Detection of micrometastases in the bone marrow in patients with gastric cancer

An immunocytochemical method using an antibody probe to recognise the epithelial membrane antigen was used to screen smears obtained surgically from bone-marrow in 88 patients with gastric cancer. Tumor cells were detected in the bone-marrow of 58 patients (65.9%). The EMA positive cells in the marrow were not correlated with the location and node status of the stomach. In the stage of TNM I, II, III and IV, the positive rates of micrometastases in the bone marrow were 42.9%, 57.1%, 73.7% and 69.0%, respectively. The results showed that the poorer differentiated lesion, the higher rate of positive cells in the bone marrow. The curative surgery and multimodality treatment after operation could result in remission of positive cells in some patients. The method can detect occult metastases in bone marrow, and may be useful to monitor patients for evidence of response. It can measure the efficacy of adjuvant therapy, and predict prognosis of the patients.     

Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.     

Value of fluorescence in situ hybridization for detecting the bcr/abl gene fusion in interphase cells of routine bone marrow specimens

A patient presented with a severe nephrotic syndrome and a renal biopsy consistent with early focal segmental glomerulosclerosis (FSGS). After a year of intensive immunosuppressive therapy proteinuria was unabated and renal function began to deteriorate. Treatment with weekly plasmapheresis combined with moderate doses of prednisone and azathioprine produced a dramatic decrease in proteinuria and serum creatinine. A marked fall in the B-cell population occurred during treatment, as well as an increase both in T-lymphocytes of the mature CD4+ helper/suppressor phenotype and in the immature/cytotoxic CD8+ phenotype. Activation of the immune system during treatment was demonstrated by an increase in the rate of spontaneous proliferation of peripheral blood mononuclear cells and an increase in T-cell expression of the interleukin-2 receptor.     

ALL R-87 protocol in the treatment of children with acute lymphoblastic leukaemia in early bone marrow relapse

Seventy-three children with acute lymphoblastic leukaemia (ALL) in first bone marrow (BM) relapse, occurring within 30 months from complete remission (CR), were enrolled in an Italian cooperative study (ALL R-87 protocol). This treatment programme consisted of an induction phase with intermediate-dose cytarabine (IDARA-C) plus idarubicin (IDA) and prednisone (PDN), followed by a multidrug consolidation therapy and bone marrow transplant (BMT). 55/73 children achieved CR (75.3%); 15 (20.5%) failed to respond and three (4.2%) died during induction. The response rate was significantly higher for children with a first CR duration > or = 12 months (P=0.0005) and for those with a white blood cell (WBC) count at relapse < 20 x 10(9)/l (P=0.004). The estimated disease-free survival (DFS +/- SE) at 82 months was 0.18 +/- 0.05 for all responders, and 0.70 +/- 0.14 for allotransplanted patients versus 0.05 +/- 0.05 for those autografted (P=0.001). The estimated probabilities of survival +/- SE and event-free survival (EFS +/- SE) at 83 months were 0.16 +/- 0.07 and 0.13 +/- 0.04, respectively. for all enrolled children. Univariate analysis showed that age < 10 years at initial diagnosis and B-lineage immunophenotype favourably influenced both DFS (P=0.001) and EFS probabilities (P=0.0014 and P=0.012, respectively), whereas a first CR duration > or = 12 months and a WBC count at relapse < 20 x 10(9)/l were associated only with a better EFS rate (P=0.026 and P=0.004, respectively). Our results show the efficacy of the IDA plus IDARA-C schedule used in the ALL R-87 protocol in high-risk relapsed ALL children. Allogeneic BMT proved effective for patients with an HLA sibling donor. In a multivariate analysis, age > or = 10 years at initial diagnosis (P=0.016) and WBC count at relapse > or = 20 x 10(9)/l (P=0.048) were independently associated with a worse disease outcome.     

Central review of bone marrow biopsy specimens from patients with neuroblastoma

In order to achieve some uniformity in histological detection of bone marrow infiltration by neuroblastoma and to provide a measure of variation in histological opinions, sections from 712 evaluable trephine biopsy cores from children in a European Neuroblastoma Study Group (ENSG) study were reviewed centrally. Biopsy specimens were graded as tumour positive or negative. Discordance between local and central review opinions was found in 5% of specimens. Only five of 165 children at presentation and nine of 256 re-staging procedures in 126 children, affecting one child each, had their diagnosis upgraded to positive. In six re-staging procedures, affecting one child each, the diagnosis was downgraded. The low discordance rate is encouraging and substantially less important than previously documented difficulties in obtaining adequate specimens.     

Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia

PURPOSE: To evaluate the 2-year clinical performance of two polyacid-modified resin composites and two resin-modified glass ionomers in Class V carious cavities. MATERIALS AND METHODS: A total of 120 Class V cavities were selected and 30 cavities were restored with one of two resin-modified glass ionomer materials (Fuji II LC Improved and Vitremer) and two polyacid-modified resin composites (Dyract and Compoglass) in Class V carious cavities after 2 years. The restorations were clinically evaluated after 1 and 2 years using the USPHS criteria. RESULTS: One-year findings revealed a significant difference in color match between Vitremer and other materials (P < 0.05) and no significant difference was found for the other criteria. Two-year results indicated a significant difference between resin-modified glass ionomers and polyacid-modified resin composite materials. The difference between Compoglass and Dyract was not statistically significant whereas the difference between Vitremer and Fuji II LC was statistically significant. Caries was not recorded at any evaluation period.     

Polyclonal/monoclonal ratio in kidney and bone marrow transplanted patients treated with cyclosporine

OBJECTIVE: To examine the hypothesis that suppression of frequent premature ventricular contractions may be associated with improvement in left ventricular function in patients with presumed idiopathic dilated cardiomyopathy. DESIGN: We conducted a retrospective case study and statistical analysis of the effect of cardiac medical therapy on outcome. MATERIAL AND METHODS: The study population consisted of 14 patients with more than 20,000 premature ventricular contractions in 24 hours recorded by Holter monitoring and associated left ventricular dysfunction (ejection fraction, 40% or less). Clinical characteristics, number of premature ventricular contractions per hour on 24-hour ambulatory Holter monitoring, and ejection fraction based on transthoracic echocardiography were compared before and after cardiac therapeutic intervention. RESULTS: Of the 14 patients, 10 had presumed idiopathic dilated cardiomyopathy, and 4 had ischemic heart disease. Of the overall study group, seven had received additional cardiac medical therapy after the index evaluation, including four patients who had amiodarone therapy. A significant reduction (75% or more from baseline) in premature ventricular contractions after medical therapeutic intervention was observed in five patients at the first follow-up examination. The mean interval to the first follow-up examination was 6 +/- 3 months. Of the five patients, four had significant improvement in clinical functional status and the ejection fraction. The mean ejection fraction of these five patients increased from 27 +/- 10% at baseline to 49 +/- 17% after medical therapy (P = 0.04). CONCLUSION: The suppression of frequent premature ventricular contractions may be associated with improvement of left ventricular function in patients with presumed idiopathic dilated cardiomyopathy.     

Magnetic resonance imaging in patients with bone marrow disorders

Magnetic resonance imaging (MRI) provides a non-invasive means to evaluate a large fraction of marrow in less than one hour. Marrow disorders produce non-specific changes in marrow signal intensities which primarily reflect changes in proportions of fat and cellular elements. The pattern of these signal changes narrows the differential diagnosis, and the combination of these features with the clinical context allows interpretations which are clinically useful in many ways. These include: 1) the diagnosis of avascular necrosis (and its distinction from other causes of joint pain), 2) detection of osteomyelitis, 3) differential diagnosis of hypoplastic disorders, 4) staging of lymphomas and myeloma, 5) selection of patients for autologous bone marrow transplant, 6) objective measures of marrow response to therapy, 7) detection of leukemic transformation, and 8) improved detection of marrow disease (primary or secondary) in patients with otherwise unexplained bone pain.     

Enzymes in peripheral and bone marrow serum in patients with cancer

Lactic dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT), and acid and alkaline phosphatase activities in bone marrow and in cubital vein serum were compared. For patients without cancer, marrow serum LDH attained levels four times as high, and GOT and alkaline phosphatase, levels twice as high as those normal for cubital vein serum; levels of acid phosphatase were the same for both sources. For patients with cancer, significant increase of enzyme levels over reference levels depends on the tumor origin and on the presence and localization of metastases. Marrow enzyme levels may become elevated with or without concurrent elevation in cubital vein serum. Concurrent elevations were found with colonic carcinoma and lymphoid leukemia, and noncurrent elevations, with prostatic cancer, myeloid leukemia, and myeloma. A nonconcurrent elevation of marrow enzymes indicates that the origin of the enzyme is in the marrow, whereas with concurrent elevation, the source of the enzyme may be another organ.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Detection of neuroblastoma in the bone marrow: biopsy versus aspiration

OBJECTIVES: To determine the clinical effects of a change from postural drainage (PD) to positive expiratory pressure chest physiotherapy (PEP) in children with cystic fibrosis (CF) and symptoms of gastro-oesophageal reflux (GOR). To measure the effects of PD on GOR in children with CF. METHODS: Study 1: Six adolescents with CF and symptoms of GOR during PD were changed to upright PEP physiotherapy. The effects on lung function, reflux symptom scores and annual hospital days were measured. Study 2: Twenty-four children with CF (mean age 11 years) and symptoms suggestive of GOR underwent 24-h pH monitoring, including periods of chest physiotherapy. RESULTS: Study 1: All six patients reported a reduction in reflux symptoms during PEP therapy (P < 0.001). Lung function parameters improved during the first 6 months of PEP (P < 0.001). This improvement was sustained for a further 18 months. Annual hospital days decreased significantly (P < 0.0005). Study 2: Nine of 24 patients (37.5%) had pathological GOR. Reflux episodes were significantly increased during PD (P < 0.0001), as was fractional reflux time (P < 0.01). CONCLUSIONS: Upright PEP physiotherapy may be more appropriate than PD in selected patients with CF and symptomatic GOR. The role of GOR as a cofactor in the progression of pulmonary disease in CF needs further evaluation.     

Immunohistochemistry in apparently normal bone marrow trephine specimens from patients with nodal follicular lymphoma

AIM: To establish the role of immunohistochemistry (using a limited panel of antibodies) in detecting minimal involvement by follicular lymphoma in routinely processed bone marrow trephine specimens, which show no obvious morphological (light microscopic) evidence of lymphoma; to determine whether bcl-2 immunostaining in bone marrow distinguishes between benign and malignant infiltrates in a patient with nodal follicular lymphoma. METHODS: Twenty seven consecutively selected paraffin wax embedded, formalin fixed bone marrow trephine specimens were stained with the following antibodies: anti-bcl-2, anti-CD79a, anti-CD3, and kappa and lambda light chains, using the Streptavidin biotin complex technique. RESULTS: Five of the 27 cases, which showed no evidence of involvement by follicular lymphoma on routine stains, showed monotypic B cells on immunohistochemistry. Two of the cases were diffuse, while the remaining three showed mini-aggregates around bony trabeculae. In all five cases the lymphomatous infiltrates were strongly bcl-2 positive. Reactive B lymphoid nodules did not show the same degree of bcl-2 positivity, and negative cells could be discerned within the reactive nodules. CONCLUSIONS: There is merit in studying so-called negative bone marrows immunohistochemically in order to detect minimal involvement by follicular lymphoma. A limited panel of antibodies including anti-bcl-2, anti-CD79a and anti-CD3 is usually adequate to accomplish this. Strongly bcl-2 positive lymphoid aggregates in the bone marrow of patients with nodal follicular lymphoma are indicative of lymphoma.     

Detection of polyomaviral DNA in clinical samples from immunocompromised patients: correlation with clinical disease

Characterization of the region between HLA-B and the TNF loci in the human MHC revealed the presence of duplicated loci, named CL1 and CL2, that included repeat sequences. Development and use of a PCR typing methodology that amplified both CL microsatellites simultaneously indicated that PCR product patterns analysed on native agarose gels were allelic (Abraham et al., 1992). The purpose of the current study was to determine the molecular explanation for the unique patterns achieved. Sequence analysis of the CL1 locus from 32 chromosomes representing 10 ancestral haplotypes indicated that six alleles were present. The CL microsatellites also provided an opportunity to study the evolutionary relationships between MHC haplotypes from different racial groups. Sequence comparison of closely related ancestral haplotypes from different racial groups suggested that the CL1 microsatellite has not changed in the period since divergence.     

In-vitro growth patterns of bone marrow erythroid progenitors from patients with post-renal transplant erythrocytosis

BACKGROUND: Erythrocytosis is relatively common after renal transplantation and is associated with a higher risk of thromboembolism. Its aetiology is unclear and there is still debate about the most frequently suggested causes. The culture in vitro of erythroid progenitors is regarded as a useful tool for the differential diagnosis of patients with unclear erythrocytosis. We studied the growth in vitro of bone marrow erythroid progenitors from renal transplant patients with erythrocytosis and controls without erythrocytosis. SUBJECTS AND METHODS: Thirteen renal transplant patients with erythrocytosis and 12 normocythaemic renal transplant controls were studied. The clinical characteristics of these patients were evaluated and serum erythropoietin (Epo) and ferritin levels were determined. Bone marrow erythroid progenitors were cultured both with and without the addition of Epo to the medium. RESULTS: Samples from six polycythaemic patients and seven controls did not grow spontaneously in the absence of exogenous Epo. Three cases of post-transplant erythrocytosis and five controls produced CFU-E, but not BFU-E. A few CFU-E and BFU-E grew spontaneously in samples from four polycythaemic patients but not in samples from the controls. Addition of 1 unit per millilitre Epo caused similar increases in the number of colonies in both polycythaemic patients and controls. Of the nine patients eligible for follow-up, all four with spontaneous growth of BFU-E had transient erythrocytosis and four of the five patients with no spontaneous growth or spontaneous growth of CFU-E only had persistent erythrocytosis requiring treatment with ACE inhibitors. CONCLUSIONS: Pathophysiology of post-transplant erythrocytosis is heterogeneous. In one-third of the patients, there was unexpected, spontaneous and transient growth of BFU-E which was not predictive of permanent erythrocytosis. The results of stem-cell studies suggest that in these cases erythrocytosis may be caused by defective regulation of erythroid progenitor proliferation, possibly due to particular cellular interactions or the effect of cyclosporin on erythropoiesis.     

Busulfan-based regimens and allogeneic bone marrow transplantation in patients with myelodysplastic syndromes

Preparative regimens containing busulfan (BU) followed by allogeneic bone marrow transplantation (BMT) were used in 27 consecutive patients with myelodysplastic syndromes (MDS). The median age was 33 years (range, 4 to 54). Ten were female and 17 male. Sixteen patients had primary MDS, 11 other patients had antecedent hematologic diseases or developed MDS after cytotoxic and/or radiation therapy. Six patients had leukemic transformation and received antileukemic therapy before BMT. Pre-BMT cytogenetic studies showed complex chromosomal abnormalities in 13 patients, a simple abnormality in 5 patients, and normal chromosome in 8 patients. Three BU-based preparative regimens were used: 1 patient received BU 4 mg/kg orally (PO) daily for 4 days and cyclophosphamide (CY) 50 mg/kg intravenously (IV) daily for 4 days (BUCY-4); 24 patients received BU 4 mg/kg PO daily for 4 days, cytosine arabinoside (ara-C) 2 g/m2 IV every 12 hours for 4 doses, and CY 60 mg/kg IV daily for 2 days (BAC); and 2 patients with preceding Fanconi anemia received BU 2 mg/kg PO daily for 4 days followed by total lymphoid irradiation of 5 Gy. Seventeen of 27 patients are alive with no evidence of disease. Ten patients have died: 2 from hepatic veno-occlusive disease, 3 from sepsis, 1 from a cerebral bleed, 1 from a massive gastrointestinal (GI) bleed associated with acute graft-versus-host disease, 1 from hemolytic uremic syndrome with adult respiratory distress syndrome, 1 from bronchiolitis obliterans, and the only patient who did not engraft died from acute myeloid leukemia. Regimen-related toxicities (RRT) include GI tract (diarrhea, 14; stomatitis, 11), liver (9), cardiac (1), and skin (5). Patients who received a genotypically matched marrow graft had a significantly better disease-free survival (DFS) than patients who received a nongenotypic marrow graft (P = .02). The Kaplan-Meier analysis projects an overall DFS of 56% +/- 13% and 78% +/- 10% for patients who received a genotypically matched marrow graft. With the exception of a child who did not engraft, there was no relapse of MDS or leukemia. Excellent DFS, acceptable RRT, and the ease of administration are advantages of this regimen.     

Tumor angiogenesis and micrometastasis in bone marrow of patients with early gastric cancer

In a subset of patients with early gastric cancer, there were recurrences of the disease after a curative resection had been done. Direct evidence of tumor seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap because it is a feature of epithelial cells that would not normally be present in bone marrow. From 1994-1997, the bone marrow of 45 patients with early gastric cancer was examined for tumor cells, using immunocytochemical techniques and an antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. Intratumoral microvessels were stained with anti-CD31 monoclonal antibody. Clinicopathological characteristics were determined for subjects with cytokeratin-positive cells in the bone marrow. Of these 45 patients, 9 (20.0%) had cytokeratin-positive cells in the bone marrow at the time of primary surgery. These positive findings were not related to tumor advance-related factors of lymph node metastasis and distinct lymphatic and vascular invasion. Microvessel density in the primary tumor exceeded 2-fold in cytokeratin-positive cells, compared with findings in negative cells (P < 0.05). Tumor cells in bone marrow are indicative of the general disseminative metastasis in patients with early gastric cancer, and the metastatic potential was closely related to angiogenesis in the primary tumor.