Airway Mucus Hyperconcentration in Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.

CD169(+) macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer.

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune-competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169(+) macrophages using a suicide gene knock-in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer-induced bone changes. In human bone metastasis specimens, CD68(+) macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune-competent mouse model, CD169(+) macrophage ablation significantly inhibited prostate cancer-induced woven bone formation, suggesting that CD169(+) macrophages within pathological woven bone are integral to tumour-induced bone formation. In contrast, pan-phagocytic cell, but not targeted CD169(+) macrophage depletion resulted in increased tumour mass, indicating that CD169(-) macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour-induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased urinary podocytes following indomethacin suggests drug-induced glomerular injury.

BACKGROUND: Preterm infants are delivered while glomerulogenesis is ongoing and may be exposed to insults, including medications that may affect renal development. Podocytes detected in the urine are an indicator of glomerular injury. The aims of this study were to determine whether preterm and term infants excrete podocytes in their urine and whether exposure to gentamicin and indomethacin increase podocyte excretion in their urine. METHODS: Preterm infants <33 weeks gestation had urine collected each day while receiving either gentamicin or indomethacin. Preterm and term control infants had urine collected for 3 days. The number of casts and podocytes present in the urine of infants receiving indomethacin and gentamicin were compared with preterm and term control infants. RESULTS: Forty-two neonates were included in the study. Podocytes were present in small numbers (< 2) in the urine of both preterm and term control neonates. The number of podocytes in the preterm group receiving indomethacin was significantly higher than in all other groups (p=0.02) ,as was urinary albumin (p=0.02). CONCLUSIONS: Increased number of podocytes in preterm neonates receiving indomethacin and higher excretion of albumin suggest glomerular injury is occurring. It is unknown whether injury to glomeruli during glomerulogenesis in preterm neonates has long-term sequelae for renal development and function into adulthood.

Disruption of Glycogen Utilization Markedly Improves the Efficacy of Carboplatin against Preclinical Models of Clear Cell Ovarian Carcinoma.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

CDCP1 enhances Wnt signaling in colorectal cancer promoting nuclear localization of ß-catenin and E-cadherin.

Elevated CUB-domain containing protein 1 (CDCP1) is predictive of colorectal cancer (CRC) recurrence and poor patient survival. While CDCP1 expression identifies stem cell populations that mediate lung metastasis, mechanisms underlying the role of this cell surface receptor in CRC have not been defined. We sought to identify CDCP1 regulated processes in CRC using stem cell populations, enriched from primary cells and cell lines, in extensive in vitro and in vivo assays. These experiments, demonstrating that CDCP1 is functionally important in CRC tumor initiation, growth and metastasis, identified CDCP1 as a positive regulator of Wnt signaling. Detailed cell fractionation, immunoprecipitation, microscopy, and immunohistochemical analyses demonstrated that CDCP1 promotes translocation of the key regulators of Wnt signaling, ß-catenin, and E-cadherin, to the nucleus. Of functional importance, disruption of CDCP1 reduces nuclear localized, chromatin-associated ß-catenin and nuclear localized E-cadherin, increases sequestration of these proteins in cell membranes, disrupts regulation of CRC promoting genes, and reduces CRC tumor burden. Thus, disruption of CDCP1 perturbs pro-cancerous Wnt signaling including nuclear localization of ß-catenin and E-cadherin.

Role of immunohistochemistry in staging diffuse large B-cell lymphoma (DLBCL).

The use of immunohistochemistry (IHC) in staging bone marrow in non-Hodgkin's lymphoma (NHL) is largely limited to ambiguous cases, particularly those with lymphoid aggregates. Its role in routine clinical practice remains unestablished. This study aimed to determine whether the routine use of IHC in diffuse large B-cell lymphoma (DLBCL) would improve the detection of lymphomatous involvement in the bone marrow. It also sought to determine the impact of IHC on predicting survival compared with routine histological diagnosis using hematoxylin and eosin (H&E), Giemsa, and reticulin staining. The bone marrow trephines of 156 histologically proven DLBCL cases were assessed on routine histology, and IHC using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a), and kappa and lambda light chains. IHC detected lymphomatous involvement on an additional 11% cases compared with histology alone. Although both routine histology and IHC were good predictors of survival, IHC was better at predicting survival on stepwise multivariate Cox regression analysis. IHC performed routinely on bone marrow trephines has the ability to improve detection of occult lymphoma in experienced hands. Furthermore, it is a better predictor of survival compared with routine histological examination alone.

Occult bone marrow involvement in patients with diffuse large B-cell lymphoma: results of a pilot study.

AIMS: It is known that advanced stage disease in diffuse large B-cell lymphoma (DLBCL) confers a poor prognosis, and staging investigations are routinely performed at diagnosis, including a bone marrow (BM) biopsy. However, examination of the BM is usually limited to routine light microscopy, with the role of ancillary investigations remaining unestablished. The aim of this pilot study was to estimate the incidence of occult marrow involvement using flow cytometry, immunohistochemistry and immunoglobulin heavy chain (IgH) gene rearrangements, and to determine the impact on survival. METHODS: Clinical and pathological data were obtained on 36 patients diagnosed with DLBCL. Immunohistochemistry using CD3, CD45RO, CD20 and CD79a, and polymerase chain reaction (PCR) to look for IgH gene rearrangements were performed on formalin fixed BM trephines. RESULTS: Nine patients had morphologically apparent BM involvement. Occult marrow involvement was found in seven of 36 (19.4%) patients using the additional diagnostic modalities. When these cases were included with morphologically apparent cases in a proposed new definition of marrow involvement, the median survival of patients with BM involvement was statistically worse (p=0.02) than those without involvement. CONCLUSIONS: The results indicate that use of additional tests on BM at diagnosis can upstage disease for a proportion of patients, which appears to correlate adversely with survival.

Pericellular regulation of prostate cancer expressed kallikrein-related peptidases and matrix metalloproteinases by cell surface serine proteases.

We provide evidence of a pericellular network of proteases that are elevated and co-expressed in prostate cancer. The network involves the membrane bound serine proteases hepsin and TMPRSS2, the secreted kallikrein-related peptidases KLK4 and KLK14, and the secreted matrix metalloproteinases MMP-3 and MMP-9. Western blot analysis of cell lysates, conditioned cell culture media, immunoprecipitates and cell surface proteins, demonstrates a network of interactions centred largely at the plasma membrane, with the Arg/Lys specific proteases hepsin and TMPRSS2 key regulators of the network. Our data demonstrate that like TMPRSS2, hepsin is able to autoactivate. Active hepsin degrades KLK4, generating a cell associated degradation product with corresponding reduction in levels of cell-free KLK4. In contrast hepsin activates KLK14. TMPRSS2 appears to cleave amino terminal to the KLK4 activation site such that it is available for further processing to generate the active KLK4 protease. In contrast with hepsin, TMPRSS2 degrades KLK14. In addition to these direct mechanisms of regulation, hepsin and TMPRSS2 indirectly modulate KLK4 activity by cleaving the KLK4-activating protease MMP-3. Hepsin and TMPRSS2 also activate MMP-9, which similar to MMP-3, associates with the cell surface. Interestingly our data also show that proteolysis occurs between the membrane spanning and catalytic domains of hepsin and TMPRSS2. Hepsin cleavage occurs via an autoproteolytic mechanism, whereas TMPRSS2 cleavage is mediated by KLK14. Hepsin and TMPRSS2 are not shed from the cell surface but proteolysis likely disrupts domains that regulate the proteolytic activity of these proteases. Immunocytochemical analyses demonstrate that hepsin and TMPRSS2 colocalize on the cell surface with the secreted serine proteases KLK4 and KLK14, only in membrane protrusions, suggesting that reciprocal proteolytic interactions occur in defined cellular structures that are important during cancer dissemination for cell migration, invasion and survival. Also of note, immunohistochemical analysis of serial sections of prostate tumor demonstrated significant overlapping expression of the six proteases in vivo. Collectively these data suggest the possibility that the novel proteolytic network identified by us, will be most important during active dissemination of prostate cancers, and that its disruption could inhibit metastasis.

Cancer stem cell markers in prostate cancer: an immunohistochemical study of ALDH1, SOX2 and EZH2.

The aims of this study were to investigate the immunohistochemical expression and potential prognostic significance of putative cancer stems cell markers ALDH1, EZH2 and SOX2 in prostate cancer.A total of 142 consecutive radical prostatectomies submitted to one laboratory with a diagnosis of prostatic adenocarcinoma between 2008 and 2012 were retrieved and retrospectively studied. Immunohistochemistry for the three markers was performed in each case and both univariate and multivariate analyses were undertaken to evaluate the correlation between the staining patterns and known histopathological prognostic features.ALDH1 showed a statistically significant association with tumour stage p < 0.001), extraprostatic extension (p < 0.001) and lymphovascular invasion (p = 0.001). EZH2 correlated with Gleason score (p = 0.044) and lymph node metastases (p = 0.023). SOX2 showed a statistically significant correlation with lymphovascular invasion only (p = 0.018) in both univariate and multivariate analyses.Cancer stem cell markers are variably expressed in prostate adenocarcinoma and immunohistochemical staining for ALDH1 and EZH2 may have a role in predicting tumour aggressiveness before treatment of prostate cancer.

The placenta in Beckwith-Wiedemann syndrome: genotype-phenotype associations, excessive extravillous trophoblast and placental mesenchymal dysplasia.

AIMS: Placental mesenchymal dysplasia (PMD) is a rare condition which is associated with the disparate fetal outcomes of Beckwith-Wiedemann syndrome (BWS), fetal growth restriction or intrauterine and neonatal death. We aimed to investigate the potential epigenetic/genetic anomalies associated with PMD and their relationship with the different causes of BWS. METHODS: Eight archival cases in which PMD, BWS or both were diagnosed were investigated by correlating morphology with p57 Kip2 expression, XY fluorescence in situ hybridisation (FISH) analysis and DNA genotyping. RESULTS: Placentae from BWS cases caused by aberrant IC2 methylation, leading to abnormal p57 Kip2 expression, did not show PMD but had a striking excess of extravillous trophoblast. PMD in the absence of BWS was caused by androgenetic/biparental mosaicism. The single case of BWS with PMD was due to mosaic uniparental disomy of 11p15.5. In the latter two aetiologies, our results indicate that the uniparental disomy is confined to the villous mesenchyme. CONCLUSIONS: These results suggest that the link between PMD and BWS is uniparental disomy of genes confined to the telomeric IC1 region of 11p15.5. A strong candidate gene is IGF2, a known growth factor of placental mesenchyme.

Immunohistochemical expression of EGFR in colorectal carcinoma correlates with high but not low level gene amplification, as demonstrated by CISH.

AIM: To assess and compare immunohistochemical expression of epidermal growth factor receptor (EGFR) with gene amplification as demonstrated by chromogenic in situ hybridisation (CISH), in colorectal adenocarcinoma. METHODS: Sections from 100 consecutive colorectal cancer resection specimens were stained for EGFR using immunohistochemistry and CISH. Immunohistochemical assessment was independently performed at two laboratories, using the same antibody and protocols. RESULTS: With immunohistochemistry, strong circumferential membrane staining (3+ staining) was demonstrated in only 5% of cases, and this was only focal in three of five cases. At one laboratory, weak or incomplete staining (1+ or 2+) was observed in five further cases (5%), which had been negative at the other laboratory. CISH demonstrated high level gene amplification (>10 copies/nucleus) in the same five cases which had demonstrated 3+ staining with immunohistochemistry, and in those cases where the staining was focal, the amplification was demonstrated in the same foci of the tumour. Five further cases (5%) had low level amplification (5-10 copies per nucleus); these cases did not exhibit significant positive staining with immunohistochemistry. All the cases which demonstrated gene amplification (high or low level) arose in the distal colon. There was no correlation between gene amplification status and a variety of other variables, including stage at diagnosis, mucinous differentiation, neuroendocrine differentiation, or loss of expression of mismatch repair proteins. CONCLUSIONS: Immunohistochemical expression of EGFR is variable between laboratories, even using standardised protocols. 3+ staining is predictive of high level gene amplification, but correlates very poorly with low level amplification, which may still be clinically significant. In some cases gene amplification was only focal, offering a potential explanation for poor response to targeted therapy in patients with EGFR positive tumours.