The evaluation of minimal residual disease in multiple myeloma by fluorescent molecular beacons in real time PCR of IgH gene rearrangements and correlation with flow cytometry.

PURPOSE: Multiple myeloma (MM) patients relapse after a period of time despite longer disease-free survival due to novel treatment options. In this study we aimed to assess the value of real-time polymerase chain reaction (RT-PCR) for detecting the immunoglobulin heavy chain (IgH) gene rearrangement using allele-specific molecular beacons as fluorescence probes to quantify minimal residual disease (MRD) and also to correlate post-treatment flow cytometric detection of plasma cells' (PCs) expression of CD19, CD38, CD45, CD56 and CD138 in MM. METHODS: After diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify IgH's clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. Examined were also the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in bone marrow aspirates of patients with MM by flow cytometry. RESULTS: Detection of MRD was positive in 13 (76%) of 17 patients by RT-PCR. The infiltration ratio was significantly correlated with CD138 expression (p=0.009). Significant correlation was also found between RT-PCR detection of MRD and CD138 expression (p=0.006). Nevertheless, no correlation was observed among other surface antigens (CD38, CD45, CD56). CONCLUSION: Our results indicated that RT-PCR with specific molecular beacons provide a feasible, accurate and reproducible method for the determination of MRD in MM. Flow cytometry detection of CD138 expression may be used as a disease marker in addition to RT-PCR.

Mucormycosis-associated fungal infections in patients with haematologic malignancies.

Among patients with haematologic disorders, mucormycosis most commonly occurs in those with acute leukaemia or lymphoma who have developed neutropenia due to malignancy or to chemotherapy, and in transplanted patients receiving immunosuppressive treatment. Here, we aim to present a retrospective study conducted over a 5-year period (2001-2005). The study included 20 patients with haematologic malignancies with a proven mucormycosis admitted in Medical Oncology Divisions in Cukurova University Hospital. The most frequent sites of infection were paranasal sinuses (95%) and lung (5%). Antifungal treatment was empirically administered in 18 (90%) patients; 18 patients underwent radical surgical debridement (90%). The therapy was successful for only eight patients (40%). Eleven patients died within 1 months of the diagnosis of fungal infection: the cause of death was only by mucormycosis in four patients (36.6%), mucormucosis and systematic inflamatuar response syndrome (SIRS) in two patients (18.2%) and progression of haematologic disease in five patients (45.5%). At univariate analysis, the factors that correlated with a positive outcome from infection were the following: amphotericin B treatment, neutrophil recovery from postchemotherapy aplasia. At multivariate analysis, the factors that significantly correlated with recovery from infection were the liposomal amphotericin B treatment (p = 0.026), doses of L-AmB (p = 0.008) and the length of the treatment (p = 0.01), respectively. It seems to have increased in recent years. Although a reduction of mortality has been observed recently, the mortality rate still remains high. Extensive and aggressive diagnostic and therapeutic procedures are essential to improve the prognosis in these patients.

Expression of c-kit protooncogen in hepatitis B virus-induced chronic hepatitis, cirrhosis and hepatocellular carcinoma: has it a diagnostic role?

AIM: There are more than 350 million people worldwide chronically infected with hepatitis B virus (HBV), who are at high risk for the development of hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Because of the conflicting results about c-kit expression in HCC and the key role played by c-kit in gastrointestinal stromal tumours (GIST) and other solid tumours, the aim of this study was to determine c-kit expression in the course of hepatitis B infection. MATERIALS AND METHODS: Paraffin-embedded tissues in Cukurova University Faculty of Medicine Department of Pathology between January 2002 and February 2006 were searched restrospectively to investigate this issue. We performed immunohistochemistry on biopsies of 125 patients with HBV infection, grouped as: mild, moderate and severe hepatitis, cirrhosis and HCC, 25 patients in each of them, using anti c-kit monoclonal antibody. The severity of parenchymal inflammation and of interface hepatitis was semiquantitatively graded on a haematoxylin and eosin stained paraffin sections. Additionally, 50 more HCC, formed on HBV basis, were studied to determine the prevalence of c-kit overexpression. RESULTS: In cirrhotic liver, lower intensity of staining and rarely c-kit positivity were present. The greatest number of the c-kit positivity and higher intensity of staining was found in the livers of patients with severe hepatitis and HCC. In chronic hepatitis B infection, the staining intensity was parallel with the grade and stage of the disease. In the areas where fibrosis was seen, c-kit positivity was rare or absent. In the HCC specimens, c-kit positivity appeared both inside and around the cancerous nodes. C-kit expression was observed in 62 of 75 HCC tissue specimens (82%) (p < 0.001). CONCLUSIONS: C-kit positivity was observed in the mitotic, proliferating and also dysplastic hepatic cells. These results suggest that c-kit expression may be used as an early diagnostic indicator for HBV induced HCC.

Expression of mesenchymal, hematopoietic, and biliary cell markers in adult rat hepatocytes after partial hepatectomy.

BACKGROUND AND PURPOSE: It has been suggested that liver regeneration can occur either by differentiated adult hepatocytes which retain the capability for several rounds of replication or by hepatic progenitor cells, depending on the number of hepatocytes lost. We sought to study the differentiation potential of hepatocytes following partial hepatectomy (PH) in rats. METHODS: Using immunohistochemistry and confocal microscopy we studied the distribution of cytokeratin 7 (CK7), CK19, vimentin, desmin, CD34, and c-kit among adult rat liver hepatocytes after PH at various times just after hepatectomy and after 8, 16, 24, 36, 48, and 60 hour and 6 and 16 days. RESULTS: Vimentin, c-kit, and desmin positivity were observed in regenerating hepatocytes in the early stages. Desmin and vimentin staining were also demonstrated in stellate cells. Staining enhancement in stellate cells progressed from day 3 to day 6. No liver sections were stained positive for CD34, CK19, or CK7. CONCLUSION: After PH, mature hepatocytes revealed their potential to regain the markers that they do not express when they are quiescent. This result supported the plasticity and differentiation potential of adult hepatocytes during regeneration.