Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system.

OBJECTIVES: Automated cell counters have replaced manual enumeration of cells in blood and most body fluids. However, due to the unreliability of automated methods at very low cell counts, most laboratories continue to perform labor-intensive manual counts on many or all cerebrospinal fluid (CSF) samples. This multicenter clinical trial investigated if the GloCyte System (Advanced Instruments, Norwood, MA), a recently FDA-approved automated cell counter, which concentrates and enumerates red blood cells (RBCs) and total nucleated cells (TNCs), is sufficiently accurate and precise at very low cell counts to replace all manual CSF counts. METHODS: The GloCyte System concentrates CSF and stains RBCs with fluorochrome-labeled antibodies and TNCs with nucleic acid dyes. RBCs and TNCs are then counted by digital image analysis. Residual adult and pediatric CSF samples obtained for clinical analysis at five different medical centers were used for the study. Cell counts were performed by the manual hemocytometer method and with the GloCyte System following the same protocol at all sites. The limits of the blank, detection, and quantitation, as well as precision and accuracy of the GloCyte, were determined. RESULTS: The GloCyte detected as few as 1 TNC/µL and 1 RBC/µL, and reliably counted as low as 3 TNCs/µL and 2 RBCs/µL. The total coefficient of variation was less than 20%. Comparison with cell counts obtained with a hemocytometer showed good correlation (>97%) between the GloCyte and the hemocytometer, including at very low cell counts. CONCLUSIONS: The GloCyte instrument is a precise, accurate, and stable system to obtain red cell and nucleated cell counts in CSF samples. It allows for the automated enumeration of even very low cell numbers, which is crucial for CSF analysis. These results suggest that GloCyte is an acceptable alternative to the manual method for all CSF samples, including those with normal cell counts.

Surgery combined with controlled-release doxorubicin silk films as a treatment strategy in an orthotopic neuroblastoma mouse model.

BACKGROUND: Neuroblastoma tumour resection goal is maximal tumour removal. We hypothesise that combining surgery with sustained, local doxorubicin application can control tumour growth. METHODS: We injected human neuroblastoma cells into immunocompromised mouse adrenal gland. When KELLY cell-induced tumour volume was >300 mm(3), 80-90% of tumour was resected and treated as follows: instantaneous-release silk film with 100 µg doxorubicin (100IR), controlled-release film with 200 µg (200CR) over residual tumour bed; and 100 and 200 µg intravenous doxorubicin (100IV and 200IV). Tumour volume was measured and histology analysed. RESULTS: Orthotopic tumours formed with KELLY, SK-N-AS, IMR-32, SH-SY5Y cells. Tumours reached 1800±180 mm(3) after 28 days, 2200±290 mm(3) after 35 days, 1280±260 mm(3) after 63 days, and 1700±360 mm(3) after 84 days, respectively. At 3 days post KELLY tumour resection, tumour volumes were similar across all groups (P=0.6210). Tumour growth rate was similar in untreated vs control film, 100IV vs 100IR, and 100IV vs 200IV. There was significant difference in 100IR vs 200CR (P=0.0004) and 200IV vs 200CR (P=0.0003). Tumour growth with all doxorubicin groups was slower than that of control (P: <0.0001-0.0069). At the interface of the 200CR film and tumour, there was cellular necrosis, surrounded by apoptotic cells before reaching viable tumour cells. CONCLUSIONS: Combining surgical resection and sustained local doxorubicin treatment is effective in tumour control. Administering doxorubicin in a local, controlled manner is superior to giving an equivalent intravenous dose in tumour control.

Adaptive off-line protocol for prostate external radiotherapy with cone beam computer tomography.

PURPOSE: The goal of this work was to prepare and to evaluate an off-line adaptive protocol for prostate teleradiotherapy with kilovoltage cone beam computer tomography (CBCT). PATIENTS AND METHODS: Ten patients with localized prostate carcinoma treated with external beams underwent image-guided radiotherapy. In total, 162 CBCT images were collected. Position of prostate and pubis symphysis (PS) with respect to the isocenter were measured off-line. Using the CBCT scans obtained in the first three fractions the average position of prostate in relation (AvPosPr) to PB was calculated. On each CBCT scan, the position of prostate with respect to AvPosPr was calculated and cumulative histogram of prostate displacement with respect to AvPosPr was prepared. Using this data, the adaptive protocol was prepared in which (1) based on the CBCT made in the first three fractions the AvPosPr to PS is obtained, (2) in all other fractions two orthogonal images are acquired and if for any direction set-up error exceeds 0.2 cm the patient's position is corrected, and (3) additionally, the patient's position is corrected if the AvPosPr exceeds 0.2 cm in any direction. To evaluate the adaptive protocol for 30 consecutive patients, the CBCT was also made in 10th and 21st fraction. RESULTS: For the first 10 patients, the results revealed that the prostate was displaced in relation to AvPosPr >0.7 cm in the vertical and longitudinal directions only on 4 and 5 images of 162 CBCT images, respectively. For the lateral direction, this displacement was >0.3 cm in one case. For the group of 30 patients, displacement was never >0.7, and 0.3 cm for the vertical and lateral directions. In two cases, displacements were >0.7 cm for the longitudinal direction. CONCLUSION: Implementation of the proposed adaptive procedure based on the on-line set-up error elimination followed by a reduction of systematic internal error enables reducing the CTV-PTV margin to 0.7, 0.7, and 0.4 cm for the vertical, longitudinal, and lateral directions, respectively.

Loss of a cohesin-linked suppressor APRIN (Pds5b) disrupts stem cell programs in embryonal carcinoma: an emerging cohesin role in tumor suppression.

Cohesins appear to have critical functions beyond mitotic cohesion. Our data on a cohesin-associated Pds5-paralog, APRIN, indicate a novel cohesin role in stem cell differentiation. APRIN/Pds5B is lost in many cancers and it is a putative tumor suppressor. Its mutations in the germ line, however, generate birth defects. We reasoned that as both cancer and birth defects share disrupted stem cell differentiation, the data suggest an APRIN/Pds5B cohesin function in stem cells. We used an embryonal carcinoma stem cell model and show here that (i) APRIN expression is precisely coordinated with stem cell differentiation; (ii) this coordination involves surface-contact and endocrine pathways; and (iii) APRIN/Pds5b coordination is critical in stem/progenitor exit decisions. APRIN knockdown disrupted Oct4, Nanog and SOX2 patterns, differentiation failed and the resulting immature proliferative cells did not progress beyond proneural progenitor phase. Furthermore, the phenotype-blocked progenitor exit (Mash-1(+)); failed E-cadherin exit (E-Cadh(low+)); incomplete N-cadherin transition (N-Cadh(low+)); retained proliferative capacity (c-myc(+)); irregular stemness (SOX2(late++)) and lost response to contact and hormonal cues-shares similarities with cancer-initiating cells. The data suggest novel APRIN/Pds5B-linked cohesin roles in stem/progenitor programs and a new mechanism in tumor suppression.

Somatostatin type 2A receptor immunoreactivity in human pancreatic adenocarcinomas.

Somatostatin and its analogs have been included in experimental treatment protocols for advanced pancreatic adenocarcinoma based on their known antisecretory and antiproliferative properties. Somatostatin receptor type 2 (sstr2A) mediates antiproliferative actions of somatostatin and has the strongest affinity to the therapeutically used somatostatin analog--octreotide. We investigated localization of sstr2A in 27 pancreatic adenocarcinomas in relation to tumor histological features and neuroendocrine differentiation confirmed by immunoreactivity for chromogranin A (CgA), chromogranin B (CgB), or somatostatin. Immunoreactivity for sstr2A generally coincided with tumor neuroendocrine differentiation demonstrated by staining for CgA and was present on the cell membranes of pancreatic islet cells and endocrine cells occasionally present in the wall of normal pancreatic ducts. Thirteen pancreatic adenocarcinomas contained cells immunoreactive for sstr2A in numbers ranging from occasional single cells, cell clusters, or carcinoma duct segments. In two cases, cells immunoreactive for sstr2A and CgA represented more than 30 and 10% of the total tumor cell population (case 1 and 15, respectively). Case 1 fulfills the diagnostic criteria of mixed ductal endocrine carcinoma. We conclude that immunohistochemical staining for a generic neuroendocrine marker such as CgA would facilitate identification of a subgroup of pancreatic adenocarcinomas expressing sstr2A receptors. Future studies need to evaluate the responsiveness of these tumors to somatostatin analogue treatment.

Clinicopathological value of somatostatin type 2A and estrogen receptor immunoreactivity in human breast carcinoma.

Somatostatin type 2A (sstr2A) and estrogen receptor (ER) are interrelated regulatory receptors present in normal breast epithelium and in a population of breast carcinomas. ER mediates growth stimulatory effects of estrogens whereas sstr2A mediates growth inhibitory actions of somatostatin. However, much work has been devoted to elucidate the biological role of ER, little is known about sstr2A in breast cancer. In the present study we examined immunoreactivity of sstr2A and ER in 64 breast carcinomas in correlation with tumor size and histological grade (HG), presence of lymph node metastasis (LNM), Nottingham prognostic index (NPI), and the patients' age. ER and sstr2A immunoreactivity were present in 78% and 63% of the breast carcinomas, respectively. Ninety percent of tumors immunoreactive for sstr2A were simultaneously immunoreactive for ER. ER immunoreactivity correlated significantly with lower HG (p = 0.03) and better NPI (p = 0.02). sstr2A immunoreactivity correlated significantly with lower HG (p = 0.012) but not with NPI (p = 0.26). There was no correlation of sstr2A immunoreactivity and tumor size, patients' chronological age or LNM. The results confirm prognostic value of ER immunohistochemistry in breast carcinoma. However sstr2A cannot substitute ER for prognostic evaluation, sstr2A immunoreactivity being significantly associated with lower HG seems to represent an independent prognostic factor in breast carcinoma.

Immunohistochemical expression of gonadotropin releasing hormone receptor in human breast carcinoma.

Gonadotropin releasing hormone (GnRH) analogs can cause regression of hormone-dependent breast carcinomas via the specific GnRH receptor (GnRH-R). In an attempt to obtain a better understanding of GnRH actions in human breast carcinoma, the expression of GnRH-R was examined immunohistochemically in 58 invasive ductal carcinomas and correlated with various clinicopathological parameters. GnRH-R was immunolocalized in the cytoplasm of carcinoma cells in 37 of 58 invasive ductal carcinoma cases (64%). Immunoreactivity for GnRH-R was also detected focally in the cytoplasm of morphologically normal glandular epithelia adjacent to the carcinoma. A significant correlation was observed between the immunohistochemical expression of GnRH-R and estrogen receptor labeling index (LI; P = 0.030) or progesterone receptor LI (P = 0.0074). There was a significant inverse correlation between GnRH-R immunoreactivity and Ki-67 LI (P = 0.012). No significant correlations were detected between GnRH-R and other clinicopathological parameters, including patient age, menopausal status, stage, tumor size, lymph node status, histological grade and prognosis. This study indicates that GnRH-R is widely distributed in human breast carcinoma cells and regulates GnRH actions locally. Breast carcinomas positive for GnRH-R maintain some hormonal regulatory mechanisms, and GnRH actions may lead to a low proliferative rate in human breast carcinoma.

Fibromatosis of the mesoappendix.

Mesenchymal tumors of the appendix are very rare. Mesenteric fibromatosis is the most common diagnosis in the case of isolated proliferative fibroblastic lesion found in this region. However, the differential diagnosis between fibromatosis and well-differentiated fibrosarcoma (Grade I) can be difficult in some borderline cases. A rare case of proliferative fibroblastic lesion involving the appendix and mesoappendix is presented, and its origin, diagnosis and differential diagnosis are discussed.

Primary hepatic carcinoid tumor: a case report.

A case of primary hepatic carcinoid tumor is reported. A 57-year-old woman had no endocrine symptoms. Light microscopic findings revealed tumor cells forming trabecular or insular structures and demonstrating diffuse positive staining by the Grimelius method. Mucin stained with Periodic acid-Schiff (PAS) and alcian-blue was partially present in the tumor cells. Immunohistochemically, most tumor cells stained positive with chromogranin A and cytokeratin stains. Intensive and careful investigation before and after the operation revealed no other origin of the tumor. We present this rare case, describe the clinical features of hepatic carcinoid, and discuss its diagnosis.

Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors.

Although chromogranin A (CgA) is widely distributed in neuroendocrine tumors, the distribution of chromogranin B (CgB) has not been elucidated. Hormones produced by tumors are sometimes prohormones and not necessarily bioactive hormones. Prohormones have to be processed into bioactive peptides by prohormone convertases (PCs), and some of them have to be amidated by peptidylglycine a-amidating monooxygenase (PGM). Whether PCs and PGM are present or not in tumors may explain why some tumors are functioning and some are nonfunctioning. We investigated 45 carcinoids and 16 pancreatic endocrine tumors. Of the carcinoids, CgA was expressed in most of the tumors, except for the rectal and ovarian carcinoids, which expressed CgB strongly. The expressions of PC2, PC3, and PGM were 31%, 100%, and 87%, respectively. In the pancreatic tumors, CgA was expressed in all tumors, whereas CgB was not expressed in any tumor. The expressions of PC2, PC3, and PGM were 63%, 88%, and 63%, respectively. PC3 was expressed in all of the functioning tumors but not in two of the four nonfunctioning tumors. PC2 and PGM were not expressed in three of the four nonfunctioning tumors. In conclusion, expression of CgA and CgB was different depending on the tumor location. High frequency of PCs and PGM may explain why even nonfunctioning tumors produce some inconspicuous peptides.

Expression of somatostatin type 2A receptor correlates with estrogen receptor in human breast carcinoma.

Somatostatin type 2A receptor (sstr2A) has been shown to be directly involved in the transduction of antiproliferative effects and also to be the most predominant sstr subtype in human normal breast epithelium, as well as in human breast carcinoma. We investigated the immunoreactivity of sstr2A in 34 cases of human breast carcinoma and correlated these findings with the immunoreactivity of the estrogen receptor (ER), epidermal growth factor receptor (EGFR), transforming growth factor alpha (TGFalpha) and insulin like growth factor I (IGF-I). We detected sstr2A immunoreactivity in normal mammary tissue, and in 27 of 34 (79%) breast carcinomas. The sstr2A immunoreactivity was localized on the cellular membrane, however, weak cytoplasmic immunoreactivity was also observed. Sstr2A immunoreactivity was heterogenously distributed in the whole tumor section. There was a statistically significant correlation between sstr2A and ER immunoreactivity in the same tumor. No statistically significant correlation was found between sstr2A immunoreactivity and immunoreactivity for EGFR, TGFalpha and IGF-I or the patients' age.

Immunohistochemical expression of somatostatin type 2A receptor in neuroendocrine tumors.

Somatostatin (SS) and SS analogues inhibit the growth of various kinds of endocrine and exocrine cells via the SS receptor (SSTR). Carcinoid tumor is representative of the tumors treatable by SS analogues. We examined the expression of SSTR2A by immunohistochemical and in situ hybridization methods with a specific antibody against a synthesized 20-amino acid peptide of the COOH terminus of human SSTR2A and oligonucleotide probes in 62 endocrine tumors of various kinds: pancreatic endocrine tumor; carcinoid; neuroendocrine carcinoma; medullary thyroid carcinoma; pheochromocytoma; and small cell carcinoma of the lung, neuroblastoma, and ganglioneuroma. SSTR2A was expressed in 87% of these tumors and at both primary and metastatic sites. The immunohistochemical reactivity of SSTR2A was strong on the cell membrane and less intense in the cytoplasm of the tumor cells. SSTR2A mRNA was also detected in the tumor cells. The results indicate the usefulness of SSTR2A analogues for the treatment of neuroendocrine tumors, even metastatic ones: metastatic carcinoids, metastatic pheochromocytomas, tumors that adhered to large vessels, and neuroendocrine carcinomas.

Primary hepatic carcinoid and neuroendocrine carcinoma: clinicopathological and immunohistochemical study of five cases.

Primary hepatic carcinoid and neuroendocrine carcinoma (NEC) are rare tumors. We experienced three carcinoids and two NEC originating in the liver during the past 25 years and attempted to elucidate the clinicopathological and immunohistochemical features of these tumors. The patients had no endocrine symptoms despite two of them having elevated plasma serotonin. Three of the five patients died of the tumor after operation with an average survival time of 20.6 months. All tumors were large (up to 26 cm in diameter), four of them solitary and one multinodular, and were not associated with liver cirrhosis. The carcinoid tumors showed insular, trabecular or glandular arrangement of argyrophilic cells, whereas in the NEC this histological pattern was distorted. Immunohistochemically the tumors showed expression of chromogranin A (all cases), chromogranin B (three cases), pancreastatin and chromostatin (four cases, respectively), prohormone convertase PC3 (three cases), carcinoembryonic antigen (CEA) and CA19-9 (two cases), cytokeratin 56 kDa (three cases), 160 kDa neurofilament (two cases) and neuron-specific enolase (two cases). Serotonin and glucagon were sporadically detected in two tumors. The most useful marker to confirm the diagnosis was chromogranin A, which was cleaved to pancreastatin and chromostatin in the tumor tissue, and was more reliable than other markers of neuroendocrine differentiation.

Immunohistochemical study of TGF-alpha, TGF-beta1, EGFR, and IGF-1 expression in human breast carcinoma.

Localization of growth factors such as transforming growth factor alpha (TGF-alpha) and beta1 (TGF-beta1), insulin-like growth factor 1 (IGF-1), and epidermal growth factor receptor (EGFR) in breast cancer tissue is controversial. We immunohistochemically investigated expression patterns of these growth factors and EGFR along with estrogen receptor (ER) status in 36 breast carcinomas (21 invasive ductal, 11 invasive lobular, 4 noninvasive ductal) and compared the results with those found in 10 fibroadenomas. Twenty-four of 36 carcinomas and all of the 10 fibroadenomas showed positivity for ER. TGF-alpha was immunoreactive in all of the carcinomas and fibroadenomas. TGF-beta1 was negative in all of the invasive ductal carcinomas and positive in all of the fibroadenomas and in five lobular carcinomas. EGFR was regularly expressed preferentially in the myoepithelial cells of mammary ducts in the fibroadenomas and in nontumorous glands. Six of the 36 carcinomas were positive for EGFR. Those tumors were negative for ER (P < .001). There was IGF-1 expression in all of the cases of carcinoma and fibroadenoma. We conclude that TGF-alpha is expressed abundantly in invasive and intraductal breast carcinomas and in fibroadenomas. EGFR expression significantly correlates with negative ER status in breast carcinoma. In breast carcinoma, IGF-1 is broadly expressed by the tumor as well as by stromal cells and might act as a growth stimulator in endocrine, paracrine, and autocrine manners.

-Cardiomyopathy as the cause of death in genetic hemochromatosis-. / Kardiomyopathie als Todesursache bei genetischer Hämochromatose.

Hemochromatosis is an autosomal-recessive disease which causes iron-overload of various organs including liver, pancreas and heart. This report analyzes the course of hemochromatosis in two patients (a 28-year-old man and a 57-year-old woman) in whom hemochromatosis was detected because of severe cardiomyopathy. Initial symptoms were edema, anasarca and dyspnea. Further examinations showed pleural effusion, decreased left-ventricular-function, skin pigmentation, diabetes mellitus and liver cirrhosis. Although phlebotomy treatment and iron-chelation therapy with deferoxamine initially resulted in some improvement, both patients died from cardiomyopathy three months after diagnosis. The reports of these two cases underline that hemochromatosis-associated cardiomyopathy is often irreversible if severe congestive heart failure is present. In cardiac decompensation heart transplantation has to be considered as early as possible.

Malignant gliomas--ploidy and DNA-content before and after therapy.

DNA ploidy and S-phase percentage from nine malignant gliomas (four glioblastomas, four anaplastic astrocytomas grade 3 and one anaplastic oligoastrocytoma grade 3) have been estimated by single cell cytophotometry on biopsy and necropsy specimens. All gliomas from biopsy material showed, with the exception of two diploid tumours, a polyploid-aneuploid DNA-pattern and stem-lines of different ploidy. The most frequent stem-lines were diploid and hyperdiploid. In necropsy material, following treatment i.e. operation and combined drug and radiation therapy, the heterogeneous nature of all malignant gliomas persisted. From seven aneuploid-polyploid gliomas four showed an elevation and three of them a decrease of DI values. Diploid tumors remained diploid. Because of marked heterogeneity of ploidy patterns and the small number of tumors investigated, ploidy changes could not be used for estimation of therapy efficacy and prognosis. Further studies will be necessary to answer this question.

[Dementia and psychotic symptoms in amyotrophic lateral sclerosis]. / Demenz und psychotische Symptome bei der amyotrophen Lateralsklerose.

We present 2 case histories of patients, treated in psychiatric clinics because of psychotic disorders, in whom further investigation revealed the presence of amyotrophic lateral sclerosis (ALS) and dementia. The causal relationship between psychotic symptoms and dementia on the one hand and ALS on the other hand is discussed. Probably in ALS other functional systems besides motor-neuron pathways are involved.