Laurette Geldenhuys

Scoring system for renal pathology in Fabry disease: report of the International Study Group of Fabry Nephropathy (ISGFN)

BACKGROUND In Fabry nephropathy, alpha-galactosidase deficiency leads to accumulation of glycosphingolipids in all kidney cell types, proteinuria and progressive loss of kidney function. METHODS An international working group of nephrologists from 11 Fabry centres identified adult Fabry patients, and pathologists scored histologic changes on renal biopsies. A standardized scoring system was developed with a modified Delphi technique assessing 59 Fabry nephropathy cases. Each case was scored independently of clinical information by at least three pathologists with an average final score reported. RESULTS We assessed 35 males (mean age 36.4 years) and 24 females (43.9 years) who mostly had clinically mild Fabry nephropathy. The average serum creatinine was 1.3 mg/dl (114.9 micromol/l); estimated glomerular filtration rate was 81.7 ml/min/1.73 m(2) and urine protein to creatinine ratio was 1.08 g/g (122.0 mg/mmol). Males had greater podocyte vacuolization on light microscopy (mean score) and glycosphingolipid inclusions on semi-thin sections than females. Males also had significantly more proximal tubule, peritubular capillary and vascular intimal inclusions. Arteriolar hyalinosis was similar, but females had significantly more arterial hyalinosis. Chronic kidney disease stage correlated with arterial and glomerular sclerosis scores. Significant changes, including segmental and global sclerosis, and interstitial fibrosis were seen even in patients with stage 1-2 chronic kidney disease with minimal proteinuria. CONCLUSIONS The development of a standardized scoring system of both disease-specific lesions, i.e. lipid deposition related, and general lesions of progression, i.e. fibrosis and sclerosis, showed a spectrum of histologic appearances even in early clinical stage of Fabry nephropathy. These findings support the role of kidney biopsy in the baseline evaluation of Fabry nephropathy, even with mild clinical disease. The scoring system will be useful for longitudinal assessment of prognosis and responses to therapy for Fabry nephropathy.

Cyclin D1 polymorphism (G870A) and risk for esophageal adenocarcinoma

To investigate individual susceptibility to gastroesophageal reflux disease, Barrett esophagus, and esophageal adenocarcinoma, the authors studied the frequency of the common G870A polymorphism of CCND1, which encodes cyclin D1, a key cell cycle regulatory protein.

Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene

Purpose: The basic fibroblast growth factor (FGF-2) gene is bidirectionally transcribed to generate overlapping sense and antisense (FGF-AS) mRNAs. FGF-AS has been implicated in the post-transcriptional regulation of FGF-2 expression. The aim of this study was to characterize FGF-2 and FGF-AS in esophageal cancer and to correlate their expression with clinicopathologic findings and outcome. Experimental Design: Reverse transcription-PCR was used to study FGF-2 and FGF-AS mRNA expression (normalized to glyceraldehyde-3-phosphate dehydrogenase) in 48 esophageal cancers relative to matched histologically normal esophageal epithelia (internal control). We used Cox proportional hazards analysis to calculate hazard ratios for recurrence and survival of patients with underexpression relative to the overexpression of FGF-2 and/or FGF-AS. Results: Overexpression of FGF-2 mRNA, by comparison with tumors underexpressing FGF-2, was associated with significantly increased risk for tumor recurrence (hazard ratio, 3.80; 95% confidence interval, 1.64-8.76) and reduced overall survival (hazard ratio, 2.11; 95% confidence interval, 1.0-4.58). When the effects of FGF-2 and FGF-AS were considered simultaneously, the association of FGF-2 mRNA overexpression with recurrence and mortality was even more pronounced, whereas FGF-AS mRNA overexpression was associated with reduced risk for recurrence and improved survival. Conclusions: Overexpression of FGF-2 mRNA is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer and that these risks are reduced in tumors coexpressing the FGF-AS mRNA. These data support the hypothesis that FGF-AS is a novel tumor suppressor that modulates the effect of FGF-2 expression and may have potential clinical application to the development of novel therapeutic strategies.

Inducible nitric oxide synthase, nitrotyrosine and p53 mutations in the molecular pathogenesis of Barrett's esophagus and esophageal adenocarcinoma.

Nitric oxide (NO) has been implicated as a potential causative factor for endogenous p53 mutations in gastrointestinal malignancy. To investigate the role of NO in esophageal adenocarcinoma (EADC), we studied patterns of p53 mutations, expression of inducible nitric oxide synthase (iNOS) and the tissue accumulation of nitrotyrosine (NTS), a stable reaction product of NO and a marker for cellular protein damage, in human premalignant and malignant esophageal epithelia. Tissues were obtained from patients with gastroesophageal reflux disease (GERD)‐induced esophagitis (n = 76), Barretts esophagus (BE; n = 119) and primary EADC (n = 54). DNA sequencing was used to characterize p53 mutations, RT‐PCR to study iNOS mRNA expression, and immunohistochemistry to study NTS. Relative to self‐matched normal epithelia, a progressive increase in iNOS mRNA expression was seen in GERD (30%; 23/76), BE (48%; 57/119), and EADC (63%; 34/54) tissues (P < 0.001). Among patients with EADC, elevated levels of NTS immunoreactivity were more frequent in tumors with p53 mutations (11/21; 52%) compared with tumors with wild‐type p53 (9/33; 27%; P = 0.063), and specifically in tumors with p53 mutations at CpG dinucleotides (10/12; 83%) compared with non‐CpG p53 mutations (1/9; 11%; P = 0.008). The increasing frequency of iNOS (mRNA) overexpression in GERD, BE and EADC supports the hypothesis that an active inflammatory process, most likely a consequence of GERD, underlies molecular progression to EADC. The highly significant association between NTS, reflecting chronic NO‐induced cellular protein damage, and endogenous p53 mutations at CpG dinucleotides, provides further evidence for a molecular link between chronic inflammation and esophageal malignancy.

Histologic examination of fascia used in colporrhaphy

OBJECTIVE To perform a histologic examination of tissue identified as fascia and used during colporrhaphy. METHODS In patients undergoing primary anterior and posterior colporrhaphy, biopsies were taken from three surgically distinct vaginal tissues types: the wall, the “fascia,” and areolar tissue. The biopsies were placed in formalin, identified numerically, and sent to pathology for staining with hematoxylin‐eosin, Masson trichrome for collagen, Movat for elastin, and immunoperoxidase stain for actin in smooth muscle. Simultaneous photographs were taken of the biopsy sites. The histologic diagnosis was compared with the surgical diagnosis. RESULTS A total of 60 samples were taken from five women. The specimens from two of these patients were disqualified. The pathologist made the following histologic diagnosis for each type of surgical specimen: vaginal wall, mucosa and underlying connective tissue; fascia, moderately dense connective tissue with smooth muscle; areolar tissue, loose connective tissue. The histologic appearance of the “fascia” was indistinguishable from the deeper aspects of the vaginal wall. It was composed of the same proportions of smooth muscle, elastin, and collagen. Using the histologic appearance as the “gold standard,” the accuracy of the surgical diagnosis was: “vaginal wall,” 12 of 12 (100%); “fascia,” seven of 12 (58%); and “areolar tissue,” eight of 12 (67%). CONCLUSIONS The surgical “fascia” used during colporrhaphy consists of moderately dense connective tissue with smooth muscle similar to the deep aspects of the vaginal wall, is the same in both the anterior and posterior compartments, and is an artifact of the surgical dissection used to separate the vaginal wall from the underlying organs.

Regulation of CDX2 expression in esophageal adenocarcinoma

Reflux of acidic gastric contents and bile acids into the lower esophagus has been identified to have a central role in esophageal malignancy and is reported to upregulate caudal‐related homologue 2 (CDX2), a regulatory gene involved in embryonic development and axial patterning of the alimentary tract. The aim of this study was to characterize the expression of CDX2 in a well‐defined series of human esophageal tissues, comprising reflux‐induced esophagitis, premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC). To explore potential molecular regulatory mechanisms, we also studied the expression of β‐catenin, SOX9, and CDX2 promoter methylation in esophageal tissues, in addition to the effect of bile acids and nitric oxide (NO) on CDX2 expression in the normal human esophageal cell line Het1A. Relative to matched normal esophageal epithelia, CDX2 was overexpressed in esophagitis (37% for RNA; cytoplasmic immunoreactivity in 48% of tissues), a high proportion (91%) of BE tissues, and in EADC (57% for RNA; cell nuclear immunopositivity in 80%). An association with β‐catenin expression was seen, but not with SOX9 or CDX2 promoter methylation. In Het1A cells, CDX2 was upregulated following exposure to bile acids and NO, alone and in combination. These results further implicate CDX2 and β‐catenin in the molecular pathogenesis of human EADC. The observed synergistic effect of NO on the efficacy of bile acid‐induction of CDX2 suggests a novel role for NO in modulating the development of the Barrett phenotype and esophageal adenocarcinogenesis. Mol. Carcinog.

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product

BackgroundGFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms.ResultsRT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization.ConclusionPrevious findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.

Sensitivity and specificity of the Pap smear for glandular lesions of the cervix and endometrium.

OBJECTIVE To investigate the sensitivity and specificity of the Pap smear for detection of adenocarcinoma in situ of the cervix (AIS), endocervical adenocarcinoma (ECAC) and endometrial adenocarcinoma (EAC) as well as the overall specificity of the smear for detection of glandular lesions in general. STUDY DESIGN Computer records of the laboratory of the QE II Health Sciences Center, Halifax, were searched for patients who had AIS, ECAC or EAC diagnosed on histology between June 1, 1999, and May 31, 2001 and who had had a Pap smear within the preceding year. Computer records were also searched for patients who had a Pap smear result consisting of suspicious or positive for AIS or adenocarcinoma (AC) with subsequent tissue diagnosis during the same time. The histologic and cytologic findings were correlated. RESULTS One hundred percent of patients with AIS, 80% with ECAC and 22% with EAC on histology had positive findings on a Pap smear performed within a year of the histologic diagnosis. One hundred percent of patients with a Pap smear result consisting of suspicious or positive for AIS or AC and follow-up histology had a lesion on histology: 13% AIS, 13% ECAC, 37% EAC, 23% other AC, 10% high grade squamous lesion and 0.3% low grade squamous lesion. CONCLUSION This study confirmed the good overall specificity of the Pap smear for glandular lesions in general. It also confirmed the good sensitivity for glandular lesions of the cervix and the poor sensitivity for glandular lesions of the endometrium. It thus confirmed that the Pap smear is not an effective screening tool for endometrial AC, and that the quest for alternative screening methods should continue.

Selective cyclooxygenase-2 inhibition suppresses basic fibroblast growth factor expression in human esophageal adenocarcinoma

Inhibition of cyclooxygenase (COX)‐2 is reported to suppress growth and induce apoptosis in human esophageal adenocarcinoma (EADC) cells, although the precise biologic mechanism is unclear. In this study we tested the hypothesis that the antitumor activity of COX‐2 inhibitors may involve modulation of basic fibroblast growth factor (FGF‐2), which is overexpressed in EADC. We evaluated the effects of NS‐398, a selective COX‐2 inhibitor, on FGF‐2 expression and proliferation of EADC cell lines that express COX‐2 and those that do not. We also correlated COX‐2 and FGF‐2 expression with clinico‐pathologic findings and outcome in a well‐characterized series of surgically resected EADC tissues. Seg‐1 cells robustly expressed COX‐2 and FGF‐2, whereas Bic‐1 cells expressed neither transcript. FGF‐2 was reduced to undetectable levels in Seg‐1 cells following NS‐398 treatment, but increased within 4 h of drug removal. NS‐398 significantly inhibited the growth of Seg‐1 cells, and this effect was ameliorated by addition of exogenous FGF‐2. In contrast, NS‐398 had no effect on Bic‐1 cell proliferation and FGF‐2 alone had no effect on proliferation of either cell line. NS‐398, or a neutralizing anti‐FGF‐2 antibody, induced apoptosis in Seg‐1 cells, and these effects were inhibited by addition of exogenous FGF‐2. COX‐2 protein was strongly expressed in 46% (10/22) of EADCs, and was associated with a trend towards reduced disease‐free survival. These findings indicate that the antitumor effects of COX‐2 inhibition in EADC cells may be mediated via suppression of FGF‐2, and that COX‐2 may be a clinically relevant molecular marker in the management of human EADC.

Canadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee for High Complexity Testing/Immunohistochemistry: guidelines for the preparation, release, and storage of unstained archived diagnostic tissue sections for immunohistochemistry.

OBJECTIVES Formalin-fixed, paraffin-embedded unstained archived diagnostic tissue sections are frequently exchanged between clinical laboratories for immunohistochemical staining. The manner in which such sections are prepared represents a type of preanalytical variable that must be taken into account given the growing importance of immunohistochemical assays, especially predictive and prognostic tests, in personalized medicine. METHODS Recommendations were derived from review of the literature and expert consensus of the Canadian Association of Pathologists-Association canadienne des pathologists National Standards Committee for High Complexity Testing/Immunohistochemistry. RESULTS Relevant considerations include the type of glass slide on which to mount the unstained sections; the thickness of the tissue sections; the time from slide preparation to testing; the environment, particularly the temperature at which the unstained sections will be maintained prior to testing; the inclusion of on-slide positive control tissue where possible; and whether patient identifier(s) should be included on slide labels. CONCLUSIONS Clear communication between requesting and releasing laboratories will facilitate the proper preparation of unstained sections and also ensure that applicable privacy considerations are addressed.