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26.  Dermal wound transcriptomic responses to Infection with Pseudomonas aeruginosa versus Klebsiella pneumoniae in a rabbit ear wound model 
Background
Bacterial infections of wounds impair healing and worsen scarring. We hypothesized that transcriptome analysis of wounds infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) would indicate host-responses associated with the worse healing of P.a.- than K.p.-infected wounds.
Methods
Wounds created on post-operative day (POD) 0 were infected during the inflammatory phase of healing on POD3 and were harvested on POD4 for microarray and transcriptome analysis. Other wounds received topical antibiotic after infection for 24 hours to promote biofilm development, and were harvested on POD6 or POD12.
Results
Wounds infected for 24 hours, relative to uninfected wounds, elevated transcripts of immune-response functions characteristic of infiltrating leukocytes. But P.a.-infected wounds elevated many more transcripts and to higher levels than K.p.-infected wounds. Coincidently, suppressed transcripts of both wounds enriched into stress-response pathways, including EIF2 signaling; however, this was more extensive for P.a.-infected wounds, including many-fold more transcripts enriching in the ‘cell death’ annotation, suggesting resident cutaneous cell toxicity in response to a more damaging P.a. inflammatory milieu. The POD6 wounds were colonized with biofilm but expressed magnitudes fewer immune-response transcripts with no stress-response enrichments. However, elevated transcripts of P.a.-infected wounds were inferred to be regulated by type I interferons, similar to a network unique to P.a.-infected wounds on POD4. On POD12, transcripts that were more elevated in K.p.-infected wounds suggested healing, while transcripts more elevated in P.a.-infected wounds indicated inflammation.
Conclusions
An extensive inflammatory response of wounds was evident from upregulated transcripts 24 hours after infection with either bacterium, but the response was more intense for P.a.- than K.p.-infected wounds. Coincidently, more extensive down-regulated transcripts of P.a.-infected wounds indicated a stronger “integrated stress response” to the inflammatory milieu that tipped more toward cutaneous cell death. Unique to P.a.-infected wounds on POD4 and POD6 were networks inferred to be regulated by interferons, which may result from intracellular replication of P.a. These data point to specific downregulated transcripts of cells resident to the wound as well as upregulated transcripts characteristic of infiltrating leukocytes that could be useful markers of poorly healing wounds and indicators of wound-specific treatments for improving outcomes.
doi:10.1186/1472-6890-14-20
PMCID: PMC4101837  PMID: 25035691
27.  A case of EDTA-dependent pseudothrombocytopenia: simple recognition of an underdiagnosed and misleading phenomenon 
Background
EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is a common laboratory phenomenon with a prevalence ranging from 0.1-2% in hospitalized patients to 15-17% in outpatients evaluated for isolated thrombocytopenia. Despite its harmlessness, EDTA-PTCP frequently leads to time-consuming, costly and even invasive diagnostic investigations. EDTA-PTCP is often overlooked because blood smears are not evaluated visually in routine practice and histograms as well as warning flags of hematology analyzers are not interpreted correctly. Nonetheless, EDTA-PTCP may be diagnosed easily even by general practitioners without any experiences in blood film examinations. This is the first report illustrating the typical patterns of a platelet (PLT) and white blood cell (WBC) histograms of hematology analyzers.
Case presentation
A 37-year-old female patient of Caucasian origin was referred with suspected acute leukemia and the crew of the emergency unit arranged extensive investigations for work-up. However, examination of EDTA blood sample revealed atypical lymphocytes and an isolated thrombocytopenia together with typical patterns of WBC and PLT histograms: a serrated curve of the platelet histogram and a peculiar peak on the left side of the WBC histogram. EDTA-PTCP was confirmed by a normal platelet count when examining citrated blood.
Conclusion
Awareness of typical PLT and WBC patterns may alert to the presence of EDTA-PTCP in routine laboratory practice helping to avoid unnecessary investigations and over-treatment.
doi:10.1186/1472-6890-14-19
PMCID: PMC4012027  PMID: 24808761
Thrombocytopenia; Laboratory hematology; Hematology analyzers
28.  Reference ranges for organ weights of infants at autopsy: results of >1,000 consecutive cases from a single centre 
Background
Infancy is the most common period for childhood death, including both neonatal deaths from obstetric or medical complications and sudden unexpected infant deaths. The weighing of organs at autopsy is an established process and is recommended in current protocols. However, minimal contemporary data is available regarding reference ranges for organ weights of infants.
Methods
Organ weight data for consecutive infant autopsies over a 14 year period performed at a single tertiary centre, including >1,000 cases, were examined in order to provide up to date reference ranges across this age range, using linear regression modelling and the standard LMS method.
Results
1,525 infant autopsies were analysed, of which 1,190 were subsequently used in the creation of linear regression models prior to performance of the LMS method. Organ weight charts were produced for the 5th, 25th, 50th, 75th and 95th centiles for the heart, lungs, liver, spleen, kidneys, pancreas, thymus gland and adrenal glands.
Conclusion
This study provides the largest single centre contemporary dataset of infant autopsies allowing provision of up-to-date ‘normal’ ranges for all major organ weights across this age range.
doi:10.1186/1472-6890-14-18
PMCID: PMC4017708  PMID: 24822034
Infant; Autopsy; Postmortem; Organ weights
29.  Pleomorphic lobular carcinoma of the male breast with axillary lymph node involvement: a case report and review of literature 
Background
Carcinoma of the male breast is responsible for less than 1% of all malignancies in men but the incidence is rising. Invasive ductal carcinoma is the most common histological subtype while invasive lobular carcinoma is responsible for only 1.5% of the total cases of which pleomorpic lobular carcinoma is an extremely rare variant. We report the case of a gentleman with node positive, pleomorphic lobular carcinoma of the breast.
Case presentation
An elderly gentleman with a past history of type 2 diabetes and long term ethanol use presented to us with a self-discovered palpable lump in the left breast. Physical examination revealed bilateral gynaecomastia along with a well circumscribed subareolar mass and palpable lymphadenopathy in the ipsilateral axilla. The breast nodule revealed atypical cells on fine needle aspiration biopsy and the patient underwent a modified radical mastectomy after systemic surveillance was negative for metastatic disease. The lesion was reported as grade III pleomorphic lobular carcinoma with a lack of E-cadherin expression on immunohistochemistry and the neoplastic cells exhibited strong positivity for estrogen receptor in the absence of Her2 gene amplification. Six out of the eleven dissected regional lymph nodes showed evidence of disease. The patient completed 4 cycles of adjuvant chemotherapy without evidence of recurrent disease and was subsequently lost to follow up.
Conclusions
Although invasive lobular carcinomas comprise 12% of all female breast cancers, they are very rare in males due to lack of acini and lobules in the normal male breast. Pleomorphic lobular carcinoma, an aggressive variant of ILC is even rarer in males.
Chronic consumption of ethanol by our patient may have resulted in some degree of hepatic impairment with resultant hyperestrogenism. This in theory may have been the cause of his gynaecomastia, resultant breast cancer and is a plausible explanation for development of the invasive lobular subtype in a male. The prognosis and clinicopatholocial features of pleomorphic lobular carcinoma in men are less clearly defined due to its rarity. Additional studies are hence necessary to improve our understanding of this disease in males.
doi:10.1186/1472-6890-14-16
PMCID: PMC4008413  PMID: 24795533
Male breast cancer; Pleomorphic lobular carcinoma; E-cadherin
30.  Diagnostic testing managed by hematopathology specialty and other laboratories: costs and patient diagnostic outcomes 
Background
Successful management of patients with hematologic malignancies depends upon accurate and timely diagnosis, which frequently requires integration and interpretation of multiple tests. Our retrospective analysis compared diagnostic uncertainty, resource utilization, and costs for patients with diagnostic bone marrow (BM) tests managed by commercial laboratories.
Methods
Patients with BM biopsies and suspected hematologic cancer/condition were identified from claims (2005–2011) within a large US health plan (coverage ≥6 pre- and ≥3-months post-biopsy). Cohorts defined by laboratories performing BM morphologic assessment/directing testing sequence: Genoptix (GX, specialty hematology-testing laboratory), large commercial laboratories (LL), other laboratories (OL). One-year post-biopsy changes in diagnosis or treatments, tests performed, and diagnostic/treatment medical costs (measured as per-patient-per-month [PPPM]) were examined.
Results
The study population included 1,387 GX, 4,162 LL, and 19,115 OL patients with suspected hematologic malignancy/disease and BM morphology assessment. GX had lower diagnostic uncertainty measured between 2 time periods by diagnostic stability (no conditions the same; 6.16% GX, 8.04% LL, 9.73% OL; p < 0.001) and changes (≥1 condition different; 7.88% GX, 11.19% LL, and 14.08% OL; p < 0.001), fewer repeat BM biopsies, and fewer chemotherapy changes (30-days and 60-days post-initiation). One-year PPPM costs adjusted for patient characteristics differences were $8,202 GX, $7,711 LL, and $10,302 OL (p < 0.05); adjusted PPPM costs (excluding testing period) were $6,019 GX, $6,649 LL, and $7,801 OL (p < 0.05).
Conclusions
Our data suggests that a hematopathology specialty laboratory may result in earlier final diagnosis, fewer subsequent diagnosis changes, reduced need for follow-on testing requiring repeat biopsy procedures, and may result in lower downstream healthcare costs. Further evaluations using medical chart abstractions or registries will be valuable.
doi:10.1186/1472-6890-14-17
PMCID: PMC4016629  PMID: 24817828
Diagnostic laboratory tests; Diagnostic costs; Hematopathology; Leukemia; Lymphoma
31.  Galectin-1 and Galectin-3 mRNA expression in renal cell carcinoma 
Background
Galectins are known to regulate cell differentiation and growth as well as cell adhesion and apoptosis. Galectins have been discussed as possible prognosticators for survival in renal cell cancer (RCC) and other urological tumors. They might also play an emerging role as possible new marker-proteins for RCC. In this study, we analyzed the expression of galectin-1 and galectin-3 mRNA in order to further investigate their clinical significance in RCC.
Methods
Tissue samples were obtained from 106 patients undergoing surgery for RCC. The expression of galectin-1 and galectin-3 mRNA in normal kidney and corresponding cancer tissue was analyzed using quantitative real time PCR. Differences in expression levels of paired tissue samples were assessed using paired two-sample tests. Associations of relative mRNA expression levels in tumor tissues with clinical findings were analyzed using univariate logistic regression.
Results
The expression of galectin-1 (p < 0.001) and -3 (p < 0.001) mRNA were significantly higher in RCC when compared to the adjacent normal kidney tissue. For clear cell RCC, an association of male gender with higher galectin-1 and galectin-3 mRNA expression (p = 0.054, p = 0.034) was detected. For all RCCs, galectin-1 mRNA expression failed to show a significant association with advanced disease as well as a higher rate of lymph node metastases (p = 0.058, p = 0.059).
Conclusion
The mRNA expression of galectin-1 and galectin-3 is significantly increased in RCC cancer tissue. The higher mRNA expression in tumor tissue of male patients raises the question of a functional connection between galectins and the higher prevalence of RCC in men. Associations with advanced disease might lead to new ways of identifying patients at higher risk of recurrent disease and might even facilitate early metastasectomy with curative intent.
doi:10.1186/1472-6890-14-15
PMCID: PMC4026056  PMID: 24708743
Galectin; Renal cell carcinoma; Biomarker; Prognosis
32.  Clinico-pathological features of tuberculosis due to Mycobacterium tuberculosis Uganda genotype in patients with tuberculous lymphadenitis: a cross sectional study 
Background
Tuberculous lymphadenitis is next to pulmonary tuberculosis as the most common cause of tuberculosis. Uganda genotype, one of the sub-lineages of Mycobacterium tuberculosis, is the most prevalent cause of pulmonary tuberculosis in Uganda. We here investigate the clinicopathological characteristics of patients with tuberculous lymphadenitis infected with M. tuberculosis Uganda genotype compared with those infected with M. tuberculosis non-Uganda genotype strains.
Methods
Between 2010 and 2012, we enrolled 121 patients (mean age 28.5 yrs, male 48%; female 52%) with tuberculous lymphadenitis, and categorized them by their M. tuberculosis genotypes. The clinical features and lymph node cytopathological parameters were compared between patients in the Uganda and non-Uganda categories using a crude and multivariable logistic regression model with adjustment for confounding factors.
Results
Of the 121participants, 56 (46%) were infected with strains of Uganda genotype. Patients infected with this genotype had significantly lower frequency of abdominal lymphadenopathy (odds ratio 0.4, p = 0.046) after adjusting for sex, age and HIV. Abdominal lymphadenopathy was also significantly associated with abnormal chest X-ray (p = 0.027).
Conclusion
Tuberculous lymphadenitis patients infected with M. tuberculosis Uganda genotype were significantly less prone to have abdominal lymphadenopathy indicating potential reduced ability to disseminate and supporting the concept that differences in M. tuberculosis genotype may have clinical implications.
doi:10.1186/1472-6890-14-14
PMCID: PMC3974774  PMID: 24690344
Tuberculosis; Extra pulmonary; Mycobacterium; Genotype; Abdominal lymphadenopathy; Uganda
33.  Ultrastructural features of neuroblastic tumours in relation to morphological, and molecular findings; a retrospective review study 
Background
Neuroblastoma is the most common solid tumour of infancy and is responsible for 15% of childhood cancer deaths. Presence of amplified MYCN in neuroblastoma is associated with poor prognosis and rapid tumour progression. The aim of this study was to examine and compare the ultrastructural features of high-risk MYCN amplified neuroblastomas, with lower-risk non-MYCN amplified tumours.
Methods
This was a retrospective study evaluating archival diagnostic tissue samples, in which Fluorescence in-situ hybridisation (FISH) had been used at diagnosis to establish MYCN status. 22 (11 MYCN amplified tumours and 11 non-MYCN amplified) tumours of similar light microscopic appearance (poorly differentiated neuroblastoma) were then selected for ultrastructural examination.
Results
There is a relationship between ultrastructural features in neuroblastoma and MYCN status, although with marked overlap between groups. MYCN amplified tumours generally exhibited a ‘less differentiated’ ultrastructural phenotype, with significantly smaller neurotubules (NT) in the cell body (p < 0.002). Non-MYCN amplified tumours show increased features of neuronal differentiation, with fewer neurosecretory granules (NSG) and NT in the cytoplasm.
Conclusions
MYCN amplification is associated with a less differentiated ultrastructural phenotype, and lack of MYCN amplification with relative ultrastructural neuronal differentiation.
doi:10.1186/1472-6890-14-13
PMCID: PMC4012145  PMID: 24679140
Neuroblastoma; Ultrastructure; Electron microscopy; Molecular biology; MYCN
34.  Colorectal cancer in patients seen at the teaching hospitals of Guadeloupe and Martinique: discrepancies, similarities in clinicopathological features, and p53 status 
Background
In Guadeloupe and Martinique, two French Overseas Departments, colorectal cancer (CRC) has become an essential public health issue. However, little is known about CRC characteristics and the p53 status in these populations, particularly in Guadeloupe, whereas certification of a cancer registry has been recently validated.
Methods
This was a descriptive retrospective study of 201 patients who, between 1995 and 2000, underwent surgery for CRC in the Guadeloupe Teaching Hospital (GlpeTH; 83 patients) and in the Martinique Teaching Hospital (MqueTH; 118 patients). The clinicopathological features and the p53 expression, evaluated with immunohistochemistry, were compared at the time of diagnosis. A relationship between these parameters and the p53 expression was also studied. Data were analysed, using the SPSS computer software version 17.0.
Results
No statistical difference was found between the two groups of patients regarding age (p = 0.60), percentage of young patients (≤50 years; p = 0.94)), sex (p = 0.47), histological type (p = 0.073) and tumour sites (p = 0.65), although the GlpeTH patients were diagnosed with more distal colon cancers (54.2%) than the Mque TH patients (47.4%). By contrast, a significant difference was found regarding the tumour grade (p < 0.0001), the pTNM stage (p = 0.045) and the pT stage (p < 0.0001). Regarding p53 expression, solely for the MqueTH patients, nuclear expression was associated with pTNM, the percentage of p53 negative tumours increasing with the progression of the pTNM stages (p = 0.029).
Conclusions
For the first time, this study reveals discrepancies in clinicopathological features and in the p53 status between the two groups of patients. The GlpeTH patients were diagnosed with more moderated CRCs but with few CRCs at pTNM IV stage. By contrast, the MqueTH patients were diagnosed with more differentiated tumours, but with many more CRCs at pTNM IV stage. This paradox may be due to differences in tumour location (distal vs proximal), multiplicity of the genetic profiles of patients, or patients getting treatment elsewhere. Although our study is limited due to its small size, it emphasizes the originality of our results.
doi:10.1186/1472-6890-14-12
PMCID: PMC3973004  PMID: 24679126
Colorectal cancer; Guadeloupian patients; Martinican patients; Clinicopathology; p53 status; Discrepancy
35.  DKK-1 in prostate cancer diagnosis and follow up 
Background
Dickoppf-1 (DKK-1) is a negative regulator of bone formation with tumorigenic potential. The up-regulation of DKK-1 is an early event in prostate cancer (PCa) development, thus we investigated its role as a marker in the diagnosis and prognosis of PCa.
Methods
We retrospectively enrolled 159 patients who underwent prostate biopsy, either for elevated PSA or suspect digital rectal examination, between 2003 and 2010. During the biopsy, one serum sample was collected from all patients; PSA and DKK-1 were measured by ELISA technique. Amongst the biopsy of 159 patients 75 were affected by PCa and 84 were not the mean period of follow-up for these patients was 5 years; a new biopsy was performed in case of PCa suspicion.
Results
PSA performed better than DKK-1 in detecting PCa (0.63 vs 0.51 respectively). Differently from PSA DKK-1 was significantly higher in patients who developed PCa during follow-up than in cancer-free ones, thus DKK-1 performed better than PSA in detecting these patients (0.67 vs 0.55). DKK-1 was significantly lower in patients with bone metastases, whereas PSA was not significantly different in patients with different outcomes.
Conclusions
DKK-1 might be predictive for patients negative at first biopsy who will develop PCa and in the prognosis of bone metastases. It performed worse than PSA in the early diagnosis of Pca.
doi:10.1186/1472-6890-14-11
PMCID: PMC3976154  PMID: 24655661
Prostate cancer; PSA; Dickoppf-1; DKK-1; Bone metastases
36.  Expression of histone deacetylases 1, 2 and 3 in urothelial bladder cancer 
Background
Histone deacetylases (HDACs) are known to be associated with an overexpression in different types of cancer such as colon and prostate cancer. In this study we aimed to evaluate the protein expression of class I HDACs in urothelial carcinoma of the bladder.
Methods
A tissue microarray containing 348 tissuesamples from 174 patients with a primary urothelial carcinoma of the bladder was immunohistochemically stained for HDAC 1, 2 and 3. Intensity of staining was evaluated and the association with clinico-pathological features and prognosis was assessed.
Results
High HDAC expression levels were found in 40 to 60% of all investigated urothelial carcinomas (HDAC-1: 40%, HDAC-2: 42%, HDAC-3: 59%).
HDAC-1 and HDAC-2 were significantly associated with higher tumour grades.
Although all three markers could not predict progression in univariate analyses, high HDAC-1 expression was associated with a trend toward poorer prognosis. Patients with high-grade tumours and high expression levels of HDAC-1 were more likely to progress compared to all other patients (p < 0.05).
Conclusions
High-grade noninvasive papillary bladder tumours are associated with high expression levels of HDAC-1 and HDAC-2. High grade tumours in combination with high expression of HDAC-1 showed a worse prognosis than the other tumours. The high expression levels of HDACs observed particularly in high grade urothelial bladder cancer clearly warrant subsequent studies on the potential use of HDAC inhibitors as a novel therapeutic approach.
doi:10.1186/1472-6890-14-10
PMCID: PMC3995609  PMID: 24624923
Class I HDACs; Urothelial cancer; Molecular markers
37.  High tumor cell expression of microRNA-21 in node positive non-small cell lung cancer predicts a favorable clinical outcome 
Background
MicroRNA (miR)-21 has been revealed as an oncogene in cancer development, and is one of the miRNAs closely connected to angiogenesis. We aimed to explore the impact of miR-21 expression in both tumor and stromal compartments of non-small cell lung cancer (NSCLC), and correlations between miR-21 and angiogenic protein markers.
Methods
From 335 unselected stage I to IIIA NSCLC carcinomas, duplicate tumor and tumor-associated stromal cores were collected in tissue microarrays (TMAs). In situ hybridization (ISH) was used to detect the expression of miR-21 separately in tumor cells and stromal cells of the tumor, and immunohistochemistry (IHC) was used to detect the expression of the protein markers protein kinase B (Akt), phosphatidylinositol-3-kinase (PI3K), hypoxia induced factor 1 (HIF1α) and vascular endothelial growth factor-A (VEGF-A).
Results
In univariate analyses, high tumor cell expression of miR-21 in patients with lymph node metastasis was a positive prognostic factor (P = 0.024). High stromal miR-21 expression had a negative prognostic impact (P = 0.022). In the multivariate analysis, low tumor mir-21 expression in node positive patients was an independent adverse prognostic factor (HR 2.03, CI 95% 1.09-3.78, P = 0.027).
Conclusions
In patients with lymph node metastasis, miR-21 expression in tumor cells is an independent positive prognostic factor. High stromal miR-21 expression is a negative prognostic factor.
doi:10.1186/1472-6890-14-9
PMCID: PMC3931486  PMID: 24524655
NSCLC; Stage I-IIIA; Survival; Prognostic impact; miR-21; miRNA
38.  Identification of potential serum peptide biomarkers of biliary tract cancer using MALDI MS profiling 
Background
The aim of this discovery study was the identification of peptide serum biomarkers for detecting biliary tract cancer (BTC) using samples from healthy volunteers and benign cases of biliary disease as control groups. This work was based on the hypothesis that cancer-specific exopeptidases exist and that their activities in serum can generate cancer-predictive peptide fragments from circulating proteins during coagulation.
Methods
This case control study used a semi-automated platform incorporating polypeptide extraction linked to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to profile 92 patient serum samples. Predictive models were generated to test a validation serum set from BTC cases and healthy volunteers.
Results
Several peptide peaks were found that could significantly differentiate BTC patients from healthy controls and benign biliary disease. A predictive model resulted in a sensitivity of 100% and a specificity of 93.8% in detecting BTC in the validation set, whilst another model gave a sensitivity of 79.5% and a specificity of 83.9% in discriminating BTC from benign biliary disease samples in the training set. Discriminatory peaks were identified by tandem MS as fragments of abundant clotting proteins.
Conclusions
Serum MALDI MS peptide signatures can accurately discriminate patients with BTC from healthy volunteers.
doi:10.1186/1472-6890-14-7
PMCID: PMC3923428  PMID: 24495412
Cholangiocarcinoma; Biliary tract cancer; PSC; Serum peptide biomarker; MALDI-TOF MS
39.  Ephrin (Eph) receptor A1, A4, A5 and A7 expression in human non-small cell lung carcinoma: associations with clinicopathological parameters, tumor proliferative capacity and patients’ survival 
Background
Ephrin (Eph) receptors are frequently overexpressed in a wide variety of human malignant tumors, being associated with tumor growth, invasion, metastasis and angiogenesis. The present study aimed to evaluate the clinical significance of EphA1, A4, A5 and A7 protein expression in non-small cell lung carcinoma (NSCLC).
Methods
EphA1, A4, A5 and A7 protein expression was assessed immunohistochemically in tissue microarrays of 88 surgically resected NSCLC and was analyzed in relation with clinicopathological characteristics and patients’ survival.
Results
Elevated EphA4 expression was significantly associated with low histopathological stage and presence of inflammation (p = 0.047 and p = 0.026, respectively). Elevated EphA7 expression was significantly associated with older patients’ age, presence of fibrosis and smaller tumor size (p = 0.036, p = 0.029 and p = 0.018, respectively). EphA1, A5 and A7 expression were positively associated with tumor proliferative capacity (p = 0.047, p = 0.002 and p = 0.046, respectively). Elevated EphA4, A5 and A7 expression were identified as predictors of favourable patients’ survival at both univariate (Log-rank test, 0 = 0.019, p = 0.006 and p = 0.012, respectively) and multivariate levels (Cox-regression analysis, p = 0.029, p = 0.068 and p = 0.044, respectively).
Conclusions
The present study supported evidence that Ephs may be involved in lung cancer progression, reinforcing their utility as clinical biomarkers for patients’ management and prognosis, as also as potential targets for future therapeutic interventions.
doi:10.1186/1472-6890-14-8
PMCID: PMC4234387  PMID: 24495444
Ephrin receptors; Non-small cell lung carcinoma; Clinicopathological parameters; Prognosis; Immunohistochemistry
40.  Should prophylactic thrombolysis be routine in clinical practice? Evidence from an autopsy case of septicemia 
Background
Central venous catheters provide easy access for intravenous infusion and nutrition, but they can bring about complications such as catheter-related infections. Infected central venous catheters often cause nosocomial bloodstream infections with high morbidity and mortality. However, most of the morphological data that have been published are derived from in vitro and in vivo studies and few reports of direct evidence obtained from patient-derived samples have been described. Here we present visual evidence of catheter-related candidemia. To our knowledge, this is the first reported conventional histopathological evidence of a Candida-infected intraluminal thrombus in a patient’s central venous catheter.
Case presentation
A 62-year-old Japanese female with obstructive jaundice, gastrointestinal bleeding, and liver metastasis from pancreatic head cancer was given an implantable subcutaneous central venous port for nutrition and chemotherapy administration. High fever ensued on day 16 after the central venous port insertion and blood cultures revealed Candida albicans. Although the patient was given 300 mg/day of fosfluconazole according to the suggestion of the infection control team, she died from respiratory failure. Postmortem computed tomography revealed findings consistent with acute respiratory distress syndrome, suggesting that the patient’s course was complicated by catheter-related sepsis. Autopsy revealed a subcutaneous abscess around the port, from which C. albicans was cultured. However, no catheter-adherent thrombus, thrombosis of the great central veins, or endocardial vegetations were detected in the patient. Histological analysis revealed scattered abscesses in several organs including lungs and kidneys. Hyaline membrane formation and Candida colonies were found in the lungs. The central venous port tube, together with the part of the subclavian vein into which it had been inserted, was involved in an intraluminal fibrin thrombus containing neutrophils and macrophages, indicating that the thrombus existed while the patient was alive. Histopathological examination following use of the periodic acid-Schiff reagent and the Grocott stain revealed scattered Candida in the thrombus.
Conclusions
Prophylactic thrombolysis should be encouraged to prevent central venous catheter-related candidiasis in clinical practice.
doi:10.1186/1472-6890-14-6
PMCID: PMC3909761  PMID: 24475758
Autopsy; Candida spp; Central venous catheter; Sepsis; Thrombus; Pathological staining
41.  The VEGF- and PDGF-family of angiogenic markers have prognostic impact in soft tissue sarcomas arising in the extremities and trunk 
Background
Soft-tissue sarcomas are rare malignant tumors of mesenchymal lineage that can arise in any part of the body. Prognosis, and hence also treatment may vary according to histologic subtype and localization. Angiogenesis is the process of forming new blood vessels from pre-existing ones. The deregulation of this process is thought to be an important step in malignant transformation. This study investigates the prognostic impact of platelet derived growth factor- (PDGF), vascular endothelial growth factor- (VEGF) and fibroblast growth factor (FGF) families in soft-tissue sarcomas of the extremities & trunk (ET) and visceral & retroperitoneal (VR) locations.
Methods
Tumor samples from 181 patients (115 ET and 66 VR) with resected soft tissue sarcomas were collected and tissue microarrays were constructed. Immunohistochemistry was used to evaluate angiogenic marker expression. Recurrence-free survival (RFS), metastasis-free survival (MFS) and disease-specific survival (DSS) were used as endpoints in prognostic impact assessment.
Results
In univariate analyses, almost all investigated angiogenic markers had prognostic impact in the ET group. In contrast, only FGFR-1 showed any significant prognostic impact in the VR group. In the multivariate analyses, PDGF-D (HR = 1.863, 95% CI = 1.057-3.283, P = 0.031), VEGFR-1 (HR = 2.106, 95% CI = 1.038-4.272, P = 0.039) and VEGF-A (HR 2.095, 95% CI 1.028-4.271, P = 0.042) were independent negative prognosticators for DSS, MFS and RFS, respectively, in the ET group. FGFR-1 was an independent positive prognosticator for DSS (HR = 0.243, 95% CI = 0.095-0.618, P = 0.003) in the VR group.
Conclusions
Angiogenic molecules from the PDGF and VEGF families have prognostic impact in soft-tissue sarcomas arising in the ET, but not in VR locations. In the latter histological grade and resection margins are the most important prognostic factors.
doi:10.1186/1472-6890-14-5
PMCID: PMC3898209  PMID: 24444363
Angiogenesis; Sarcoma; Extremity; Trunk; FGF; PDGF; VEGF; Visceral; Retroperitoneal
42.  Annual acknowledgement of manuscript reviewers 
Contributing reviewers
The editors of BMC Clinical Pathology would like to thank all of our reviewers who have contributed to the journal in Volume 13 (2013).
doi:10.1186/1472-6890-14-2
PMCID: PMC3897993  PMID: 24433578
43.  First North American case of Hemoglobin Shepherds Bush (β 74[E18] Gly → Asp) in a central Pennsylvania family 
Background
Hemoglobin Shepherds Bush (Human Genome Variation Society name: HBB:c.224G > A) is an unstable hemoglobin variant resulting from a β 74 GGC to GAC mutation (Gly to Asp) that manifests clinically as hemolytic anemia or gall bladder disease due to chronic subclinical hemolysis.
Case presentation
We report a Pennsylvania family of English descent with this condition, first noticed in a 6-year-old female. The proband presented with splenomegaly, fatigue, dark urine and an elevated indirect bilirubin. Hemoglobin identification studies and subsequent genetic testing performed according to a systematic algorithm elucidated the diagnosis of Hb Shepherds Bush.
Conclusions
This is the first case of this rare hemoglobin variant identified in North America to our knowledge. It was identified using a systematic algorithm of diagnostic tests that should be followed whenever considering a rare hemoglobinopathy as part of the differential diagnosis.
doi:10.1186/1472-6890-14-4
PMCID: PMC3898211  PMID: 24428873
Hemoglobin; Shepherds bush; Hemolytic anemia
44.  Increased fatty acid synthase expression in prostate biopsy cores predicts higher Gleason score in radical prostatectomy specimen 
Background
Fatty acid synthase (FAS) is highly expressed in various types of cancer, and elevated expression of FAS has been suggested to be a predictor of tumor aggressiveness and poor prognosis. We examined whether FAS expression in prostate biopsy cores could predict the pathological characteristics of radical prostatectomy (RP) specimens.
Methods
Paraffin-embedded prostate biopsy cores, obtained from 102 patients who subsequently underwent RP, were immunostained with polyclonal anti-FAS antibody. The staining intensity was categorized into non-staining, weak, moderate, and strong. Tumors with moderate or strong immunostaining were considered to show high FAS expression, and other tumors were considered to show low FAS expression. The relation between the FAS expression status in biopsy cores and pathological parameters in RP specimens was analyzed.
Results
The FAS expression in the biopsy cores of 64 of the 102 tumors (63%) was high, whereas it was low in the biopsy cores of the other 38 tumors (37%). High FAS expression was significantly associated with Gleason Score (GS) ≥ 7 in RP specimens (p< 0.0001). In multivariable logistic regression analyses, GS ≥7 in biopsy cores (p <0.0001), higher preoperative PSA (p = 0.0194), and high FAS expression (p = 0.0004) were independent predictors of GS ≥ 7 in the RP specimen.
Conclusions
Increased FAS expression in prostate biopsy cores could be a novel parameter for predicting higher GS in RP specimens. The treatment strategy for patients with high FAS expression in prostate biopsy cores should be carefully determined.
doi:10.1186/1472-6890-14-3
PMCID: PMC3898069  PMID: 24422874
Fatty acid synthase; Prostate cancer; Biopsy core; Gleason score
45.  Sulfotransferase 1A1 (SULT1A1) gene expression is regulated by members of the NFI transcription factors in human breast cancer cells 
Background
Sulfotransferase 1A1 (SULT1A1) gene expression is tissue specific, with little to no expression in normal breast epithelia. Expression in breast tumors has been documented, but the transcriptional regulation of SULT1A1 in human breast tissue is poorly understood. We identified Nuclear Factor I (NFI) as a transcription factor family involved in the regulation of SULT1A1 expression.
Methods
Transcription Factor Activation Profiling Plate Array assay was used to identify the possible transcription factors that regulate the gene expression of SULT1A1in normal breast MCF-10A cells and breast cancer ZR-75-1 cells. Expression levels of NFI-C and SULT1A1 were determined by real-time RT-PCR using total RNA isolated from 84 human liver samples. Expression levels of SULT1A1, NFI-A, NFI-B, NFI-C, and NFI-X were also determined in different human breast cancer cell lines (MCF-7, T-47D, ZR-75-1, and MDA-MB-231), in the transformed human epithelial cell line MCF-10A, and in ZR-75-1 cells that were transfected with siRNAs directed against NFI-A, NFI-B, NFI-C, or NFI-X for 48 h. The copy numbers of SULT1A1 in cell lines ZR-75-1, MCF-7, T-47D, MDA-MB-231, and MCF-10A were determined using a pre-designed Custom Plus TaqMan® Copy Number kit from Life Technologies.
Results
In normal human liver samples, SULT1A1 mRNA level was positively associated with NFI-C. In different human breast cancer and normal epithelial cell lines, SULT1A1 expression was positively correlated with NFI-B and NFI-C. SULT1A1 expression was decreased 41% and 61% in ZR-75-1 cells treated with siRNAs against NFI-A and NFI-C respectively. SULT1A1 gene expression was higher in cells containing more than one SULT1A1 copy numbers.
Conclusions
Our data suggests that SULT1A1 expression is regulated by NFI, as well as SULT1A1 copy number variation in human breast cancer cell lines. These data provide a mechanistic basis for the differential expression of SULT1A1 in different tissues and different physiological states of disease.
doi:10.1186/1472-6890-14-1
PMCID: PMC3913331  PMID: 24393253
SULT1A1; NFI; Human breast cancer cell lines; Gene expression regulation; siRNA transfection; Gene copy number
46.  Efficient and reproducible identification of mismatch repair deficient colon cancer: validation of the MMR index and comparison with other predictive models 
Background
The identification of mismatch-repair (MMR) defective colon cancer is clinically relevant for diagnostic, prognostic and potentially also for treatment predictive purposes. Preselection of tumors for MMR analysis can be obtained with predictive models, which need to demonstrate ease of application and favorable reproducibility.
Methods
We validated the MMR index for the identification of prognostically favorable MMR deficient colon cancers and compared performance to 5 other prediction models. In total, 474 colon cancers diagnosed ≥ age 50 were evaluated with correlation between clinicopathologic variables and immunohistochemical MMR protein expression.
Results
Female sex, age ≥60 years, proximal tumor location, expanding growth pattern, lack of dirty necrosis, mucinous differentiation and presence of tumor-infiltrating lymphocytes significantly correlated with MMR deficiency. Presence of at least 4 of the MMR index factors identified MMR deficient tumors with 93% sensitivity and 76% specificity and showed favorable reproducibility with a kappa value of 0.88. The MMR index also performed favorably when compared to 5 other predictive models.
Conclusions
The MMR index is easy to apply and efficiently identifies MMR defective colon cancers with high sensitivity and specificity. The model shows stable performance with low inter-observer variability and favorable performance when compared to other MMR predictive models.
doi:10.1186/1472-6890-13-33
PMCID: PMC3878549  PMID: 24341444
Pathology; Colorectal cancer; Microsatellite instability; Mismatch repair; Prediction model
47.  An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas 
Background
Inactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies.
Methods
We analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression.
Results
50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene.
Conclusions
The results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.
doi:10.1186/1472-6890-13-32
PMCID: PMC4028812  PMID: 24330579
Liposarcoma; MDM2; MDMX; P53; Targeted therapy
48.  The relationship between lymphovascular invasion and angiogenesis, hormone receptors, cell proliferation and survival in patients with primary operable invasive ductal breast cancer 
Background
Several well-established tumour prognostic factors are used to guide the clinical management of patients with breast cancer. Lymphovascular invasion and angiogenesis have also been reported to have some promise as prognostic factors. The aim of the present study was to examine the prognostic value of tumour lymphovascular invasion and microvessel density compared with that of established prognostic factors in invasive ductal breast cancer.
Methods
In addition to hormone receptor status and Ki-67 proliferative activity, lymphovascular invasion and microvessel density and their relationship with survival were examined in patients with invasive ductal breast cancer. Full sections and tissue microarrays (n = 384 patients) were utilised to assess these factors and were scored by appropriate methods.
Results
On univariate analysis tumour size (P < 0.05), lymph node involvement (P < 0.01), lymphovascular invasion (P < 0.05), microvessel density (P < 0.05) and local- regional treatment (P < 0.01) were associated with poorer survival in ER negative tumours. On multivariate analysis in ER negative tumours lymph node involvement (P < 0.01) and local- regional treatment (P < 0.05) were independently associated with poorer cancer-specific survival. On univariate analysis tumour grade (P < 0.05), lymph node involvement (P < 0.001), HER-2 (P < 0.05), Ki-67 (P < 0.01) and lymphovascular invasion (P < 0.001) were associated with poorer survival in ER positive tumours. On multivariate analysis lymph node involvement (P < 0.001), Ki-67 (P < 0.001) and lymphovascular invasion (P < 0.05) were independently associated with poorer cancer-specific survival in ER positive tumours.
Conclusion
Lymphovascular invasion but not microvessel density was independently associated with poorer survival in patients with ER positive but not ER negative invasive ductal breast cancer.
doi:10.1186/1472-6890-13-31
PMCID: PMC4175483  PMID: 24274633
Primary invasive breast cancer; Prognostic factors; Lymphovascular invasion; Angiogenesis; Survival
49.  Metabolic markers GAPDH, PKM2, ATP5B and BEC-index in advanced serous ovarian cancer 
Background
A deregulated energy metabolism is a hallmark of malignant disease that offers possible future targets for treatment. We investigated the prognostic value of the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase type M2 (PKM2), mitochondrial β-F1-ATPase (ATP5B) and the bioenergetic cellular (BEC) index in advanced ovarian cancer.
Methods
Fresh tumor samples were prospectively collected from 123 patients undergoing primary surgery for suspected advanced ovarian cancer. Of these, 57 met the eligibility criteria; stage IIC-IV, serous or endometrioid subtype, specimens containing ≥ 50% tumor cells and patients receiving platinum-based chemotherapy. An adequate amount of mRNA could be extracted in all but one case, with a resultant study population of 56 patients. Eighty-six percent of cases had serous tumors, and 93% were grade 2–3. GAPDH, PKM2 and ATP5B mRNA- and protein expression was assessed by real-time PCR and immunohistochemistry. We estimated the association with platinum-free interval (PFI) and overall survival (OS) by Cox proportional hazards models. Median follow-up was 60 months.
Results
High GAPDH mRNA levels (HR 2.1, 95% CI 1.0-4.5) and low BEC-index (HR 0.47, 95% CI 0.23-0.95) were both independently associated with shorter PFI. Median PFI for patients with high GAPDH mRNA was 5.0 months compared to 10.1 months for low expression cases (p = 0.031). Similarly, median PFI for patients with low BEC-index based on mRNA was 5.3 months compared to 9.8 months for high BEC-index cases (p = 0.028).
Conclusions
High GAPDH or low BEC-index mRNA expression indicate early disease progression in advanced serous ovarian cancer.
doi:10.1186/1472-6890-13-30
PMCID: PMC3874631  PMID: 24252137
Metabolic markers; GAPDH; PKM2; ATP5B; BEC; Ovarian cancer; Immunohistochemistry; Real-time PCR
50.  A mill based instrument and software system for dissecting slide-mounted tissue that provides digital guidance and documentation 
Background
Dissection of specific Areas Of Interest (AOIs) of slide-mounted tumor samples is often used to enrich for cancer cells in order to generate better signal to noise ratios in subsequent biochemical characterization. Most clinical laboratories utilize manual dissection for practical reasons and to avoid the expense and difficulties of laser microdissection systems. Unfortunately, manual methods often lack resolution and process documentation. The goal of this project was to design a dissection system for slide-mounted tissue with better precision than manual methods that also provides digital image guidance and electronic process documentation.
Methods
An instrument that is essentially a micro tissue mill was developed. It employs a specialized disposable mill bit that simultaneously dispenses liquid, cuts tissue from the slide surface, and aspirates the liquid along with the displaced tissue fragments. A software package was also developed that is capable of transferring digitally annotated AOIs between images of serially cut tissue sections to guide dissection and generate an electronic record of the process.
Results
The performance of this “meso” dissection system was tested using post dissection visual examination for resolution and accuracy, fluorescence based DNA quantitation for recovery efficiency, and dissection of closely situated mouse-human tissue sections followed by PCR amplification for purity determination. The minimum resolution is a dissected circle smaller than 200 microns in diameter, edge dissection accuracy is tighter than 100 microns, recovery efficiency appears greater than 95%, and recovery purity is greater than 99% relative to a different tissue located 100 microns from the dissection boundary. The system can dissect from both paraffinized and deparaffinized FFPE tissue sections that are mounted on plain glass slides, and it is compatible with DNA, RNA, and protein isolation.
Conclusions
The mesodissection system is an effective alternative to manual dissection methods and is applicable for biomarker analysis of anatomical pathology samples, where enrichment of AOIs from the tissue section is helpful, but pure cell populations are not required.
doi:10.1186/1472-6890-13-29
PMCID: PMC3826833  PMID: 24188417
Slide-mounted tissue dissection; Microdissection; Mesodissection; Macrodissection; Digital pathology; Anatomic pathology tracking; FISH

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