Detection of global hypermethylation in well-differentiated thyroid neoplasms by immunohistochemical (5-methylcytidine) analysis.

PURPOSE: While global hypomethylation of DNA has been found in several malignancies, studies on thyroid tumours have shown controversial results using different techniques. To help resolve this issue, we assessed methylation status using two different techniques in papillary thyroid carcinomas (PTC) and follicular adenomas (FA) and carcinomas (FTC), comparing adjacent non-neoplastic thyroid tissue. METHODS: A series of 15 FA, 18 FTC and 17 PTC were assessed by: (1) measurement of methylation levels of long interspersed nuclear elements (LINE-1) using a combined bisulfite restriction analysis polymerase chain reaction protocol and (2) immunostaining with an anti-5-methylcytidine antibody that detects methylated DNA regardless of the DNA sequence. Immunostaining was scored by image analysis. RESULTS: Methylation levels of LINE-1 in FA, FTC and PTC were not significantly different from adjacent normal tissue. There was no significant difference in methylation levels of LINE-1 between FA, FTC and PTC (p = 0.44). By immunohistochemical staining for methylation, the 5-methylcytidine score was significantly higher in tumours than in normal tissue counterparts, for FA (p < 0.001), FTC (p = 0.04) and PTC (p = 0.02). PTC showed the highest 5-methylcytidine expression amongst all tumours which was significantly different from FTC (p = 0.015), but not FA (p = 0.09). There was no correlation in methylation level between LINE-1 and 5-methylcytidine scores for each group and overall. CONCLUSIONS: Well-differentiated thyroid neoplasms (FA, FTC and PTC) were not found by two independent methods to undergo global hypomethylation as part of an oncogenic sequence from normal tissue to carcinoma. Instead, hypermethylation was detected in all types of tumours, implying that this epigenetic event may contribute to oncogenic development of thyroid neoplasms (both benign and malignant).

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab.

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA.

Analysis of segmental duplications, mouse genome synteny and recurrent cancer-associated amplicons in human chromosome 6p21-p12.

It has been proposed that regions of microhomology in the human genome could facilitate genomic rearrangements, copy number transitions, and rapid genomic change during tumor progression. To investigate this idea, this study examines the role of repetitive sequence elements, and corresponding syntenic mouse genomic features, in targeting cancer-associated genomic instability of specific regions of the human genome. Automated database-mining algorithms designed to search for frequent copy number transitions and genomic breakpoints were applied to 2 publicly-available online databases and revealed that 6p21-p12 is one of the regions of the human genome most frequently involved in tumor-specific alterations. In these analyses, 6p21-p12 exhibited the highest frequency of genomic amplification in osteosarcomas. Analysis of repetitive elements in regions of homology between human chromosome 6p and the syntenic regions of the mouse genome revealed a strong association between the location of segmental duplications greater than 5 kilobase-pairs and the position of discontinuities at the end of the syntenic region. The presence of clusters of segmental duplications flanking these syntenic regions also correlated with a high frequency of amplification and genomic alteration. Collectively, the experimental findings, in silico analyses, and comparative genomic studies presented here suggest that segmental duplications may facilitate cancer-associated copy number transitions and rearrangements at chromosome 6p21-p12. This process may involve homology-dependent DNA recombination and/or repair, which may also contribute towards the overall plasticity of the human genome.

Identification of pathogens causing invasive fungal rhinosinusitis in surgical biopsies using polymerase chain reaction.

BACKGROUND: Invasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis. METHODS: Tissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced. RESULTS: Polymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species. CONCLUSION: Polymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

Co-amplification of MYCN and a DEAD box gene (DDX1) in primary neuroblastoma.

DEAD box proteins are putative RNA helicases that have been implicated in cellular processes involving alteration of RNA secondary structure, such as translation initiation and splicing. These proteins share eight conserved amino acid motifs, including Asp(D)-Glu-(E)-Ala(A)-Asp(D) which is part of a more extended motif. Recently, we have shown that the novel DDX1 gene containing a DEAD box motif maps to the same chromosome band as MYCN at 2p24 and is co-amplified with MYCN in retinoblastoma cell lines. Here, we show that the DDX1 gene is co-amplified with the MYCN gene in 2 of three neuroblastoma cell lines and that DDX1 RNA levels correlate with DDX1 gene copy number. Since amplification of MYCN is an indicator of poor prognosis in neuroblastoma, it was of interest to determine whether co-amplification with DDX1 occurred in clinical samples of neuroblastoma and whether such a finding carried any additional prognostic significance. We determined the gene copy number of DDX1 in 32 neuroblastoma patient samples (representative of all stages): 13 were MYCN amplified and 19 had normal copy numbers of the MYCN gene. Of the 13 neuroblastomas that were MYCN amplified, seven were also DDX1 amplified. Of the 19 that were not MYCN amplified, none were DDX1 amplified. This is the first example of a gene that is co-amplified with MYCN at a high frequency in neuroblastoma. While there was a trend towards a worse clinical outcome with co-amplification, the numbers were too small to reach significance.

Immunohistochemical detection of P-glycoprotein: prognostic correlation in soft tissue sarcoma of childhood.

Increased expression of P-glycoprotein is associated with multidrug resistance (MDR) in many cell lines. Significant levels of P-glycoprotein have been detected in a number of human tumors. The purpose of this study was to determine whether P-glycoprotein expression correlates with both response to chemotherapy and prognosis in soft tissue sarcoma of childhood. In a retrospective study, biopsy samples from 30 cases of rhabdomyosarcoma (RMS) and undifferentiated sarcoma (US) treated at The Hospital for Sick Children in Toronto were analyzed using a semiquantitative immunohistochemical procedure. P-glycoprotein was detected in nine patients, four at diagnosis, and five at subsequent biopsy. All nine patients relapsed after a clinical response (complete [CR] 55%, partial [PR] 45%) to chemotherapy. Twenty of 21 patients with consistently P-glycoprotein-negative tumors received chemotherapy and they all responded clinically (CR 80%, PR 20%). Only one of these 20 patients has relapsed. The probability of relapse-free survival was significantly different (P less than .000000012) in chemotherapy-treated patients whose tumors contained detectable levels of P-glycoprotein (n = 9), compared with those whose tumors contained no detectable P-glycoprotein (n = 20). The overall probability of survival was also significantly different in these two groups (P less than .0000267). Both relapse-free and overall survivals remained statistically different in the two groups of patients when analyzed by the log-rank method, after adjustment for differences in stages and sites. The incidence of other adverse prognostic factors in the two groups, for example, younger and older ages, low pretreatment lymphocyte counts, large tumors, and unfavorable histology were not significantly different. Thus, detectable P-glycoprotein appears to be an important adverse prognostic factor in children with soft tissue sarcoma, and consistent absence of the protein is associated with a favorable prognosis.

Prognostic significance of age, MYCN oncogene amplification, tumor cell ploidy, and histology in 110 infants with stage D(S) neuroblastoma: the pediatric oncology group experience--a pediatric oncology group study.

PURPOSE: Although a high rate of spontaneous regression is observed in infants with stage D(S) neuroblastoma (NB), survival is not uniform. To determine the prognostic relevance of age at diagnosis, therapy, and tumor biology in infants with stage D(S) NB, we reviewed the Pediatric Oncology Group (POG) experience. PATIENTS AND METHODS: A review of patients diagnosed with stage D(S) NB registered on POG protocols was performed. Survival according to age at diagnosis, treatment, and tumor biology was determined. RESULTS: Between 1987 and 1996, 110 infants with stage D(S) NB had an estimated 3-year survival rate of 85% +/- 4%; survival rate was 71% +/- 8% for infants 2 months of age or younger, and 68% +/- 12%, 44% +/- 33%, and 33% +/- 19% for patients with diploid, MYCN-amplified, and unfavorable histology tumors, respectively. Survival rates were similar for patients who received adjuvant chemotherapy versus those who did not (82% +/- 5% v 93% +/- 6%, respectively; P = .187). Furthermore, there was no statistical difference in survival rate for patients who underwent complete resection of their primary tumor compared with those who underwent partial resection or biopsy only (90% +/- 5% v 78% +/- 7%, respectively; P = .083). CONCLUSION: Our review confirmed that the survival of infants with stage D(S) NB is excellent. However, subsets of patients with poor prognosis can be identified by young age and unfavorable biologic factors. More effective therapy is needed for the group of stage D(S) infants who show unfavorable clinical and biologic features.

Effectiveness of the angiogenesis inhibitor TNP-470 in reducing the growth of human neuroblastoma in nude mice inversely correlates with tumor burden.

Angiogenesis plays an important role in the growth and metastasis of malignant tumors. We have previously reported that in children with neuroblastoma (NB), tumor vascularity directly correlates with metastatic disease, MYCN amplification, and poor outcome. The angiogenesis inhibitor TNP-470 has been shown to reduce the rate of NB growth in rodents with macroscopic tumors without ultimately impacting survival. To investigate whether TNP-470 could more effectively inhibit NB growth in animals with a low tumor burden, we treated 30 nude mice with minimal disease with this angiogenesis inhibitor (supplied by TAP Pharmaceuticals, Inc.). Therapy was initiated before tumors were clinically evident after s.c. inoculation of 5 x 10(6) cells from the MYCN-amplified NB cell line NBL-W-N. TNP-470 was administered 3 days/week, and after 12 weeks of treatment, 53% of the treated mice remained tumor free, whereas 100% of the control mice developed tumors (P < 0.0001). To further assess the relationship between the efficacy of TNP-470 treatment and tumor burden, TNP-470 was also administered s.c., 3 days/week, to mice with clinically evident small (<400 mm3; n = 15) and large (>400 mm3; n = 11) tumors. For animals with small tumors, the mean rate of growth was significantly decreased in the treated mice compared to the controls (P = 0.02). In contrast, there was no difference in the mean rate of tumor growth between animals with large tumors treated with TNP-470 and controls (P = 0.64). Our studies demonstrate that the effectiveness of TNP-470 inversely correlates with tumor burden. We speculate that TNP-470 may most effectively inhibit NB tumor growth in children with a low tumor burden.

Atypical and suspicious categories in fine needle aspiration cytology of the breast: histological and mammographical correlation and clinical significance.

INTRODUCTION: This study aims to correlate fine-needle aspiration specimens diagnosed as C3 (atypical, probably benign) and C4 (suspicious, probably malignant) with histology and mammography, and to evaluate these two cytology categories in terms of diagnostic usefulness and patient management. METHODS: All fine-needle aspiration (FNA) specimens in categories C3 or C4 at the Maharaj Nakorn Chiang Mai Hospital, Thailand between 2000-2004 were reviewed. Results were correlated with available histological and mammographical studies. RESULTS: 148 FNA specimens were identified, comprising 43 category C3 and 105 category C4. Histology was available in 90 cases. 14 (64 percent) C3 cases showed benign histology on biopsy and eight (36 percent) were malignant. 13 (19 percent) C4 cases were benign on biopsy, whereas 55 (81 percent) were malignant. Mammographical studies were available in 56 of the histologically-proven cases. All seven cases with benign mammograms had benign histology, and all 26 cases called "highly suggestive of malignancy" were malignant on histology (five C3 and 21 C4). Of the 23 cases called "suspicious abnormality" on mammography, 14 turned out to be malignant on biopsy (one C3 and 13 C4). CONCLUSION: Our study supports maintaining cytology categories C3 and C4. About two-thirds of C3 cases were benign on biopsy whereas 81 percent of C4 cases were malignant (p-value is less than 0.001). There was complete correlation between histological and mammographical studies except those with equivocal mammograms. Our study supports the combined use of clinical, mammographical and cytological findings for optimal patient management. This is especially important for patients with C3 aspiration results, in order to avoid unnecessary surgery for benign lesions.

Focal segmental glomerulosclerosis and progressive renal failure associated with a unilateral kidney.

Persistent proteinuria, chronic renal failure, and focal segmental glomerulosclerosis developed in three children with solitary kidneys. Two of these children were born with unilateral kidneys. The third had bilateral reflux and underwent a unilateral nephrectomy and reimplantation of the remaining ureter; persistent proteinuria developed 7 years later. It is postulated that hyperperfusion of a critical number of glomeruli during childhood may be the mechanism responsible for the production of focal segmental glomerulosclerosis in these patients.

Localized scleroderma in childhood: a report of 30 cases.

Localized scleroderma (LS), a rare disease that occurs primarily in the pediatric age group, differs from systemic sclerosis (SSc) in that it is usually limited to the skin and subcutaneous tissue and is only rarely associated with systemic manifestations. The authors' experience with pediatric LS seen in 30 patients at a tertiary care center was reviewed: 26 had linear scleroderma, 19 on an extremity and 7 on the face; 3 had morphea; and 1 had generalized morphea. Antinuclear antibodies were present in 76% and rheumatoid factor in 39%. Five of 19 patients with linear scleroderma that involved an extremity had growth failure in that limb, and 1 required surgery. Sclerodermatous involvement over a joint resulted in limited range of movement in 6 patients, and 1 required surgery. One of the 30 patients developed SSc and polymyositis. There was difficulty in evaluating disease activity and hence in evaluating treatment. This experience with a large patient population suggests that LS, although usually a self-limiting disease, can result in significant morbidity.

Iris sector heterochromia as a marker for neural crest disease.

A 6-month-old female infant with biopsy-proved Hirschsprung's disease had associated sector heterochromia of the irides. The association between sector heterochromia and Hirschsprung's disease has been previously reported and both conditions have been ascribed to neural crest defects. Histologic characteristics of the ocular involvement have not previously been reported, to our knowledge. Histopathologic examination of the globes revealed decreased iris stroma, decreased pigmentation in the anterior stroma, and reduced numbers of pigment-producing cells in the affected areas. Both the ocular and gastrointestinal findings reflect abnormalities in tissues of neural crest origin.

Distinct keratin patterns demonstrated by immunoperoxidase staining of adenocarcinomas, carcinoids, and mesotheliomas using polyclonal and monoclonal anti-keratin antibodies.

The authors assessed whether distinct patterns for keratin could be demonstrated in 10 adenocarcinomas, 10 carcinoids, and 4 mesotheliomas by an immunoperoxidase reaction using 3 polyclonal and 3 monoclonal antibodies to keratin. When color development in diaminobenzidine (DAB) was allowed to proceed for less than 2 minutes, distinct patterns for keratin could be demonstrated using two polyclonal and two monoclonal antibodies; these were plasma membrane and/or web-like in the adenocarcinomas, punctate or crescentic in the carcinoids, and perinuclear in the mesotheliomas consisting of tumor cells with abundant cytoplasm. Immunoelectron microscopy using protein A colloidal gold confirmed these results. When color development in DAB was allowed to proceed for more than 2 minutes, only diffuse staining was seen. The distinct patterns of immunostaining for keratin obtained with the shorter color development were helpful in differentiating adenocarcinomas, carcinoids, and mesotheliomas.

Bilateral congenital midureteral adynamic segments.

Two cases of congenital midureteral adynamic segments are presented. Both children were successfully managed with excision of the lesions and primary reanastomosis. Pathologic examination revealed probe-patent ureters with muscular disarray, suggesting functional obstruction. Appropriate management of the anomaly was dependent on precise radiographic localization of the area of narrowing, and for this retrograde urography was essential. Primary ureteroureterostomy was successful in both cases. In this report we discuss the diagnosis, embryology, radiographic evaluation, and management of this rate situation.

Multidrug resistance. Clinical opportunities in diagnosis and circumvention.

Increased P-glycoprotein expression has been shown to be the molecular cause of multidrug resistance in tumor cell lines. Sensitive immunohistochemical and molecular biologic techniques have been developed to detect P-glycoprotein/mdr1 mRNA expression in clinical samples of tumors. We have reviewed the tools now available for assessment of P-glycoprotein expression in the clinic, the current evidence for a relevant role of the protein in mediation of resistance to chemotherapy, and one strategy used to overcome therapeutic failures due to multidrug resistance. It is now recognized that low levels of increased P-glycoprotein/mdr1 mRNA can occur at diagnosis and during the course of treatment in some cases of acute myelogenous leukemia, non-Hodgkin's lymphoma, multiple myeloma, breast carcinoma, rhabdomyosarcoma and undifferentiated sarcoma of children, neuroblastoma, and retinoblastoma, and these relatively low levels of mdr1 overexpression appear to be associated with poor prognosis. In contrast, it has not been established whether a multidrug resistance mechanism is the rate-limiting factor in response to chemotherapy in carcinomas that arise from tissues normally expressing increased P-glycoprotein. Clinical trials have been initiated to determine whether pharmacologic chemosensitization improves the outcome of chemotherapy-treated malignancies. Preliminary results suggest that chemosensitizers can modulate the effects of increased P-glycoprotein in low-expressing tumors for which effective multiagent chemotherapy is available. Further research is needed for more potent chemosensitizers or combinations of agents that can be used more effectively. The successful circumvention of chemotherapy failure by chemosensitizers will ultimately establish the clinical relevance of the P-glycoprotein efflux mechanism.

Xanthogranulomatous cholecystitis and cholecystoduodenal fistula formation associated with total parenteral nutrition in a six year old child.

A unique complication of florid xanthogranulomatous cholecystitis with cholecystoduodenal fistula formation is described in a 6 yr old male. The patient, who had a short gut syndrome, had been maintained on lifelong total parenteral nutrition (TPN) following extensive neonatal ischemic bowel necrosis secondary to gastroschisis. Endoscopic duodenal mucosal biopsy demonstrated a granulomatous inflammatory infiltrate surrounding bile casts suggesting the possibility of a fistula between the biliary tract and duodenum. Additional clinical and radiological evidence of a cholecystoduodenal fistula prompted surgical intervention. At laparotomy the gallbladder was firmly bound to the duodenum by dense fibrous adhesions. Histologic examination showed xanthogranulomatous inflammation in association with fragments of bile that were present both within the gallbladder wall and within a fistulous tract in the adjacent connective tissue.

Characterization of the NC1 domain of collagen type IV in glomerular basement membranes (GBM) and of antibodies to GBM in a patient with anti-GBM nephritis.

Anti-glomerular basement membrane (GBM) nephritis is associated with production of antibodies to the Goodpasture antigen (GPA) component of the NC1 domain of collagen type IV. We studied a patient with anti-GBM nephritis with regard to 1) reactivity of the anti-GBM antibodies in his serum, plasmapheresis fluid (PPF), and an eluate prepared from GBM of his nephrectomy specimen, and 2) electrophoretic and immunologic properties of the NC1 domain extracted by collagenase digestion from GBM of his nephrectomy specimen. Antibodies to different NC1 determinants in serum, PPF and eluate were detected by immunofluorescence of glomerular capillaries of normal kidney. In addition, the antibody in PPF, but not in the eluate, reacted strongly in a plate-binding radioimmunoassay with NC1 domain extracted from normal human GBM, and bound by Western blotting to both dimer (46 kD and 49 kD) and monomer (24 kD, 26 kD and 28 kD) components of the NC1 domain. Analysis of the NC1 domain in the patient's GBM by SDS-PAGE showed a number of abnormalities, including an absence of monomer bands. Moreover, there was diminished reactivity of the patient's NC1 domain by the radioimmunoassay and Western blotting, using his PPF and a rabbit anti-NC1 antiserum. These findings indicated that there were different types of antibodies to NC1 domain in PPF and eluate, associated with an abnormal NC1 domain in GBM. These results have allowed us to speculate on the pathogenesis of anti-GBM nephritis in this patient.

Porphyric neuropathy: an ultrastructural and quantitative case study.

We report a case of acute neuropathy in a 46 year old female with porphyria variegata. Histologic, electron microscopic, and quantitative examinations of peripheral nerves were performed at onset of the neuropathy and at autopsy. The results revealed severe qualitative and quantitative changes in myelinated and unmyelinated fibers showing features indicative of an axonopathy with a distribution in keeping with a dying-back phenomenon.