Comparative study of Rb1, cyclin D1 and p16 immunohistochemistry expression to distinguish lung small-cell carcinoma and large-cell neuroendocrine carcinoma.

AIMS: Large-cell neuroendocrine carcinoma (LCNEC) and small-cell carcinoma (SCLC) of lung encompass high-grade neuroendocrine tumour category and share several fundamental features. As both tumours may respond to different treatment modalities and show unique molecular alterations distinction between the two is clinically relevant, but can be challenging due to sampling and fixation issues and shared morphological features. METHODS: Surgically resected primary SCLC (n = 129) and LCNEC (n = 27) were immunohistochemically stained with Rb1, cyclin D1 and p16 using tissue microarray (TMA), and expression patterns of the proteins were compared between the two to identify the discriminatory pattern. RESULTS: All markers had high diagnostic accuracy; Rb1 was the highest followed by p16 and cyclin D1. The majority of SCLC had the pattern Rb1-/p16+/cyclin D1- and more than half of LCNEC had Rb1+/p16-/cyclin D1+. Overall, the expression pattern Rb1- and cyclin D1- was strongly associated with the diagnosis of SCLC, while the pattern Rb1+ and/or cyclin D1+ was strongly associated with LCNEC. The use of this simplified expression pattern leads to a diagnostic accuracy of 97.3%. p16 did not add to further discrimination. The heterogeneity in Rb1, cyclin D1 and p16 expression was insignificant in SCLCs compared with LCNECs. CONCLUSIONS: Use of Rb1, cyclin D1 and p16 immunohistochemistry can distinguish the two with high accuracy. Notably, the Rb1-/cyclin D1- pattern in given tumour sample would confirm the diagnosis of SCLC. Our results could be extrapolated and applied to routine diagnostic samples such as biopsies and cytology samples.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.

BACKGROUND: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). PATIENTS AND METHODS: CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. RESULTS: The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. CONCLUSIONS: This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

Diagnostic SOX10 gene signatures in salivary adenoid cystic and breast basal-like carcinomas.

BACKGROUND: Salivary adenoid cystic carcinoma (ACC) is an insidious slow-growing cancer with the propensity to recur and metastasise to distant sites. Basal-like breast carcinoma (BBC) is a molecular subtype that constitutes 15-20% of breast cancers, shares histological similarities and basal cell markers with ACC, lacks expression of ER (oestrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2), and, similar to ACC, metastasises predominantly to the lung and brain. Both cancers lack targeted therapies owing to poor understanding of their molecular drivers. METHODS: Gene expression profiling, immunohistochemical staining, western blot, RT-PCR, and in silico analysis of massive cancer data sets were used to identify novel markers and potential therapeutic targets for ACC and BBC. For the detection and comparison of gene signatures, we performed co-expression analysis using a recently developed web-based multi-experiment matrix tool for visualisation and rank aggregation. RESULTS: In ACC and BBC we identified characteristic and overlapping SOX10 gene signatures that contained a large set of novel potential molecular markers. SOX10 was validated as a sensitive diagnostic marker for both cancers and its expression was linked to normal and malignant myoepithelial/basal cells. In ACC, BBC, and melanoma (MEL), SOX10 expression strongly co-segregated with the expression of ROPN1B, GPM6B, COL9A3, and MIA. In ACC and breast cancers, SOX10 expression negatively correlated with FOXA1, a cell identity marker and major regulator of the luminal breast subtype. Diagnostic significance of several conserved elements of the SOX10 signature (MIA, TRIM2, ROPN1, and ROPN1B) was validated on BBC cell lines. CONCLUSION: SOX10 expression in ACC and BBC appears to be a part of a highly coordinated transcriptional programme characteristic for cancers with basal/myoepithelial features. Comparison between ACC/BBC and other cancers, such as neuroblastomaand MEL, reveals potential molecular markers specific for these cancers that are likely linked to their cell identity. SOX10 as a novel diagnostic marker for ACC and BBC provides important molecular insight into their molecular aetiology and cell origin. Given that SOX10 was recently described as a principal driver of MEL, identification of conserved elements of the SOX10 signatures may help in better understanding of SOX10-related signalling and development of novel diagnostic and therapeutic tools.

Self-assessed approach to improving school health in Niger.

INTRODUCTION: Award schemes and self-evaluation systems have been developed to implement the 'Health-Promoting School (HPS)' concept in European and Asian countries. While there have been many successes in these regions, the implementation of HPS in African countries has been minimal. This study evaluated the impact of a self-evaluation system on school health in Niger. METHODS: A school health activity guide was developed and distributed to 1999 primary schools in the Niger Tahoua region to raise awareness and solve problems related to school health and hygiene. The number of schools that planned or implemented health-related activities, and the budget and implementation status of their activities was compared over 3 years (before, soon after, and 1 year after distribution). Focus group discussions (FGDs) were also conducted targeting Conseillers Pédagogiques (CPs), who supervise primary schools and teachers, primary school principals and members of Comité de Gestion des Etablissement Scolaire (COGES), which is a type of school steering committee. RESULTS: The number of schools planning at least one health-related activity increased from 47% to 79% soon after distribution of the guide (p <0.001).The number of schools implementing activities increased from 44% to 65% one year after distribution (p <0.001). Health-related budget per school also increased after distribution (p <0.0001) and increases were maintained 1 year after the intervention (p=0.8414). Fulfilment or partial fulfilment rates for health-related activities were lower compared with other (non-health) activities in all three years (80%, 77% & 84% in health-related activities vs 88%, 90% & 91% in others; p <0.001, p <0.001, & p=0.004, respectively). Most FGD participants expressed a positive impression of the program and noted the usefulness of the guide. However, some respondents reported difficulties, especially in relation to budget. CONCLUSION: The introduction of a health activity guide for self-assessment was effective in increasing health-related activities in primary schools in Niger, where a simple monitoring system should be introduced to establish the HPS concept.

Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma.

Activating BRAF or NRAS mutations have been found in 80% of human sporadic melanomas, but only one of these genetic alterations could be detected in each tumour. This suggests that BRAF and NRAS 'double mutants' may not provide advantage for tumour growth, or may even be selected against during tumorigenesis. However, by applying mutant-allele-specific-amplification-PCR method to short-term melanoma lines, one out of 14 tumours was found to harbour both BRAFV600E and the activating NRASQ61R mutations. On the other hand, analysis of 21 melanoma clones isolated by growth in soft agar from this tumour indicated that 16/21 clones harboured a BRAFV600E, but were wild-type for NRAS, whereas the remaining had the opposite genotype (NRASQ61R/wild-type BRAF). When compared to BRAFV600E clones, NRASQ61R clones displayed reduced growth in soft agar, but higher proliferative ability in vitro in liquid medium and even in vivo after grafting into SCID/SCID mice. These data suggest that NRAS and BRAF activating mutations can coexist in the same melanoma, but are mutually exclusive at the single-cell level. Moreover, the presence of NRASQ61R or BRAFV600E is associated with distinct in vitro and in vivo growth properties of neoplastic cells.

Evaluation of diagnostic and prognostic significance of Ki-67 index in pulmonary carcinoid tumours.

BACKGROUND: Pulmonary carcinoid (PC) tumours are classified as either typical (TC) or atypical (AC) according to mitotic index (MI) and presence of necrosis. The aim of this study was to analyse the diagnostic and prognostic values of the Ki-67 index in PC. METHODS/PATIENTS: Between January 2001 and March 2015, we evaluated 94 consecutive patients with a confirmed diagnosis of TC (n = 75) or AC (n = 19) at our institution. Diagnostic histology was centrally reviewed by a local expert neuroendocrine pathologist, with assessment of Ki-67, MI, and necrosis. RESULTS: Median patient follow-up was 35 months. Eighty-four patients who underwent curative surgical resection were included in the survival analysis for identification of prognostic factors. Ki-67 index showed high diagnostic accuracy to predict histological subtype when assessed by receiver operator characteristic curves with an area under the curve of 0.923 (95% CI 0.852-0.995, p < 0.001). Multivariate analysis showed that MI, Ki-67 index, and the presence or absence of necrosis were independent prognostic factors for relapse-free survival. Combination of MI, Ki-67, and necrosis led to the classification of patients into four different prognostic groups (very low, low, intermediate, and high risks of relapse). CONCLUSIONS: The current study proposes the incorporation of Ki-67 index in the prognostic classification of PC tumours. Due to the limited number of patients and length of follow-up, the current model needs validation by larger cohort studies. Nevertheless, our results suggest that Ki-67 index and MI have continuous effect on prognosis. Prognostic models incorporating multiple cutoffs of Ki-67 and MI might better predict outcome and inform clinical decisions.

Paralytic ileus due to strongyloidiasis: case report and review of the literature.

We report a rare case of a patient with ileus due to Strongyloides infection that occurred four times within a six-month period. The ileus was improved by treatment with ivermectin and there has not been a recurrence of the symptoms within the last two years.

Plasma (1-->3)-beta-D-glucan measurement and polymerase chain reaction on sputum as practical parameters in Pneumocystis carinii pneumonia.

We report a case of Pneumocystis carinii pneumonia in a patient with acquired immunodeficiency syndrome diagnosed and monitored with polymerase chain reaction (PCR) for Pneumocystis carinii on sputum and measurement of plasma (1-->3)-beta-D-glucan (G-test). Results of both studies paralleled the clinical and radiographic improvement. However, the plasma (1-->3)-beta-D-glucan level remained higher than normal when PCR for Pneumocystis carinii became negative in sputum. Both PCR for Pneumocystis carinii on sputum and measurement of plasma (1-->3)-beta-D-glucan are useful for noninvasive diagnosis and monitoring of Pneumocystis carinii, although further investigation is necessary to quantify their relationship.

Epithelioid haemangioma: a rare cause of painful erections and sleep deprivation.

Epithelioid haemangioma of the penis is a rare condition which usually presents a solid single nodule. We report a case in a 43-year-old man who presented with painful erections and sleep disturbance with two palpable penile nodules. Magnetic resonance imaging with an artificially induced erection revealed these as individual lesions, and local excision was successfully undertaken. Pathological diagnosis of epithelioid haemangioma was confirmed with positive staining for CD31. Although rare, penile epithelioid haemangioma should be considered as a differential in an atypical penile mass. Induction in of an artificial erection prior to MRI can aid diagnosis and treatment is typically with surgical excision.

Multicystic and well-differentiated papillary peritoneal mesothelioma treated by surgical cytoreduction and hyperthermic intra-peritoneal chemotherapy (HIPEC).

BACKGROUND: Multicystic peritoneal mesothelioma (MPM) and well-differentiated papillary peritoneal mesothelioma (WDPPM) are exceedingly uncommon lesions with uncertain malignant potential and no uniform treatment strategy. The aim of the current study was to review our experience with cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC) in these clinical settings. METHODS: Four women with MPM and eight with WDPPM underwent 13 procedures of cytoreduction and close-abdomen HIPEC with cis-platin and doxorubicin. Seven patients had recurrent disease after previous debulking (one operation in five patients, two in one, four in one). Potential clinicopathological prognostic factors were assessed. RESULTS: Optimal cytoreduction (residual tumor nodules

CDX-2 expression in pseudomyxoma peritonei: a clinicopathological study of 42 cases.

AIMS: CDX-2 is a highly sensitive and specific marker of intestinal epithelial cells and their neoplastic counterparts. CDX-2 status in pseudomyxoma peritonei (PMP) has been barely reported. The aim of this study was to investigate the clinicopathological features of 42 cases of PMP with a special emphasis on CDX-2. METHODS AND RESULTS: All patients were treated by cytoreduction. Immunohistochemistry was performed for CDX-2, MUC-2, MUC-5AC, cytokeratin (CK) 7 and CK20. Statistical correlation was evaluated for age, sex, completeness of cytoreduction and histological subtype with overall and progression-free survival (OS and PFS). PMP consisted of 32 cases of disseminated peritoneal adenomucinosis and 10 cases of peritoneal mucinous carcinomatosis. The appendix evaluated in 25 cases showed two mucinous adenocarcinomas and 21 low-grade appendiceal mucinous neoplasms. CDX-2 was diffusely positive in 40 cases, with the remaining two cases being focally positive. All cases demonstrated diffuse reactions to CK20 and MUC-2, and variable reactions to MUC-5AC, while CK7 was variably positive in 38 cases. Five-year OS was 97%. Histological type was significantly correlated with PFS (P=0.02). CONCLUSIONS: CDX-2 is diffusely and strongly positive in PMP. This is a useful marker to confirm an appendiceal origin of PMP, particularly when used in conjunction with CK7, CK20, MUC-2 and MUC-5AC.

p53, epidermal growth factor, and platelet-derived growth factor in uterine leiomyosarcoma and leiomyomas.

Uterine leiomyosarcoma (ULMS) is an aggressive gynecological disease. Although ULMS are often found in association with benign leiomyoma (LMA), little is known regarding the relationship between these benign and malignant smooth muscle neoplasms. The objective of this study was to evaluate the expression of epidermal growth factor (EGFR), platelet-derived growth factor (PDGFR), and p53 in ULMS specimens, their prognostic relevance, and the expression of these molecular markers when compared to benign LMA specimens. Between 1991 and 2001, 25 patients were identified with high-grade primary ULMS and for whom tissue was available. Tissue microarray was created with three representative cores from each of the ULMS cases as well as from 19 patients with benign uterine leiomyomata. Immunohistochemical (IHC) staining was performed for EGFR, PDGFR, and p53. Negative and positive IHC staining was scored for each marker. Outcome analysis was performed only for ULMS. Survival was determined from the time of initial diagnosis to last follow-up. Twelve (48%) ULMS expressed p53 compared to none of the LMA (P < 0.001), and 15 (60%) ULMS cases showed PDGFR expression compared to 32% of LMA samples (P= 0.08). EGFR expression did not differ between ULMS and LMA groups. ULMS patients with p53 expression had a poorer survival compared to ULMS patients with negative expression (P= 0.07). ULMS tumor stage had the strongest association with overall survival (P= 0.05). Our study supports previous investigations indicating that p53 expression may serve as a prognostic marker for ULMS patients. The difference in PDGFR expression between ULMS and LMA demonstrated a trend toward significance. EGFR was not commonly expressed in ULMS. These uniquely expressed markers may assist in stratifying patients by survival and identify novel therapeutic markers. Clearly, further investigation is needed.

So-called inflammatory myofibroblastic tumour: a proliferative lesion of fibroblastic reticulum cells?

The term inflammatory pseudotumour was originally used in a generic fashion for any lesion which simulated a neoplastic condition at a clinical, macroscopic and microscopic level but which was thought to have an inflammatory/reactive pathogenesis. In more recent times, the term has been employed in a more restrictive sense for a mass lesion characterized microscopically by the proliferation of a spindle cell component against a heavy inflammatory infiltrate of mixed composition but usually with a predominance of mature lymphocyte and plasma cells. The spindle cell component has generally been regarded as being of mesenchymal nature and having morphological and phenotypical features consistent with fibroblasts or myofibroblasts, the latter cell being clearly preferred over the former in the more resent reports. The term inflammatory myofibroblastic tumour (IMFT) is the one currently favoured, which proposes the myofibroblastic nature of the process. It is the purpose of this review to bring forth some evidence that the neoplastic spindle cell component of IMFT may be instead derived from the subtype of cells of the accessory immune system that have been variously called fibroblastic reticulum cells, myoid cells, and dictyocytes.

Evolution of Hoxa-11 in lineages phylogenetically positioned along the fin-limb transition.

HOXA11 is a transcription factor implicated in paired appendage development. To identify signatures of evolutionary change in the structural, and putative functional, domains of HOXA11, we studied its evolution in tetrapod and nontetrapod lineages that represent approximately 1.5 billion years of evolutionary time. Here, Hoxa-11 gene proper sequences were determined for frog (Xenopus tropicalis), coelacanth (Latimeria chalumnae), common zebrafish (Danio rerio; Hoxa-11a and Hoxa-11b paralogs), and giant zebrafish (D. aequipinnatus; Hoxa-11b) and aligned against previously published Hoxa-11 sequences of human, mouse, chick, and newt. Based on aligned Hoxa-11 amino acid sequences, the protein was demarcated into three segments: Domains I (N-terminal) and III (homeobox + C-terminal), which varied slightly in rates and patterns of evolution, and a variable, overall hydrophilic region (HyD), which partially overlaps with Domain I. As judged by character reconstructions of HOXA11 Domains I and III, no significant changes in rates of coding sequence evolution occurred in tetrapods (frog and chick), relative to coelacanth (a lobe-finned fish), i.e., across the fin-limb transition. Accelerated rates of Hoxa-11 coding sequence evolution were observed for the mammalian and newt lineages. This was shown to be a gene-specific phenomenon. The duplicated Hoxa-11a and Hoxa-11b genes of zebrafish exhibited accelerated rates of evolution and accumulated substitutions at sites that are conserved among coelacanth and all tetrapods examined. Amino acid sequence comparisons of the HyD of HOXA11 suggested that a putative repressor subdomain, containing stretches of consecutive alanine residues, emerged within the tetrapods. A high degree of nucleotide conservation in the 5' half of the Hoxa-11 intron was observed for tetrapod and nontetrapod lineages. Using electrophoretic mobility shift assays, a 35-bp intron sequence, which is 100% conserved in all Hoxa-11 loci except for the zebrafish Hoxa-11a paralog, was found to bind protein(s) in HeLa and chick whole-cell extracts.

Heterogeneity in fetal akinesia deformation sequence (FADS): autopsy confirmation in three 20-21-week fetuses.

Fetal akinesia deformation sequence (FADS) is a rare condition characterized by intrauterine growth retardation (IUGR), congenital limb contractures, pulmonary hypoplasia, hydramnios and craniofacial abnormalities. The present report comprises an autopsy study of three fetuses to illustrate the variable clinical manifestations and neuropathological findings. Fetus 1 had arthrogryposis and no movement on fetal ultrasound examination. Aborted at 21 weeks, the fetus showed micrognathia, bilateral joint contracture with pterygia at the elbow and axilla. Growth retardation and pulmonary hypoplasia were not major features. Neuropathologic examination revealed anterior horn cell loss and lateral corticospinal tract degeneration in spinal cord, with marked muscular atrophy. Fetus 2, 20 weeks' gestation, had fetal akinesia, nuchal thickening, left pleural effusion, and Dandy-Walker malformation on ultrasound examination. Autopsy showed low-set ears, ocular hypertelorism, cleft palate, flexion contractures with pterygia over axilla, elbow and groin, pulmonary hypoplasia, Dandy-Walker malformation, unremarkable spinal cord and skeletal muscle. Fetus 3, 21 weeks' gestation, was aborted for fetal akinesia, neck and limb webbing and severe arthrogryposis. At autopsy, similar facial abnormalities, contracture and pterygia in neck and multiple major joints were found. Borderline pulmonary hypoplasia and severe lumbar scoliosis were also present. The brain, spinal cord and muscle were unremarkable. In these three fetuses, the prenatal ultrasound and autopsy findings were characteristic of FADS. Neurogenic spinal muscular atrophy was the basis of fetal akinesia in Case 1. Dandy-Walker malformation was present in Case 2, but the pathogenetic mechanism of fetal akinesia was not clear as spinal cord and muscle histology appeared normal. The etiology of akinesia was undetermined in Case 3; no extrinsic or intrinsic cause was identified.

Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase. Substrate analogue that inhibits compound I formation in a competetive manner.

The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H(2)O(2) in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).

[Pneumatosis cystoides intestinalis associated with intravenous pulse cyclophosphamide treatment for systemic lupus erythematosus].

Pneumatosis cystoides intestinalis (PCI) is an uncommon disease manifestation characterized by the presence of air in the bowel wall. PCI is sometimes observed in patients with progressive systemic sclerosis or mixed connective tissue disease but extremely rare in patients with systemic lupus erythematosus (SLE). We here report a patient with SLE who developed PCI after the treatment with intravenous cyclophosphamide (IVCY). This is the first case that association between IVCY and PCI was suggested. A 51-year-old woman with a 24-year history of SLE was admitted to our hospital because of skin ulcers in the lower legs. She had been receiving prednisolone orally. Laboratory findings on the present admission showed a elevated titer of anti-double stranded DNA antibody and positive LE test. She was successfully treated with three pulses of methylprednisolone followed by two IVCY together with vasodilators for her disease activity of SLE including skin manifestation. Just after the second IVCY, abdominal distention was gradually developed without any other abdominal symptoms, including abdominal pain. Abdominal radiography and computed tomography revealed pneumoperitoneum and multiple intramural air collections which involved the ascending colon primarily. Gastrointestinal series, however, showed no evidence of intestinal perforation. The diagnosis of PCI was made radiologically. After she was treated with a combined therapy with intravenous hyperalimentation and breathing with high concentration of oxygen for three weeks, PCI and pneumoperitoneum disappeared. It would be necessary that IVCY is carefully administrated, especially for the patients under the risk of PCI, such as collagen diseases.

Gelatinous bone marrow transformation complicated by self-induced malnutrition.

A 48-year-old woman with a history of anorexia nervosa was admitted to our hospital because of malaise, anorexia and edema in the face and legs. She was diagnosed with gelatinous bone marrow and iron deficiency anemia due to severe malnutrition. She was intravenously treated by saccharated ferric oxide and her anemia was improved, but her bone marrow still showed much gelatinous material.