been launched in basal BCs. Although no proof of evidence has yet been reported, basal tumors are currently assimilated to and selected as triple-negative (TN) BCs in these trials because of their frequent immunohistochemical (IHC) negativity for hormone and ERBB2 receptors. Here, we have assessed the degrees of correlation and of homogeneity of the TN phenotype (IHC-based definition) and the basal subtype (gene expression-based definition). We analyzed 172 TN BCs defined by gene expression profile as basal (123 cases) and nonbasal (49 cases). Conversely, 160 tumors were defined as basal by their gene expression profile and included 123 TN and 37 non-TN samples. Uni- and multivariate analyses revealed that TN BCs represent a more heterogeneous group than basal BCs, including basal and nonbasal tumors very different both at the histoclinical and molecular level, notably for mRNA expression of molecules targeted by specific therapies under evaluation in clinical trials. These results call for caution in the interpretation of ongoing trials and selection of patients in future trials. They also warrant the identification of molecular markers for basal BCs more clinically applicable than gene expression profiles.

PET imaging in neuroendocrine tumors: current status and future prospects.

Neuroendocrine tumors (NET) are relatively rare diverse group of tumors that possess several unique characteristics and occur most commonly in the gastrointestinal tract. An important feature of these tumors is that they express somatostatin (SST) receptors, and thus can be successfully targeted with specific radiolabeled SST receptor related compounds. This has led to the introduction of multitude of single photon emission computed tomography (SPECT) and positron emission tomography (PET) tracers, which have significantly improved the ability for imaging these neoplastic lesions with high spatial resolution in the abdomen and elsewhere in the body. The introduction of Gallium-68 labeled radiopharmaceuticals as viable PET agents have added a new dimension to the management of patients with NET by providing high quality images compared with planar or SPECT with single photon emitting preparations. (18)F-labeled DOPA has demonstrated impressive results in differentiating between focal and diffuse disease in hyperinsulinism of the newborn which appears to be of crucial clinical benefit and has altered the management of these patients significantly. With regard to other types of NET, the current experience with such agents is relatively limited and therefore prospective studies are required to further define the role of PET in this complicated group of patients.

A case of multiple endocrine neoplasia type II accompanied by thyroid medullary carcinoma and pheochromocytomas expressing corticotropin-releasing factor and urocortins.

A 38-year-old woman with RET gene mutation presented with tumors in her thyroid and bilateral adrenal glands. I-metaiodobenzylguanidine scintigraphy revealed accumulation of the radioisotope in both adrenal glands. Both plasma adrenaline and noradrenaline levels were elevated. The circadian rhythms for plasma adrenocorticotropic hormone (ACTH) and cortisol levels were disturbed. Plasma ACTH and cortisol levels failed to be suppressed by an overnight dexamethasone test, suggesting autonomic secretion of ACTH and cortisol, although the patient had no typical Cushingoid features, hypertension, or impaired glucose tolerance. Pathological examination showed that these tumors were pheochromocytoma and thyroid medullary carcinoma, respectively, both of which highly expressed corticotropin-releasing factor, urocortin1, and urocortin3. Together with the endocrinological and pathological observations, the patient was diagnosed as multiple endocrine neoplasia type II with corticotropin-releasing factor- and urocortin-producing tumors that stimulated ACTH and glucocorticoid secretion.

Medullary thyroid carcinoma metastatic to the pituitary gland: an unusual site of metastasis.

We present a case of metastatic medullary thyroid carcinoma involving the pituitary gland of a 23-year-old woman with multiple endocrine neoplasia type 2b who presented with diabetes insipidus and visual loss. The diagnostic features, including cytomorphology and immunohistochemistry, used to differentiate pituitary adenoma from metastatic medullary carcinoma are discussed. Pituitary metastases and tumor-to-tumor metastases in this region are also highlighted.

Molecular characterization of the desmoplastic tumor stroma in medullary thyroid carcinoma.

Medullary thyroid carcinomas are aggressive neoplasias that metastasize very early to loco-regional lymph nodes, and tumors with a desmoplastic stromal reaction have a higher incidence of lymph node metastasis. In order to characterize the desmoplastic response in thyroid cancers, we evaluated the expression pattern of three molecular markers of activated fibroblasts/myofibroblasts, namely, fibroblast activation protein alpha (FAPalpha), tenascin-C (Tn-C), and alpha-smooth muscle actin (alpha-SMA), as well as the endothelial markers endoglyx-1, CD34 and CD31 in a series of 28 metastatic and non-metastatic medullary thyroid cancers. Immunohistochemical studies demonstrated that the three fibroblast activation markers (FAPalpha, Tn-C, alpha-SMA) are consistently expressed in the peritumoral and intratumoral stromal compartment of medullary thyroid carcinomas and expression of FAPalpha and Tn-C correlated with the degree of desmoplasia determined histologically (p=0.001 for FAPalpha and p<0.001 for Tn-C). Moreover, the extent of desmoplasia as well as the expression of FAPalpha and Tn-C correlated with the presence of lymph node (LN) metastases (p=0.002, p=0.005 and p=0.002, respectively). No correlation was found between the microvessel density (neoangiogenesis) in the tumor stroma, assessed with the endoglyx-1, CD34 and CD31 markers, and the degree of desmoplasia or incidence of LN metastases. Using a bioinformatics-based search of the BioExpresstrade mark database we found in a series of 48 thyroid cancers a significant correlation between FAPalpha RNA expression and incidence of LN metastases also in papillary cancers. These findings suggest that the link between specific molecular markers of tumor stromal reaction and locoregional metastasis extends from medullary to other thyroid cancer types.

Role of FNA cytology and immunochemistry in the diagnosis and management of medullary thyroid carcinoma: report of six cases and review of the literature.

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine thyroid malignancy. This study retrospectively reviewed 10 fine-needle aspiration samples from six MTC patients. Aspirated specimens were from thyroid (3), cervical lymph nodes (5), left lung (1), and anterior chest wall (1). Cytomorphology consisted predominantly of plasmacytoid cells (3 cases), spindle cells (2 cases), and epithelioid cells (1 case). However, all specimens had a mixture of other cell types and "salt and pepper" chromatin. Only one specimen showed Congo-red-positive amyloid. Calcitonin was expressed in 7/7 specimens. Four patients underwent surgical excision and MTC was confirmed in all four. Follow-up studies included serum calcitonin (3/6 cases) and imaging (2/6 cases). One patient had MTC associated with multiple endocrine neoplasia IIA syndrome and one had familial MTC with a history of MTC in mother. In conclusion, the cytomorphology of MTC is typical and calcitonin immunostain is a reliable method for confirming primary or metastatic MTC. Early cytological diagnosis of MTC positively impacted patient management. Follow-up with serum calcitonin and imaging is helpful in the early detection of recurrences.

Hormone receptor status and survival of medullary breast cancer patients. A Turkish cohort.

OBJECTIVES: To analyze the relationship between clinical features, hormonal receptor status, and survival in patients who were diagnosed with medullary breast cancer (MBC). Methods: Demographic characteristics, histopathological features, and survival statuses of 201 patients diagnosed with MBC between 1995 and 2015 were retrospectively recorded. Survival analyses were conducted with uni- and multivariate cox regression analysis. RESULTS: Median follow-up time was 54 (4-272) months. Median patient age at the time of diagnosis was 47 years old (26-90). Of the patients, 91.5% were triple negative. Five-year recurrence free survival time (RFS) rate was 87.4% and overalll survival (OS) rate 95.7%. For RFS, progesterone receptor (PR) negativity, atypical histopathological evaluation, absence of lymphovascular invasion, smaller tumor, lower nodal involvement were found to be favourable prognostic factors by univariate analysis (p less than 0.05). The PR negativity and smaller tumor were found to be favourable factors by univariate analysis (p less than 0.05). However, none of these factors were determined as significant independent prognostic factors for OS (p greater than 0.05).  Conclusion: Turkish MBC patients exhibited good prognosis, which was comparable with survival outcomes achieved in the literature. The PR negativity was related to a better RFS and OS rates.

Medullary Carcinoma of the Penis: A Distinctive HPV-related Neoplasm: A Report of 12 Cases.

A third to half of penile invasive squamous cell carcinomas are human papillomavirus (HPV) related. Warty (condylomatous), warty-basaloid, and basaloid carcinomas are the most common subtypes associated with HPV. Less frequent are clear cell and lymphoepithelioma-like carcinomas. Here we report a novel penile tumor associated with HPV. Twelve cases were selected from 1010 penile carcinomas, part of an international HPV detection study conducted at the Institut Català d'Oncologia, Barcelona, Spain. Immunostaining with p16 was performed on all cases, and HPV-mRNA detection was also performed. En bloc full tumor staining was the utilized criteria for positivity of p16. For HPV-DNA detection, whole-tissue section polymerase chain reaction analysis was performed by SPF10-DEIA-LiPA25 (version 1). The patients' ages ranged from 42 to 92 years (average, 71 y). The tumor was most commonly located in the glans. A characteristic microscopic finding was the presence of a moderate to dense tumor-associated inflammatory cell infiltrate composed of neutrophils, lymphocytes, plasma cells, or eosinophils. Tumors grew in large solid sheets, nests, or had a trabecular pattern. Cells were large and poorly differentiated or anaplastic. Keratinization was minimal or absent. Nuclei were large with prominent nucleoli. Mitoses were numerous. Tumor necrosis was common. Deep invasion of the corpora cavernosa was frequent. p16 and HPV-DNA were positive in all cases, whereas mRNA detection was positive in 9 cases only. The prevalent genotype was HPV16 (9 cases, 75%). Other genotypes were HPVs 58, 33, and 66. Medullary carcinomas of the penis are morphologically distinctive HPV-related high-grade neoplasms affecting older individuals. More studies are necessary to delineate the epidemiological, clinical, and molecular features of this unusual penile neoplasm.

Diagnosing colorectal medullary carcinoma: interobserver variability and clinicopathological implications.

Colorectal medullary carcinoma, recognized by the World Health Organization as a distinct histologic subtype, is commonly regarded as a specific entity with an improved prognosis and unique molecular pathogenesis. A fundamental but as yet unaddressed question, however, is whether it can be diagnosed reproducibly. In this study, by analyzing 80 colorectal adenocarcinomas whose dominant growth pattern was solid (thus encompassing medullary carcinoma and its mimics), we provided a detailed description of the morphological spectrum from "classic medullary histology" to nonmedullary poorly differentiated histologies and demonstrated significant overlapping between categories. By assessing a selected subset (n=30) that represented the spectrum of histologies, we showed that the interobserver agreement for diagnosing medullary carcinoma by using 2010 World Health Organization criteria was poor; the κ value among 5 gastrointestinal pathologists was only 0.157 (95% confidence interval, 0.127-0.263; P=.001). When we arbitrarily classified the entire cohort into "classic" and "indeterminate" medullary tumors (group 1, n=19; group 2, n=26, respectively) and nonmedullary poorly differentiated tumors (group 3, n=35), groups 1 and 2 were more likely to exhibit mismatch repair protein deficiency than group 3 (P<.001); however, improved survival could not be detected in either group compared with group 3. Our findings suggest that the diagnosis of medullary carcinoma, as currently applied, may only serve as a morphological descriptor indicating an increased likelihood of mismatch-repair deficiency. Additional evidence including a more objective classification system is needed before medullary carcinoma can be regarded as a distinct entity with prognostic relevance. Until such evidence becomes available, caution should be exercised when making this diagnosis, as well as when comparing results across different studies.

Renal cell carcinoma, unclassified with medullary phenotype: poorly differentiated adenocarcinomas overlapping with renal medullary carcinoma.

Renal medullary carcinoma (RMC) is a highly aggressive renal cell carcinoma arising in the collecting system and requiring careful correlation with status of sickle cell trait. A panel of international experts has recently proposed provisional diagnostic terminology, renal cell carcinoma, unclassified, with medullary phenotype, based on encountering an extraordinarily rare tumor with RMC morphology and immunophenotype in an individual proven not to have a hemoglobinopathy. Herein, we extend this observation to a cohort of 5 such tumors, morphologically similar to RMC, lacking SMARCB1 expression by immunohistochemistry, but each without evidence of a hemoglobinopathy. The tumors arose in 4 men and 1 woman with a mean age of 44 years, occurring in 3 left and 2 right kidneys. Clinically, aggression was apparent with involvement of perinephric adipose tissue in all 5 cases, nodal metastasis in 4 of 5 cases, and death of disease in 4 of 5 cases within 3-27 months. Histologic sections showed poorly differentiated adenocarcinoma, often with solid and nested growth patterns, as well as infiltrative glandular, tubulopapillary, cribriform, or reticular growth. Rhabdoid and sarcomatoid cytomorphology was seen in a subset. All tumors showed PAX8 nuclear positivity and SMARCB1 loss, with OCT3/4 expression in 4 of 5 cases. In summary, this first series of renal cell carcinoma, unclassified, with medullary phenotype documents tumors with morphologic, immunophenotypic, and prognostic features of RMC occurring in individuals without sickle cell trait. Although greater biologic and molecular understanding is needed, the available evidence points to these cases representing a sporadic counterpart to sickle cell trait-associated RMC.

Somatostatin receptor imaging for neuroendocrine tumors.

Tumors and metastases that express the somatostatin receptor subtypes sst2 sst3 or sst5 can be visualized in vivo after injection of radiolabeled octapeptide somatostatin analogs, like (111)In-pentetreotide. (111)In-pentetreotide scintigraphy also allows for more accurate staging of the disease by demonstrating tumor sites, which were not shown by conventional imaging. (111)In-pentetreotide scintigraphy may also detect resectable tumors that would have remained unrecognized using conventional radiological imaging techniques; it may prevent surgery with curative intent in those patients whose tumors have metastasized to a greater extend than could be detected with conventional radiological imaging and it may be used to select patients for treatment with the currently available octapeptide somatostatin analogs or with tumor targeted radioactive treatment with radiolabelled somatostatin analogs. (111)In-pentetreotide scintigraphy has also been used to select patients with pituitary tumors for medical treatment with octapeptide analogs, but its clinical usefulness for this purpose seems to be limited. It further allows scar tissue to be differentiated from tumor recurrence after the pituitary surgery or radiotherapy. However, a large variety of lesions in and around the pituitary region also express somatostatin receptors and, therefore, can be visualized by (111)In-pentetreotide scintigraphy.

Medullary carcinoma in the colorectum: a systematic review and meta-analysis.

Medullary carcinoma (MC) is a very rare variant of colorectal carcinoma (CRC). Its clinicopathologic findings are not fully elucidated. The aim of this study was to investigate the clinicopathological characteristics of MC in the colorectum through a systematic review and meta-analysis. The meta-analysis examined the incidence, age, sex, site, mismatch repair deficiency (MMRd), MMR protein expression, ARID1A expression, BRAF(V600E) mutation, KRAS mutation, and survival rate of MC. The 21469 CRCs included 462 MCs in 16 eligible studies, representing an estimated incidence of MC of 0.027 (95% confidence interval [CI] 26 0.016-0.045). MC frequently occurred in female patients and in the right colon. Lymph node metastasis of MC was significantly lower than that of poorly differentiated adenocarcinoma/undifferentiated adenocarcinoma (PDA/UDA). In addition, MC had a higher MMRd rate (0.892, 95% CI 0.758-0.956), higher BRAF(V600E) mutation rate (0.652, 95% CI 0.143-0.954) and lower KRAS mutation rate (0.171, 95% CI 0.065-0.378) than PDA/UDA and conventional adenocarcinoma. Patients with MC had significantly better overall survival rate compared to patients with PDA/UDA (hazard ratio 0.441, 95% CI 0.262-0.742). However, there was no significant difference of overall survival rate between MC and conventional adenocarcinoma patients. MC predominantly occurred in females and in the right colon, and had different molecular characteristics and behaviors compared to PDA/UDA and conventional adenocarcinoma.

Sporadic medullary carcinoma of the colon: a clinicopathologic comparison with nonhereditary poorly differentiated enteric-type adenocarcinoma and neuroendocrine colorectal carcinoma.

We studied 68 sporadic colorectal carcinomas (CRCs) with medullary features (MCRCs) and compared them with 35 poorly differentiated purely "enteric" CRCs (ECRCs) and 15 purely neuroendocrine carcinomas (NECs) of grades II and III, all in patients lacking a family history of CRC. Potential clinicopathologic differences between the study groups were assessed. MCRCs were significantly more common in the ascending colon than were ECRCs, but there was no significant dissimilarity to NECs. ECRCs occurred more often in the rectosigmoid than MCRCs or NECs. MCRCs arose in older patients, and a marked sex difference also was noted. Despite an infiltrative growth pattern, MCRC was less likely than ECRC to manifest with stage III or IV disease, but there was no stage-related difference from NECs. Although the histologic images of MCRCs were evocative of neuroendocrine differentiation, chromogranin positivity and synaptophysin reactivity in that group did not differ meaningfully from that of ECRCs but was dissimilar to the 100% labeling of NECs. p53 immunolabeling was similar in the 3 tumor groups. Follow-up data in the study cases showed that 5-year mortality was 40% (27/68) for MCRC, 59% (19/32) for ECRC, and 93% (14/15) for NEC. Medullary CRC seems to be a distinct clinicopathologic variant of CRC, which does not have a neuroendocrine lineage. The biologic behavior of MCRC was better than that of ECRC or NEC.

Expression of adhesion molecules and epithelial-mesenchymal transition factors in medullary carcinoma of the colorectum.

Medullary carcinoma (MC) of the colorectum is known as a rare variant with favorable prognosis despite its poorly differentiated morphology. The mechanism of its favorable behavior has been unclear. Here, we compared the expressions of adhesion molecules and epithelial-mesenchymal transition (EMT)-related proteins in the central portion and invasive front between 43 MCs and 30 poorly differentiated adenocarcinomas (PDAs). The expressions of membranous E-cadherin (P < .0001), ß-catenin (P < .0001) and claudin 1 (P = .0036) were significantly preserved in the invasive front of the MCs compared to those in the invasive front of the PDAs. E-cadherin membranous expression was also significantly preserved in the central portion of the MCs (P = .0178). Nuclear ß-catenin expression in both the central portion (P = .0463) and invasive front (P = .0346) of the MCs was significantly less frequent compared to that in the PDAs. Snail (P = .0035) and Twist1 (P = .0463) expressions in the invasive front of the MCs were significantly less frequent compared to the PDAs, suggesting that the EMT phenomenon may occur rarely in colorectal MC. Reduced membranous E-cadherin expression in the MC central portion was significantly correlated with poor clinical outcome (P = .0086). Our immunohistochemical results indicate that preserved adhesion molecule protein and less frequent expression of EMT-related transcription factors in the invasive front contribute to the favorable prognosis of colorectal MCs. We suggest that a reduced expression of E-cadherin in the central portion might be a good biomarker for an unfavorable prognosis in cases of MC.

C-Cells and their Associated Lesions and Conditions: A Pathologists Perspective.

This paper updates the histopathology and cytopathology of thyroid tumors and proliferations derived from the para-follicular or C cells. Beginning with an historical over view, including the recognition of medullary thyroid carcinoma as a distinct histologic entity, its relationship to the hormone, calcitonin, (which was discovered in the same decade) and to thyroid C cells, medullary carcinoma and its variants are reviewed. The molecular biology of the tumors and the associated mutations in the tumors (somatic mutations) are discussed. Additionally the genetic features (germline mutations) including familial clusters and associations with other endocrine and neuroendocrine lesions are reviewed. Screening for the tumor and its precursors is included with a review of the latest American Thyroid Association guidelines for treatment as well as timing and approach to surgery. Tabular data of specific germline mutations and their relationships to tumor virulence, and prognosis are illustrated. Precursor and early C cell lesions such as C-cell hyperplasia and micro-medullary carcinoma are discussed. Difficulties and controversies in the definition of C-cell proliferations which are neoplastic and those which are "reactive" are reviewed. The entity of medullary microcarcinoma or medullary microcarcinoma is illustrated and the distinction between C cell nodules and microcarcinoma is defined using the latest available criteria. Finally the latest approved chemotherapeutic agents and their results in metastatic medullary thyroid carcinoma are included.

Pituitary adenylate cyclase-activating polypeptide receptors of types I and II and glucagon-like peptide-I receptors are expressed in the rat medullary carcinoma of the thyroid cell line 6/23.

The expression of the messenger RNAs coding for glucagon-like peptide-I (GLP-I) receptor, VIP receptor, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptor as well as the expression of the receptor proteins were demonstrated in the rat medullary carcinoma of thyroid cell line 6/23 by the following experiments: 1) RNA extraction, reverse transcriptase, and polymerase chain reaction with specific primers; 2) binding of the radiolabeled ligands [125I]GLP-I-(7-36)-NH2, [125I]PACAP-(1-27), and [125I]VIP and inhibition by, respectively, unlabeled GLP-I-(7-36)-NH2, PACAP-(1-27), and VIP; and 3) study of adenylate cyclase activation by the peptides and selective inhibition of the VIP/PACAP response by the antagonist [D-Phe2]VIP. Besides the highly selective GLP-I receptor, PACAP receptors of types I and II were present on the cell line and coupled to adenylate cyclase. PACAP stimulated the adenylate cyclase through type I and II receptors, whereas VIP interacted with type II receptors only. Messenger RNA analysis indicated that at least three splice variants of the PACAP type I receptor may be expressed in 6/23 cells.