Hemodynamic support by left ventricular assist devices reduces cardiomyocyte DNA content in the failing human heart.

BACKGROUND: Whether adult cardiomyocytes have the capacity to regenerate in response to injury and, if so, to what extent are still issues of intense debate. In human heart failure, cardiomyocytes harbor a polyploid genome. A unique opportunity to study the mechanism of polyploidization is provided through the setting of hemodynamic support by left ventricular assist devices. Hence, the cardiomyocyte DNA content, nuclear morphology, and number of nuclei per cell were assessed before and after left ventricular assist device support. METHODS AND RESULTS: In 23 paired myocardial samples, cardiomyocyte ploidy was investigated by DNA image cytometry, flow cytometry, and in situ hybridization. Nuclear cross-sectional area and perimeters were measured morphometrically, and the binucleated cardiomyocytes were counted. The median of the cardiomyocyte DNA content and the number of polyploid cardiomyocytes both declined significantly from 6.79 c to 4.7 c and 40.2% to 23%, whereas a significant increase in diploid cardiomyocytes from 33.4% to 50.3% and in binucleated cardiomyocytes from 4.5% to 10% after unloading was observed. CONCLUSIONS: The decrease in polyploidy and increase in diploidy after left ventricular assist device suggest a numeric increase in diploid cardiomyocytes (eg, through cell cycle progression with completion of mitosis or by increased stem cells). The cardiac regeneration that follows may serve as a morphological correlate of the recovery observed in some patients after unloading.

Combined analysis of hypoxia-inducible factor 1 alpha and metallothionein indicates an aggressive subtype of colorectal carcinoma.

PURPOSE: Hypoxia-inducible factor 1 (HIF-1) is a hypoxia-induced transcription factor that regulates gene expression in critical pathways involved in tumour growth and metastasis. Metallothionein (MT) is a group of small molecular weight cysteine-rich proteins with a broad variety of functions. The present study aimed to analyse the prognostic impact of HIF-1alpha and MT expression in colorectal cancer and to evaluate a possible link of combined HIF-1alpha and MT expression with colorectal cancer progression. MATERIALS AND METHODS: We investigated the relationship of HIF-1alpha and MT with each other and clinicopathological parameters including proliferative activity (Ki67) and apoptosis (terminal desoxyribonucleotide transferase-mediated dUTP nick-end labelling) using immunohistochemistry. RESULTS: HIF-1alpha expression was identified as an independent prognostic parameter in multivariate survival analysis and characterised an aggressive cancer phenotype. In addition, HIF-1alpha was significantly linked to an increased expression of MT. CONCLUSIONS: HIF-1alpha expression qualified as an independent prognostic and characterised an aggressive cancer phenotype associated with an increased expression of MT. Our study suggests that MT can be added to the complex biological pathways induced by hypoxia in human cancer tissue.

Myositis ossificans mimicking metaplastic breast cancer on core needle biopsy.

Myositis ossificans (MO) is an uncommon myofibroblastic proliferation that may closely mimic sarcoma. A 33-year old woman presented with a 2-cm breast mass. A core needle biopsy showed highly cellular spindle cell proliferations with atypia and high proliferation activity. Focal areas of immature osteoid-like matrix were seen. After immunohistochemical analysis, a diagnosis of metaplastic breast cancer was made. Because of the proximity of the tumor to the rib, resection of the tumor including partial resection of the rib was carried out. Complete examination of the excised lesion allowed the correct diagnosis of MO. Only the resection specimens contained a predominance of mature organoid bone tissue admixed with variably cellular myofibroblastic proliferations with the typical zonal pattern, characteristic of MO. The diagnosis of MO was not possible on the initially performed core needle biopsy because biopsy material reflected only the central proliferative portion of the lesion showing increased pleomorphism and early osteoid formation. This case highlights the pitfall related to this unusual presentation of a benign lesion and points to the necessity to include MO in the differential diagnosis of triple-negative metaplastic breast cancer.

Elevated expression of cyclooxygenase-2 is a negative prognostic factor for overall survival in intrahepatic cholangiocarcinoma.

The production of prostaglandins is regulated by cyclooxygenases (COXs), which also have a role in tumour development and progression in various human malignancies, including cholangiocarcinoma. Limited information is available of the correlation of COX-2 protein expression and prognosis in intrahepatic cholangiocarcinoma (ICC). The aim of the present study was to determine the clinical significance of COX-2 expression in ICC. In addition the correlation of COX-2 expression and apoptosis/proliferation was analysed. COX-2 expression was determined immunohistochemically in 62 resected ICCs. Proliferation was assessed using Ki67-immunohistochemistry, and apoptosis was measured with the TdT-mediated dUTP nick-end-labelling technique. COX-2 was identified as an independent prognostic factor (P = 0.028) in resected ICC by survival analysis. High levels of COX-2 expression were found to be associated both with reduced apoptosis and increased proliferation of tumour cells. This study demonstrates the independent prognostic value of the COX-2 expression in resected ICC, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors.

Survival and recurrence rates after resection for hepatocellular carcinoma in noncirrhotic livers.

BACKGROUND: Hepatocellular carcinoma occurring in noncirrhotic livers is rare. The purpose of this article was to evaluate the outcomes and prognostic factors after hepatectomy in this setting. STUDY DESIGN: Between June 1998 and May 2005, 83 patients underwent liver resection for hepatocellular carcinoma in noncirrhotic livers at our institution. Preoperative treatment data, intraoperative details, pathologic findings, and information on tumor recurrence, treatment of recurrence, and survival were available for 80 of these patients. RESULTS: Postresection, the 3- and 5-year-survival rates were 48% and 30%, respectively. After R0 resection (n=66), the calculated 3- and 5-year-survivals were 54% and 39%, compared with 23% and 0%, respectively, after R1/2-resection (p<0.005). After a median followup of 25 months, tumor recurred in 40 of 63 (63%) patients after R0 resection. In univariate analysis, Union Internationale Contre le Cancer (UICC) stage, vascular invasion, and tumor grading were identified as important findings for recurrence and poor survival after R0 resection. For tumors without vascular invasion, the 3- and 5-year-survivals were 79% and 65%, respectively, which compared favorably with 21% and 7%, respectively, for tumors with vascular invasion (p<0.0001). Similarly, 3- and 5-year-survival rates (95% each) were considerably better for G1 tumors than the corresponding 36% and 22% rates in G2 and 60% and 30% in G3 tumors, respectively. CONCLUSIONS: The 3- and 5-year survivals of 54% and 39%, respectively, after R0 resections suggest that surgery is an option in hepatocellular carcinoma arising in noncirrhotic livers. Longterm results, however, are hampered by high recurrence rates. Union Internationale Contre le Cancer stage, vascular invasion, and tumor grades are predictors of tumor recurrence and diminished survival, and may help to identify candidates for potential adjuvant therapies.

High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome.

INTRODUCTION: Focal adhesion kinase (FAK) regulates multiple cellular processes including growth, differentiation, adhesion, motility and apoptosis. In breast carcinoma, FAK overexpression has been linked to cancer progression but the prognostic relevance remains unknown. In particular, with regard to lymph node-negative breast cancer it is important to identify high-risk patients who would benefit from further adjuvant therapy. METHODS: We analyzed 162 node-negative breast cancer cases to determine the prognostic relevance of FAK expression, and we investigated the relationship of FAK with major associated signaling pathways (HER2, Src, Akt and extracellular regulated kinases) by immunohistochemistry and western blot analysis. RESULTS: Elevated FAK expression did not predict patient outcome, in contrast to tumor grading (P = 0.005), Akt activation (P = 0.0383) and estrogen receptor status (P = 0.0033). Significant positive correlations were observed between elevated FAK expression and HER2 overexpression (P = 0.001), as well as phospho-Src Tyr-215 (P = 0.021) and phospho-Akt (P < 0.001), but not with phospho-ERK1/2 (P = 0.108). Western blot analysis showed a significant correlation of FAK Tyr-861 activation and HER2 overexpression (P = 0.01). CONCLUSIONS: Immunohistochemical detection of FAK expression is of no prognostic significance in node-negative breast cancer but provides evidence that HER2 is involved in tumor malignancy and metastatic ability of breast cancer through a novel signaling pathway participating FAK and Src.

Intramural carcinoma of the oesophagogastric junction.

Most oesophageal carcinomas (>70%) are squamous cell carcinomas (SCC) arising from the surface epithelial lining. In the gastro-oesophageal junction (GEJ) adenocarcinomas occur in the setting of Barrett oesophagus (BE). These carcinomas typically present with surface ulceration. The authors report a rare case of a SCC of the GEJ with completely intramural growth underneath intact non-dysplastic oesophageal squamous epithelium and BE without dysplasia. The sharply demarcated tumour was located in the muscularis propia with infiltration of perioesophageal tissue nearly reaching the diaphragm. Intramural squamous carcinomas are very rare with only three reported cases. In this study, the authors discuss this finding and review the relevant literature. Intramural carcinomas are a diagnostic challenge for clinicians and pathologist, since preoperative biopsies mostly fail to establish the diagnosis and surgical explorations must be considered.

Reversible regulation of the retinoblastoma protein/E2F-1 pathway during "reverse cardiac remodelling" after ventricular unloading.

BACKGROUND: Cyclin D1, the retinoblastoma (Rb) protein, and the E2F transcription factors are involved in the pathogenesis of cardiac hypertrophy. Cyclin D1/cdk4 complexes, by phosphorylation, inactivate Rb, thereby abrogating its growth-inhibitory effect. Ventricular unloading is associated with reversible regulation of numerous cardiomyocyte molecular systems and decreased hypertrophy. Accordingly, the hypothesis whether the Rb/E2F-1 pathway is altered by ventricular unloading was tested, and correlations with the cyclin D1 protein expression and cardiomyocyte diameters were explored. METHODS: In 21 paired myocardial samples (before and after unloading) from patients with congestive heart failure (CHF), cyclin D1, phosphorylated Rb (pRb), its homologues p107 and p130 (pocket proteins), and E2F-1 were immunohistochemically investigated and morphometrically quantified. Cardiomyocyte diameters were morphometrically determined. RESULTS: Cyclin D1 and the proteins of the Rb/E2F-1 pathway were significantly increased during CHF compared with controls and were significantly decreased after unloading. Cyclin D1, pRb, and p130 protein expression correlated significantly with cardiomyocyte diameters. A significant positive correlation was noted between the pocket proteins, E2F-1, and cyclin D1. CONCLUSION: Increased protein expression of phosphorylated (inactivated) Rb and the pocket proteins is associated with cardiomyocyte hypertrophy in CHF. Rb inactivation might be explained by phosphorylation by increased numbers of cyclin D1/cdk4 complexes associated with cardiomyocyte hypertrophy. However, ventricular unloading can reversibly regulate this process. These data underscore the importance of cell cycle regulatory proteins in the pathogenesis of CHF-associated (maladaptive) cardiomyocyte hypertrophy and might offer novel clues for pharmacologic approaches of congestive heart failure.

Cardiomyocyte survivin protein expression is associated with cell size and DNA content in the failing human heart and is reversibly regulated after ventricular unloading.

BACKGROUND: Mechanical support in congestive heart failure (CHF) by a left ventricular assist device (LVAD) is associated with decreased cardiac hypertrophy and altered cardiomyocyte molecular pathways. Survivin initiates cell cycle progression by increased cyclinD1/cdk4 complexes by abrogation of the inhibitory effect of p16(INK4a) on cdk4. Accordingly, the role of survivin in CHF and after unloading was explored. METHODS: In 20 myocardial samples from patients with terminal CHF (before and after LVAD), the protein expression of survivin, cyclin D1, cdk4, p16(INK4a), and proliferating cell nuclear antigen (PCNA) was immunohistochemically investigated and morphometrically quantified by calculating the percentage of positive cardiomyocytes per visual field. These data were correlated with cardiomyocyte size and DNA content. RESULTS: The mean percentage of cardiomyocytes immunoreactive against survivin, cyclin D1, cdk4, p16(INK4a), and PCNA was significantly increased in CHF compared with controls and significantly decreased after unloading (57.6% to 26.6%, 42% to 18.3%, 45.4% to 15.3%, 73.0% to 60.5%, and 43.5% to 25.2%, respectively; p < 0.05). All investigated parameters, in particular survivin and cyclin D1, significantly correlated with cardiomyocyte diameters (r = 0.405; r = 0.563) and DNA content (r = 0.430; r = 0.480), both in CHF (cardiac remodelling) and after unloading (p < 0.05). CONCLUSIONS: These data indicate that survivin is reversibly regulated by ventricular unloading and might be involved in cell size/DNA content regulation and cardiomyocyte proliferation in cardiac remodelling during CHF. It is suggested that after ventricular unloading, decreased survivin protein expression might contribute to cardiac hypertrophy decrease by lowering the number of cyclin D1/cdk4 complexes.

Ventricular unloading is associated with increased 20s proteasome protein expression in the myocardium.

BACKGROUND: The ubiquitin-proteasome system (UPS) breaks down misfolded and normal proteins, including cell cycle regulatory proteins involved in cardiac hypertrophy. Because congestive heart failure (CHF) increases cardiomyocyte cellular mass, indicative of increased protein synthesis and/or impaired breakdown, and ventricular unloading decreases cardiac hypertrophy and changes regulation of multiple molecular systems ("reverse cardiac remodeling"), we tested the hypothesis that ventricular unloading alters myocardial UPS. METHODS: In 23 paired myocardial specimens (before and after unloading) ubiquitin, 20S proteasome, and cyclin D1 were investigated immunohistochemically and morphometrically quantified in relation to cardiomyocyte hypertrophy, DNA content, nuclear profile area and perimeter, and cyclin D1 protein expression. Moreover, 20S proteasome plasma concentrations were measured by enzyme-linked immunoassay (ELISA). RESULTS: In CHF, sarcoplasmic 20S proteasome protein expression was significantly decreased compared with controls, but significantly increased after unloading. In contrast, sarcoplasmic ubiquitin protein was increased in CHF but significantly decreased after unloading, and both variables were inversely correlated. Cardiomyocyte 20S proteasome expression correlated inversely with cell size, mean DNA content, and cyclin D1, whereas ubiquitin protein expression was positively correlated with these parameters. The 20S proteasome plasma concentration was significantly increased after unloading. CONCLUSIONS: Our data indicate that: (1) the UPS is depressed in CHF; and (2) this is reversed by ventricular unloading and associated with decreased cardiomyocyte hypertrophy, mean DNA content, and cell cycle regulatory proteins. The findings support the view that the UPS is involved in both the pathogenesis of cardiac hypertrophy and "reverse cardiac remodeling" after ventricular unloading.

Metallothionein overexpression and its prognostic relevance in intrahepatic cholangiocarcinoma and extrahepatic hilar cholangiocarcinoma (Klatskin tumors).

Metallothionein is a group of small molecular weight cysteine-rich proteins with a broad variety of functions. Metallothionein has been shown to regulate apoptosis and proliferation. Overexpression of metallothionein frequently occurs in human tumors and is related to prognosis as well as therapy response. However, metallothionein expression and its clinical relevance in cholangiocarcinoma have not been investigated. The present study aimed to analyze metallothionein over-expression and its possible prognostic impact in intrahepatic cholangiocarcinoma and hilar extrahepatic cholangiocarcinoma (Klatskin tumors). We investigated the relationship of immunohistochemically demonstrated metallothionein expression with various clinicopathological parameters in a series of 56 intrahepatic and 56 extrahepatic cholangiocarcinoma. In noncancerous bile duct epithelia metallothionein was only occasionally weakly expressed; strong metallothionein overexpression (>50% metallothionein -positive tumor cells) was noted in 7 (12.5%) of 56 intrahepatic cholangiocarcinoma and 14 (25%) of 56 Klatskin tumors, which was associated with poor clinical outcome in univariate Kaplan-Meier testing in both intrahepatic cholangiocarcinoma (P = .002) and Klatskin tumors (P = .034). Moreover, strong metallothionein expression was identified as an independent prognostic parameter in multivariate Cox regression analysis in both intrahepatic cholangiocarcinoma (P = .005) and Klatskin tumors (P = .035). In contrast, cholangiocarcinoma with a papillary phenotype (8/112; 7.1%) exhibited a significant lack of strong metallothionein expression in all 8 of 8 cases. Strong metallothionein expression is identified as an independent poor prognostic parameter, and determination of the metallothionein expression may serve as an additional tool for the therapeutic management of patients with cholangiocarcinoma. In comparison, lack of metallothionein expression seems to be associated with cholangiocarcinoma with a papillary phenotype, which is generally recognized to have a better prognosis.

Decreased myocardial chromogranin a expression and colocalization with brain natriuretic peptide during reverse cardiac remodeling after ventricular unloading.

AIMS: In chronic heart failure, atrial and brain natriuretic peptide expression is increased and serves as a clinical marker of cardiac hypertrophy. Chromogranin A is also up-regulated during chronic heart failure and associated with disease severity and prognosis. Significant decrease of both natriuretic peptide and hypertrophy after left ventricular assist device (LVAD) treatment was reported. This study investigated whether chromogranin A and neural cell adhesion molecule (NCAM)/CD56 are associated with cardiac hypertrophy and regulated by LVAD. METHODS: Expression of atrial and brain natriuretic peptide, chromogranin A, and NCAM/CD56 were investigated by immunohistochemistry and morphometrically quantified in 33 paired myocardial samples before and after LVAD. In a different set of patients, chromogranin A was evaluated in the plasma. Cardiomyocyte colocalization of brain natriuretic peptide and chromogranin A was visualized by immunofluorescence doublestaining. RESULTS: Natriuretic peptide and chromogranin A protein expression is significantly decreased after LVAD (p < 0.05). NCAM/CD56 expression remains unaltered by unloading. In contrast with natriuretic peptide, chromogranin A and NCAM/CD56 expression is not correlated with cardiomyocyte diameters. Although increased compared with controls, no significant differences for chromogranin A plasma levels were found before and after LVAD. Sarcoplasmic colocalization of chromogranin A and brain natriuretic peptide is considerably decreased after LVAD. CONCLUSIONS: Neither chromogranin A nor CD56 is associated with cardiac hypertrophy. Chromogranin A is significantly decreased by ventricular support. Sarcoplasmic colocalization of brain natriuretic peptide and chromogranin A is diminished after unloading. However, owing to its low expression, the negative regulation of chromogranin A is not reflected by plasma levels and thus does not appear to be an appropriate biomarker of reverse cardiac remodeling after unloading.

Roles of cyclooxygenase-2 and phosphorylated Akt (Thr308) in cardiac hypertrophy regression mediated by left-ventricular unloading.

OBJECTIVES: Cyclooxygenase-2 is associated with cardiac hypertrophy during chronic heart failure and is regulated through the PI3K/Akt pathway. Cyclooxygenase-2-induced cell growth through Akt phosphorylation was demonstrated in vitro. In chronic heart failure, left ventricular assist devices lead to hypertrophy regression and molecular changes. Therefore, the expression of cyclooxygenase-2, phosphorylated Akt (p-Akt), and p-Erk 1/2, as well as cardiac hypertrophy before and after left ventricular assist device insertion, was investigated. METHODS: In myocardial tissue before and after left ventricular assist device insertion, the expression of cyclooxygenase-2, p-Akt (Thr308), p-Akt (Ser473), and p-Erk 1/2 was demonstrated by immunohistochemistry and quantified by morphometry. Colocalization of cyclooxygenase-2 and p-Akt (Thr308) was investigated by immuno-doublestaining. RESULTS: A significant decrease of cyclooxygenase-2, p-Akt (Thr308), p-Akt (Ser473), and p-Erk 1/2 protein expression and hypertrophy regression was observed after left ventricular assist device insertion. A significant correlation between cyclooxygenase-2 and p-Akt (Thr308) expression, as well as between cyclooxygenase-2 expression and cardiomyocyte diameter, was observed before, but not after, left ventricular assist device insertion. Only cyclooxygenase-2-positive cardiomyocytes showed significant hypertrophy regression on unloading. Sarcoplasmic colocalization of cyclooxygenase-2 and p-Akt (Thr308) is present before left ventricular assist device insertion and is decreased after unloading, whereas the normal myocardium is completely devoid of it. CONCLUSIONS: Left ventricular assist device treatment is associated with a significant decrease of cyclooxygenase-2, p-Akt (Thr308), p-Akt (Ser473), and p-Erk 1/2, and cardiac hypertrophy regression of cyclooxygenase-2-positive cardiomyocytes. The significant correlation and colocalization in cardiomyocytes of cyclooxygenase-2 and p-Akt (Thr308) before left ventricular assist device insertion suggests a cross-talk between the 2 molecules in the progression of cardiac hypertrophy, which is reversibly regulated by the left ventricular assist device.

Melanotic schwannoma of the neck mimicking a malignant melanoma.

We report a case of a 42-year-old female patient who presented with a 5-month history of a right cervical swelling. A fine-needle aspiration biopsy performed elsewhere led to the diagnosis of malignant melanoma. Clinical examination revealed pigmented skin and mucous-membrane lesions of the pharynx. Diagnostic exstirpation of the cervical tumor resulted in the intraoperative diagnosis of a malignant melanoma. Subsequent pathological examination including ultrastructural analysis allowed the revision of the diagnosis to that of melanotic schwannoma, a rare, pigmented nerve sheath tumor. Further analysis of the tissue obtained from neck dissection revealed additional melanotic schwannomas. With regard to prognostic and therapeutic issues, it is necessary to differentiate these tumors from the more common metastatic malignant melanoma. The preoperative differential diagnosis is very difficult, as light microscopy and immunocytochemistry allow no discrimination in small biopsies. Although the occurrence of multiple schwannomas points to a hereditary syndrome, our patient did not fulfill the criteria of Carney's syndrome or other known syndromes, suggesting a so far unknown genetic background.

Prognostic relevance of activated Akt kinase in node-negative breast cancer: a clinicopathological study of 99 cases.

Patients with lymphnode-negative breast cancer show a 10-year tumor recurrence rate of approximately 30%. Therefore, it is important to identify high-risk patients who would benefit from further adjuvant therapy. For this purpose, we examined the activation state of two kinases important in the regulation of cell proliferation and apoptosis in a series of 99 node-negative breast cancer cases with a mean follow-up of 10 years: Akt and extracellular regulated kinase (ERK1/2). The activation of Akt and ERK1/2 was investigated by immunohistochemistry using phospho-specific antibodies. The results were correlated with HER-2/neu expression, histological grading, receptor status, overall survival (OS) as well as with cell proliferation (Ki67 immunoreactivity, mitotic count) and tumor apoptosis assessed by TUNEL staining. Activation of Akt (pAkt) but not activation of ERK1/2 (pERK1/2) correlated with HER-2/neu overexpression (P<0.05) and was related to reduced tumor apoptosis (P<0.05). No association was found between pAkt or pERK1/2 with cell proliferation assessed by Ki67 and mitotic count (MC). Survival analysis of receptor status, HER2/neu expression, histological grading, MC and pAkt immunoexpression showed a significant correlation with decreased OS, but only pAkt reached statistical significance in the multivariate Cox regression analysis (P=0.015). Activation of Akt in node-negative breast cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor of OS. The determination of pAkt status may be of value in identifying high-risk patients, who would benefit from adjuvant therapy, and gives a rationale to investigate new therapy strategies by specific inhibition of the Akt signaling pathway in breast cancer.