Suzanne Jordan

The Role of the Aryl Hydrocarbon Receptor-Interacting Protein Gene in Familial and Sporadic Pituitary Adenomas

CONTEXT Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

Number of CD4+ Cells and Location of Forkhead Box Protein P3–Positive Cells in Diagnostic Follicular Lymphoma Tissue Microarrays Correlates With Outcome

PURPOSE To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance. PATIENTS AND METHODS Immunohistochemistry was used to study numbers and location of cells staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25). RESULTS CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location. CONCLUSION This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.

Integrin expression in breast cancer cytology: a novel predictor ofaxillary metastasis

The integrins are heterodimeric transmembrane receptors of varying alpha and beta subunits that modulate cell adhesion to each other and to the extracellular matrix. Loss of integrin expression on primary breast cancer frozen sections measured by immunohistochemistry may be related to the presence of axillary metastasis. The clinical application of this finding would be increased if integrin expression could also be shown to be reliably measured on breast cancer cells obtained by fine needle aspiration cytology. Axillary operations may be planned as a single stage procedure from outpatients, and neoadjuvant therapy protocols may be developed without surgery to the axilla. Expression of the alpha 1, alpha 2, alpha 3, alpha 6, alpha v, beta 1, beta 3 and beta 5 integrin subunits were measured by immunohistochemistry and immunocytochemistry in 58 patients. Integrin measurement by both these methods were found to be closely associated using the kappa-test. Loss of expression of the alpha 1, alpha 2, alpha 3, alpha 6, alpha v, beta 1 and beta 5 integrin subunits measured by cytology and histology were each related to positive nodal status (chi(2) test). Measuring integrin expression on cytology is of clinical value and may prove to have prognostic significance.

Reduced expression of the growth hormone and type 1 insulin‐like growth factor receptors in human somatotroph tumours and an analysis of possible mutations of the growth hormone receptor

objective Clinical acromegaly is characterized by elevated GH secretion in the presence of high circulating IGF‐I levels. We hypothesized that the physiological IGF‐I/GH negative feedback loop may be reset in somatotroph adenomas, specifically in terms of the level of expression of these receptors or mutations of the GH receptor (GH‐R) in such tumours.

Increased levels of insulin-like growth factor binding protein-2 in sera and tumours from patients with colonic neoplasia with and without acromegaly.

OBJECTIVE Patients with acromegaly are at increased risk of developing colorectal carcinoma and premalignant tubulovillous adenoma. The pathogenesis of these neoplasms could involve a stimulatory effect of serum growth factors on colonic epithelial cell proliferation. The aim of this study was to evaluate changes in (1) serum IGF‐I, IGF‐II, IGFBP‐3 and IGFBP‐2 and (2) changes in local expression of IGFBPs and p53 in colonic epithelium in patients with colonic neoplasia with and without acromegaly.

Oncogene-induced senescence in pituitary adenomas and carcinomas.

OBJECTIVEThe model of ‘oncogene-induced senescence’ (OIS), resulting in cell-proliferation arrest, has recently been suggested as a possible explanation for the non-progression of pituitary tumours to malignancy. The aim of the study was to compare the expression of β-galactosidase as a molecular marker of OIS, and p21/p16 as additional markers involved in mediating OIS, in pituitary adenomas, carcinomas and normal pituitary tissue.DESIGNWe performed: a) semi-quantitative immunohistochemistry (β-galactosidase, p16, p21) in 41 pituitary adenomas [(11 GH-secreting, 9 PRL-secreting, 10 ACTH-secreting, 11 non-functioning (NFPAs)], 6 carcinomas (3 multihormonal: PRL/ACTH/GH, PRL/ACTH, PRL/GH/FSH; 1 non-functioning; 2 ACTH-secreting) and 7 normal pituitary tissues; b) quantitative PCR of mRNA (p16 and p21) in 6 GH-secreting, 6 NFPAs and 6 normal pituitary tissues.RESULTSβ-galactosidase was significantly increased in GH-secreting tumours (P=0.002), NFPAs (P=0.04), macroadenomas (P=0.03) and carcinomas (P=0.02), as compared to normal pituitary tissue. We found that p16 expression was significantly lower in all tumours (both adenomas and carcinomas) probably secondary to reduced transcription, at least for NFPAs; p21 showed a different biological behaviour, implying that p21 and p16 may play different roles in the senescence of each individual type of adenoma.CONCLUSIONSβ-galactosidase was significantly over-expressed in GH-secreting and NFPAs, and unexpectedly also in carcinomas. We speculate that the senescence pathway, which may explain the rarity of malignant progression to carcinomas in GH-secreting and NFPAs, might not be universal but cell-type specific.

p16 Immunostaining Allows for Accurate Subclassification of Vulvar Squamous Cell Carcinoma Into HPV-Associated and HPV-Independent Cases.

The aim of this study was to compare morphologic assessment and p16 immunohistochemistry (IHC) in the determination of human papilloma virus (HPV) status in vulvar squamous cell carcinoma (VSCC). A total of 201 invasive VSCC cases were classified as “HPV-associated” when warty/basaloid VSCC or high-grade squamous intraepithelial lesion (vulvar intraepithelial neoplasia 2/3) was observed, or “HPV-independent” in the presence of well-differentiated keratinizing invasive SCC or differentiated vulvar intraepithelial neoplasia. For p16 IHC, strong nuclear and cytoplasmic staining of all cells in at least the lowermost third of the epithelium was scored as positive. All cases with discrepant HPV predictions by hematoxylin and eosin morphology versus p16 IHC were further analyzed by polymerase chain reaction for HPV DNA. On the basis of hematoxylin and eosin morphologic assessment, 50/201 tumors showed features suggestive of HPV-associated, and 47 of those showed p16 immunoreactivity (94% concordance). Of the 146 cases considered HPV-independent based on hematoxylin and eosin, 115 (79%) showed negative p16 immunostaining. Thus 83% (162/196) concordance between morphologic assessment and p16 IHC was observed, overall. In 34 cases, where morphologic assessment and p16 IHC did not agree, HPV polymerase chain reaction agreed with p16 IHC in 32/34 (94%). The sensitivity and specificity of p16 IHC in classification of VSCC as HPV-independent or HPV-associated was 100% and 98.4%, respectively. Morphologic assessment and p16 IHC are concordant in classifying VSCC as HPV-independent or HPV-associated in a majority of cases (83%). Most of the discrepant cases are p16-positive well-differentiated keratinizing VSCC, and HPV polymerase chain reaction supports classification of a large majority of these (94%) as HPV-associated. p16 IHC is validated as an accurate surrogate marker for determination of HPV status in VSCC.

HPV-independent Differentiated Vulvar Intraepithelial Neoplasia (dVIN) is Associated with an Aggressive Clinical Course

Differentiated vulvar intrapeithelial neoplasia (dVIN) is an human papillomavirus (HPV)–independent precursor of squamous cell carcinoma (SCC), and the aim of this study was to better characterize its natural history. Cases of dVIN were identified from the pathology archives. Outcomes of patients with dVIN only, without associated invasive SCC, were compared with a cohort of patients with high-grade squamous intraepithelial lesion [HSIL(VIN3)]. Eighteen patients diagnosed with dVIN with adjacent invasive SCC (SCC/dVIN) and 7 patients with dVIN only, without invasive carcinoma, were identified. Mean age in both cohorts was 75 yr. All lesions but 1 were unifocal. In 35% of SCC/dVIN cases the surgical resection margins were positive for SCC, with 75% and 60% having margins positive for dVIN in the SCC/dVIN and dVIN-only cohorts, respectively. In total, 23/25 women with dVIN only or dVIN/SCC, for whom there was follow-up information, experienced either progression to or recurrence of invasive SCC, respectively, at a median of 1.1 yr, including all but 1 case of dVIN only, where the median time of progression to invasive SCC was 1.9 yr. A total of 22/25 women died of disease with a median overall survival of 3.4 yr. The outcome (i.e. progression to invasive carcinoma) of patients with dVIN only was significantly worse than that of a comparison group of 18 patients with HSIL(VIN3) (progression-free survival log-rank, P<0.001; disease-specific survival, P=0.04; overall survival, P=0.01). Six of 7 patients with dVIN only developed invasive carcinoma on follow-up, compared with 0 of 18 patients with HSIL(VIN3). The diagnosis of dVIN indicates the presence of a high-risk human papillomavirus–negative precursor of invasive SCC. These patients are likely to progress to invasive carcinoma over a relatively short period, at which point their prognosis is guarded.

Leptin in Pituitary Adenomas–A Novel Paracrine

A growing number of physiological and pathophysiological processes have been shown to be influenced by leptin apart from its first recognised role as a modulator of hypothalamic appetite and weight control centers. We investigated the presence and pattern of distribution of leptin mRNA and the mRNA of the long isoform of the leptin receptor in the normal pituitary and in different types of pituitary adenomas. We also studied leptin secretion from human pituitary tumors in culture, and the in vitro pituitary hormone release following stimulation with human leptin. Leptin mRNA expression was detected at a low level of expression in 50% of tumors but in none of the normal pituitaries. By immunohistochemistry, leptin was present in occasional scattered cells in the normal pituitary and in pituitary tumors. The leptin receptor long isoform was detected in the majority (65%) of pituitary tumors and in all normal pituitaries. It did not segregate with any particular tumor type, and varying levels of expression were detected between the tissues studied. 34% of pituitary adenomas showed leptin release into the incubation media during in vitro culture. Leptin mRNA, the mRNA of the long isoform of the receptor, or in vitro leptin release, did not correlate with tumor type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumors caused stimulation of FSH and α-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. As the co-localisation of ACTH and leptin in corticotroph cells was previously suggested, we investigated whether in vivo ACTH release is accompanied by a simultaneous plasma leptin level rise (i) in peripheral plasma samples after food intake-induced ACTH rise in healthy obese and nonobese individuals and (ii) in petrosal sinus samples after CRH injection in Cushings disease patients. Our data suggest that a rise in ACTH levels is not accompanied by detectable rise in leptin levels in peripheral and in petrosal sinus blood samples. In summary, leptin is synthesized and stored within the pituitary and may modulate other pituitary hormone secretion, although probably do not contribute to plasma leptin level changes. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function.