Enzyme replacement therapy on hypophosphatasia mouse model.

Hypophosphatasia (HPP) is an inborn error of metabolism caused by deficiency of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5-phosphate (PLP), a co-factor form of vitamin B6. Enzyme replacement therapy (ERT) for HPP by functional TNSALP is one of the therapeutic options. The C-terminal-anchorless human recombinant TNSALP derived from Chinese hamster ovary cell lines was purified. TNSALP-null mice (Akp2 (-/-) ), an infantile model of HPP, were treated from birth using TNSALP and vitamin B6 diet. Long-term efficacy studies of ERT consisted of every 3 days subcutaneous or intravenous injections till 28 days old (dose 20 U/g) and subsequently every 3 days intravenous injections for 6 months (dose 10 U/g). We assessed therapeutic effect by growth and survival rates, fertility, skeletal manifestations, and radiographic and pathological finding. Treated Akp2 (-/-) mice grew normally till 4 weeks and appeared well with a minimum skeletal abnormality as well as absence of epilepsy, compared with untreated mice which died by 3 weeks old. The prognosis of TNSALP-treated Akp2 (-/-) mice was improved substantially: 1) prolonged life span over 6 months, 2) improvement of the growth, and 3) normal fertility. After 6 months of treatment, we found moderate hypomineralization with abnormal proliferative chondrocytes in growth plate and articular cartilage. In conclusion, ERT with human native TNSALP improves substantial clinical manifestations in Akp2 (-/-) mice, suggesting that ERT with anchorless TNSALP is also a potential therapy for HPP.

Long circulating enzyme replacement therapy rescues bone pathology in mucopolysaccharidosis VII murine model.

Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme ß-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.

Columnar cell lesions: a consensus study among pathology trainees.

Columnar cell lesions of the breast are a spectrum of lesions in the terminal duct lobular units, which include columnar cell change, columnar cell hyperplasia, and columnar cell change or columnar cell hyperplasia with atypia. The latter was designated by the World Health Organization as flat epithelial atypia. We studied a group of pathology trainees (pathology residents and fellows) as a model for the impact of a training tutorial on the ability to distinguish various types of columnar cell lesions. Twenty-four test cases of columnar cell lesions, including 8 cases each of columnar cell change, columnar cell hyperplasia, and flat epithelial atypia, were prepared after an independent consensus on diagnosis by 2 senior pathologists with expertise in breast pathology. Fourteen pathology trainees were given a slide tutorial for the diagnostic criteria of columnar cell lesions at a multiheaded microscope. All 14 trainees entered their diagnoses of columnar cell lesions independently before and after the tutorial. The kappa values of columnar cell lesions, columnar cell hyperplasia, and flat epithelial atypia among 14 pathology trainees were 0.42, 0.25, and 0.39 before the tutorial and 0.56, 0.41, and 0.60 after the tutorial, respectively. The agreements on columnar cell lesions, columnar cell hyperplasia, and flat epithelial atypia after the tutorial were significantly better than those before the tutorial. No significant difference in agreement was observed on columnar cell change before and after the tutorial, but the kappa value improved. We conclude that appropriate tutorial training of diagnostic criteria can increase diagnostic agreement on columnar cell lesions among pathology residents, fellows, and presumably general pathologists in practice.

Basal-like breast carcinoma: a phenotypically distinct entity.

Gene microarray profiling of human breast carcinomas has recently categorized invasive breast carcinomas into 5 distinct subtypes; luminal A, luminal B, normal breastlike, human epithelial growth factor receptor 2 (HER2) overexpressing, and basal-like. Basal-like breast carcinomas are characterized by high expression of basal cytokeratins; low or absent expression of estrogen receptor, progesterone receptor, and HER2/neu; and expression of epidermal growth factor receptor (EGFR) and/or c-kit, and they are frequently associated with breast cancer 1 (BRCA1) mutations and poor clinical outcome. Recent studies have begun to provide insights into the molecular genetics, biology, morphology, and clinical outcome of this subtype of breast carcinoma. We reviewed the literature related to basal-like breast carcinomas to better understand this clinically significant subtype of breast carcinoma.

Number of lymph nodes examined and associated clinicopathologic factors in colorectal carcinoma.

CONTEXT: Nodal metastasis is one of the most important prognostic factors in colorectal carcinoma. The number of lymph nodes recovered and examined in resection specimens has been recently shown to be critical for proper staging and is associated with survival. OBJECTIVE: To assess the clinicopathologic factors that may be associated with the number of lymph nodes harvested from surgical resections. DESIGN: Clinicopathologic factors of 434 consecutive cases of colorectal cancers treated by surgical resection from a single tertiary medical center were retrospectively reviewed and correlated with number of lymph nodes recovered. RESULTS: Our data show that patient age, tumor location, and length of resected bowel segment were associated with number of lymph nodes harvested in surgical resections of colorectal cancer. The average number of lymph nodes was 18.2 and 17.8 for patients younger than 50 years and aged 50 through 60 years, respectively, whereas it was 14.4, 15.1, and 14.9 for patients aged 61 through 70 years, 71 through 80 years, and 80 years and older, respectively. More lymph nodes were present in resection specimens of cecum/ascending colon and descending colon cancers than in those of transverse colon, sigmoid colon, and rectal cancers. There was a linear increase in number of lymph nodes examined with increasing length of bowel resection specimens. In multivariate regression analysis, the factors that remained independent predictors of removal of 12 or more lymph nodes from resection specimens were tumor location and length of resected bowel segment. CONCLUSIONS: The number of lymph nodes obtained in resection specimens for colorectal cancer was significantly associated with the length of resected segments of bowel, patient age, and location of the tumor.

Metastatic prostatic carcinoma to testis: histological features mimicking lymphoma.

We report a case of prostatic carcinoma with testicular metastasis, which mimicked malignant lymphoma of the testis. The patient was a 71 year-old man with a history of prostate adenocarcinoma of Gleason score 9 (4+5) diagnosed in 2001 for which he received hormonal therapy. Four years later, the patient developed multiple osteoblastic bone metastases. Radiotherapy of the bone metastases was given with subsequently bilateral orchiectomy for hormonal deprivation therapy in May 2005. Grossly, one of the testes had a subcapsular rubbery 0.9 cm nodule. Microscopically, the nodule was composed of malignant discohesive cells predominantly infiltrating in the interstitium with an appearance of malignant lymphoma. However, immunohistochemical stains were positive for prostate-specific antigen and prostate acid phosphatase and negative for leukocyte common antigen, which confirmed the diagnosis of metastatic prostate adenocarcinoma.

Colorectal adenocarcinoma with micropapillary pattern and its association with lymph node metastasis.

Micropapillary carcinoma has been reported as an aggressive variant of carcinoma in several organs, where it is associated with frequent lymphovascular invasion and poor clinical outcome. This study explored the clinicopathological features of colorectal adenocarcinoma with a micropapillary carcinoma component and compared them with those of conventional colorectal adenocarcinoma. One hundred seventy-eight consecutive cases of surgically resected colorectal carcinomas were studied for tumor size, type, depth of invasion, nodal and distant metastases, tumor stage, and percentage and extent of micropapillary component. Among 178 cases of colorectal carcinoma, 34 (19.1%) cases had a micropapillary component, which ranged from 5 to 60% of the entire tumor. Lymph node metastasis was identified in 25 of 34 (73.5%) carcinomas with micropapillary component, whereas they were detected in 61 of 144 (42.4%) cases without micropapillary component (P=0.001). Lymphovascular invasion was identified more frequently in carcinoma with micropapillary component (41.2%) than carcinoma without micropapillary component (20.1%; P<0.05). Distant metastases occurred in 4 of 34 cases (11.7%) with micropapillary component and in 10 of 144 cases (6.9%) without micropapillary component (P=0.311). Multivariate regression analysis demonstrated that the presence of micropapillary component, as well as tumor stage and lymphovascular invasion are independent predictors of regional nodal metastasis.

Sexually dimorphic roles of steroid hormone receptor signaling in gonadal tumorigenesis.

Sex steroids control cellular phenotypes by binding to receptor proteins that in turn regulate downstream gene expression. They are important tropic factors in hormonally responsive tissues and have been implicated in the pathogenesis of both benign proliferations and malignancies at some of these sites. Knockout mice lacking inhibins, alpha:beta heterodimeric peptide hormones of the TGFbeta superfamily, develop gonadal tumors that produce sex steroids and depend on pituitary gonadotropin hormones. To better appreciate how sex steroid receptor signaling pathways contribute to the loss of granulosa/Sertoli cell proliferation in the ovary and testis of inhibin alpha (Inhalpha) knockout mice, we are using both pharmacologic and genetic approaches. Roles of androgens in testicular tumor development have been investigated in our previous studies using double-mutant mice lacking inhibins and carrying the null testicular feminization (tfm) mutation of the androgen receptor. Herein, we report that androgens also participate in the development of ovarian tumors, as tumor development is forestalled in mice treated with flutamide, a nonsteroidal inhibitor of androgen actions. Additionally, we generated double-mutant mice lacking estrogen receptor alpha (ERalpha) and Inhalpha or ERbeta and Inhalpha, as well as triple-mutant mice lacking ERalpha, ERbeta, and Inhalpha to determine the effects of individual and combined ER signaling pathways on tumor development. Although estrogens may have proliferative effects during follicle development and are important in specifying the granulosa cell phenotype, ERalpha and ERbeta signaling are not essential for timely granulosa cell tumor development or granulosa cell-like morphological features in ovarian tumors. However, redundant ER signaling through ERalpha and ERbeta in males is critical for testicular tumor formation, as triple-knockout, but not double-knockout, males are protected from early Sertoli cell tumorigenesis and death. Together, these studies indicate important and sexually dimorphic functions of estrogens and androgens in tumor development in this mouse model and indicate, for the first time, overlapping functions of ERalpha and ERbeta in Sertoli cell pathophysiology.

Normal reproductive function in InhBP/p120-deficient mice.

The inhibins are gonadal transforming growth factor beta superfamily protein hormones that suppress pituitary follicle-stimulating hormone (FSH) synthesis. Recently, betaglycan and inhibin binding protein (InhBP/p120, also known as the product of immunoglobulin superfamily gene 1 [IGSF1]) were identified as candidate inhibin coreceptors, shedding light on the molecular basis of how inhibins may affect target cells. Activins, which are structurally related to the inhibins, act within the pituitary to stimulate FSH production. Betaglycan increases the affinity of inhibins for the activin type IIA (ACVR2) receptor, thereby blocking activin binding and signaling through this receptor. InhBP/p120 may not directly bind inhibins but may interact with the activin type IB receptor, ALK4, and participate in inhibin B's antagonism of activin signaling. To better understand the in vivo functions of InhBP/p120, we characterized the InhBP/p120 mRNAs and gene in mice and generated InhBP/p120 mutant mice by gene targeting in embryonic stem cells. InhBP/p120 mutant male and female mice were viable and fertile. Moreover, they showed no alterations in FSH synthesis or secretion or in ovarian or testicular function. These data contribute to a growing body of evidence indicating that InhBP/p120 does not play an essential role in inhibin biology.

Loss of holocytochrome c-type synthetase causes the male lethality of X-linked dominant microphthalmia with linear skin defects (MLS) syndrome.

Girls with MLS syndrome have microphthalmia with linear skin defects of face and neck, sclerocornea, corpus callosum agenesis and other brain anomalies. This X-linked dominant, male-lethal condition is associated with heterozygous deletions of a critical region in Xp22.31, from the 5' untranslated region of MID1 at the telomeric boundary to the ARHGAP6 gene at the centromeric boundary. HCCS, encoding human holocytochrome c-type synthetase, is the only gene located entirely inside the critical region. Because single gene analysis is not feasible in MLS patients (all have deletions), we generated a deletion of the equivalent region in the mouse to study the molecular basis of this syndrome. This deletion inactivates mouse Hccs, whose homologs in lower organisms (cytochrome c or c1 heme lyases) are essential for function of cytochrome c or c1 in the mitochondrial respiratory chain. Ubiquitous deletions generated in vivo lead to lethality of hemizygous, homozygous and heterozygous embryos early in development. This lethality is rescued by expression of the human HCCS gene from a transgenic BAC, resulting in viable homozygous, heterozygous and hemizygous deleted mice with no apparent phenotype. In the presence of the HCCS transgene, the deletion is easily transmitted to subsequent generations. We did obtain a single heterozygous deleted female that does not express human HCCS, which is analogous to the low prevalence of the heterozygous MLS deletion in humans. Through the study of these genetically engineered mice we demonstrate that loss of HCCS causes the male lethality of MLS syndrome.