Kurt Grünewald

Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells

BACKGROUND Vascular endothelial cells lost from the blood-vessel endothelium through necrosis or apoptosis must be replaced. We investigated in a leukaemia model whether bone-marrow-derived endothelial cells contribute to this maintenance angiogenesis. METHODS We studied six patients with chronic myelogenous leukaemia (CML) carrying the BCR/ABL fusion gene in their bone-marrow-derived cells. We screened endothelial cells generated in vitro from bone-marrow-derived progenitor cells and vascular endothelium in myocardial tissue for the BCR/ABL fusion gene by in-situ hybridisation. For detection of donor-type endothelial cells after transplantation of haemopoietic stem cells, recipient tissue was stained with monoclonal antibodies against donor-type HLA antigens. FINDINGS We identified the BCR/ABL fusion gene in variable proportions (0-56%) of endothelial cells generated in vitro. Endothelial cells expressing the fusion gene were found in the vascular endothelium of a patient. In a recipient of an allogeneic stem-cell transplant, normal donor-type endothelial cells were detected in the vascular endothelium. INTERPRETATION These findings suggest that CML is not solely a haematological disease but originates from a bone-marrow-derived haemangioblastic precursor cell that can give rise to both blood cells and endothelial cells. Moreover, normal bone-marrow-derived endothelial cells can contribute to the maintenance of the blood vascular endothelium. The integration of bone-marrow-derived endothelial cells into the vascular endothelium provides a rationale for developing vascular targeting strategies in vasculopathies, inflammatory diseases, and cancer.

Mammaglobin gene expression : A superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19

Various molecular markers have been used for the detection of circulating breast cancer cells in blood by reverse transcriptase-polymerase chain reaction (RT-PCR). Using nested RT-PCR, we compared the specificity and sensitivity of human mammaglobin (hMAM), epidermal-growth-factor receptor (EGF-R), and cytokeratin 19 (CK-19) expression as markers for circulating carcinoma cells in the peripheral blood of patients with breast cancer. Blood samples from 12 patients with ductal carcinoma in situ, 133 patients with invasive breast cancer, 20 patients with hematological malignancies, 31 healthy volunteers, and tumor tissues from 40 patients with invasive breast cancer were screened for mRNA encoding hMAM, EGF-R, or CK-19 by nested RT-PCR. In all breast cancer tissues, mRNA for hMAM, EGF-R, and CK-19 was detectable. In blood samples from patients with invasive breast cancer, 11 (8%), 13 (10%), and 64 (48%) were positive for mRNA encoding hMAM, EGF-R, or CK-19, respectively. Blood samples from none of the healthy volunteers and patients with hematological disorders were positive for hMAM, while CK-19 mRNA was found in the blood of 12 (39%) healthy volunteers and transcripts for EGF-R and CK-19 were detectable in 5 (25%) and 2 (10%), respectively, of the patients with hematological malignancies. Only hMAM mRNA expression in blood correlated with clinical parameters such as nodal status, metastasis, and CA 15–3 serum levels. In summary, hMAM transcripts detectable in blood by RT-PCR represent the most specific molecular marker for hematogenous spread of breast cancer cells. With the nested RT-PCR method, aberrant EGF-R mRNA expression might occasionally be found in hematological malignancies, whereas CK-19 mRNA expression proved to be rather nonspecific. The prognostic value of hMAM RT-PCR–based tumor cell detection in peripheral blood should be further tested and validated in prospective studies.

Low concentrations of STI571 in the cerebrospinal fluid: a case report

Summary. We report a 53‐year‐old man with lymphoid blast crisis of Ph+ chronic myeloid leukaemia who was treated with STI571, a selective inhibitor of the enzymatic activity of BCR–ABL. He responded excellently to STI571 (600 mg/d), obtaining a complete cytogenetic remission after 3 months of therapy. Although remission in the bone marrow was sustained, the patient developed an isolated central nervous system relapse. Subsequent analyses of STI571 concentrations in the cerebrospinal fluid (CSF) revealed 2‐log lower CSF levels of STI571 than corresponding plasma levels. These are the first data demonstrating a low penetration of orally administered STI571 into the CSF in humans.

High-dose interferon-α2b treatment prevents chronicity in acute hepatitis C

Acute hepatitis C takes a chronic course in 50–80% of cases. Results with interferon treatment are conflicting. To evaluate the efficacy of high-dose interferon treatment, we initiated a pilot study in 1992 using 10 MU interferon-α2b administered subcutaneously daily until normalization of serum transaminase concentrations. Treatment was begun when a diagnosis of acute hepatitis C was established. HCV-RNA was tested using PCR prior to treatment, three times weekly during the first two weeks of treatment, and then once weekly until the end of therapy. During the 15-month follow-up, HCV-RNA tests were performed monthly up to month 6 and every two to three months thereafter. Twenty-four patients were enrolled at the time of writing; age ranged from 18 to 76 years (mean=32), and nine patients were men. All patients presented with cholestatic hepatitis; 19 were actively abusing intravenous drugs, four had no known parenteral exposure, and one was a medical laboratory technician. All patients were anti-HCV positive, HCV-RNA positive, and HIV negative. Five patients were infected with genotype 3, five with genotype 1a, five with genotype 1b, three with genotypes 3 and 2, and one with genotypes 1 and 2. All patients exhibited normalized serum transaminase concentrations within 18–43 days; HCV-RNA became negative in all patients within 4–12 days. Toxicity did not exceed grade 1 and disappeared within three days of treatment. In the follow-up period, which ranged from six to 29 months (mean=19.5±10.4), serum ALT concentrations remained normal and HCV-RNA remained negative in all patients except two dropouts and two patients who developed relapsing disease after having been HCV-RNA negative for three and eight months, respectively. In both patients, the same HCV genotype 3 reemerged. Serum ALT concentrations ranged from 531 to 1940 IU/liter (mean=1055; normal <22). Concentrations of HCV-RNA (Quantiplex; Chiron, Emeryville, California) were <3.5×105 eq/ml in nine of 14 PCR-positive patients. In the other five patients, concentrations ranged from 10.4×105 eq/ml to 131.6×105 eq/ml (mean=69.6×105). No correlation was observed between HCV-RNA concentrations and serum ALT concentrations at presentation (r=0.331;P=0.67) and total dose of interferon-α2b administered until normalization of ALT (r=−0.088;P=0.74). Twenty-two of 24 patients completed treatment (two were noncompliant). Of these, 20 achieved a complete response (HCV-RNA negative for at least six months). Two of these patients relapsed, and 18 (90%) remained HCV-RNA negative for 18.65 (±9.7) months. These findings suggest that high-dose interferon-α2b is well tolerated and effective in preventing a chronic course of hepatitis C infection.

Detection of enteroviral ribonucleic acid in myocardial biopsies from patients with idiopathic dilated cardiomyopathy by polymerase chain reaction

Infection by enteroviruses, especially by Coxsackie B viruses, has been incriminated in pathogenesis of dilated cardiomyopathy. We developed polymerase chain reaction tests for the detection of enteroviral and Coxsackie B3 genomes, respectively, in myocardial biopsies obtained from a homogeneous group of 19 patients with idiopathic dilated cardiomyopathy. To determine unambiguously the incidence of enteroviruses and Coxsackie B3 viruses in these patients, we used two primer pairs, one common to all enteroviruses and the other specific for Coxsackie B3 viruses. In six patients of the dilated cardiomyopathy group, enteroviral ribonucleic acid (RNA) could be detected; only one was subspecified as Coxsackie B3 RNA. In contrast, no enteroviral RNA could be detected in a contrast group of 21 patients with other cardiac disorders. These results suggest that enteroviruses other than Coxsackie B3 are causally linked to the pathogenesis of dilated cardiomyopathy.

Syringotropic cutaneous T‐cell lymphoma: a variant of mycosis fungoides?

We report two patients with the typical clinical (patches/plaques studded with brownish‐red papules) and histopathological (hyperplastic eccrine ducts and glands surrounded and infiltrated by lymphocytes) features of syringotropic cutaneous T‐cell lymphoma. Immunohistochemical studies confirmed the T‐cell character of the infiltrate, and gene rearrangement studies its monoclonality (in one case). Our patients did not present with visceral involvement.

Induction of HA-1-specific cytotoxic T-cell clones parallels the therapeutic effect of donor lymphocyte infusion

Summary.  Donor lymphocyte infusions (DLI) can induce a graft‐versus‐leukaemia (GvL) reaction in patients with relapsed disease. However, the mechanisms involved in remission induction are not completely known. A patient with chemotherapy‐refractory relapse 1 year after human leucocyte antigen (HLA)‐identical, unrelated stem cell transplantation (SCT) for bcr/abl‐positive common acute lymphoblastic leukaemia (ALL) received a DLI from the original donor, and achieved complete cytogenetic and molecular remission concomitantly with extensive graft‐versus‐host disease (GvHD). Seven CD8+, donor‐derived, alloreactive T‐cell clones were generated by stimulating post‐DLI remission cells with the patients pretransplant mature dendritic cells. The minor histocompatibility antigen (mHag) recognized by these T‐cell clones was identified as HA‐1, a mHag associated with acute GvHD after SCT. Our finding provides evidence of HA‐1‐associated GvL effects after DLI that paralleled the eradication of full‐blown, chemotherapy‐refractory ALL relapse after allogeneic SCT.

Urinary neopterine as marker for haematological neoplasias

Urinary neopterine levels were studied in 79 normal subjects and in 112 patients with haematological neoplasias. The mean values in 79 patients with active disease were significantly raised compared to the control group. Results obtained in 79 patients with active disease indicate that 91% had neopterine levels higher than the mean value of 79 normal individuals +3 SD. There is only a little overlap between the range of neopterine levels in cancer patients and the range in healthy subjects. No significant difference was found between the mean urinary neopterine levels of 33 patients with non-Hodgkins or with Hodgkins lymphoma in remission and the healthy group. Only 15% of these patients had elevated neopterine levels. The mean urinary neopterine levels correlated well with the tumor stage in patients with chronic lymphocytic leukaemia and with non-Hodgkins disease. In patients with chronic leukaemia those without hepatosplenomegaly excreted significantly more neopterine than controls, and patients with hepatosplenomegaly significantly more than those without hepatosplenomegaly. It is concluded that urinary neopterine levels are of value for following the progression of haematological neoplasias.

Mammaglobin expression in gynecologic malignancies and malignant effusions detected by nested reverse transcriptase-polymerase chain reaction.

The detection of micrometastatic disease remains a challenge for the diagnosis and monitoring of malignant disease. RT-PCR for human mammaglobin (hMAM) was recently shown to provide a sensitive method for assessing circulating breast cancer cells in peripheral blood. This study was aimed at investigating hMAM expression in normal and malignant tissue from the female genital tract and the prostate as well as in malignant effusions derived from gynecologic malignancies. hMAM expression was analyzed with nested RT-PCR in 152 samples of normal (n = 73) and malignant epithelial tissues (n = 79) and in 33 specimens of various normal mesenchymal tissue types. We found hMAM expression was not restricted to the normal mammary gland and breast carcinoma but was also detectable in most specimens of benign and malignant epithelial tissue from the ovary (97% versus 95%), uterus (both 100%), and cervix (91% versus 90%). Notably, hMAM expression was also found in benign prostatic hyperplasia (45%) and in prostate cancer (55%). A much lower expression rate was found in various normal and benign mesenchymal tissues (12%). In keeping with our previous data, hMAM expression was absent in all control samples (n = 124) of peripheral blood and bone marrow from healthy volunteers and patients with hematologic malignancies. In pleural or peritoneal effusions (n = 42) from patients with carcinomas of the breast, endometrium, or ovary, hMAM positivity was noticed in the majority of cases (74%), whereas only 52% of the specimens were cytologically positive for tumor cells. In conclusion, hMAM expression assessed by nested RT-PCR is a sensitive molecular marker for detecting micrometastatic tumor spread into pleural effusions and ascites from patients with breast cancer and various other gynecologic neoplasms.

Combination of Cytology, Fluorescence In Situ Hybridization for Aneuploidy, and Reverse-Transcriptase Polymerase Chain Reaction for Human Mammaglobin/Mammaglobin B Expression Improves Diagnosis of Malignant Effusions

PURPOSE The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. The aim of this study was to improve tumor cell detection in effusions by molecular approaches. MATERIALS AND METHODS A total of 157 effusions from patients with tumors and 72 effusions from patients without a history or evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, fluorescence in situ hybridization (FISH) for aneuploidy, and reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of human mammaglobin (hMAM) and mammaglobin B (hMAM-B). RESULTS In effusions from patients with tumors, the sensitivities of tumor cell detection by cytology, FISH, and hMAM and hMAM-B detection were 46.2%, 53.3%, 36.4%, and 57.7%, respectively. The corresponding specificities were 94.4%, 97.0%, 87.1%, and 88.6%. Notably, a high percentage of effusions containing malignant cells were in fact transudates, indicating the necessity for molecular diagnostic work-up of transudates collected from patients with tumors. Dependent on the tumor type, the use of appropriate marker combinations improved tumor cell detection in effusions significantly. By combining all four diagnostic tests, a positive test result indicating the presence of malignancy was achieved in 81.1%, with a fairly good specificity of 70.1%. CONCLUSION Molecular techniques are definitely useful to detect malignancy in cytologically negative effusions. Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. This finding should help to improve tumor staging, prognostic assessment, and treatment monitoring.