Loss of red cell A, B, and H antigens is frequent in myeloid malignancies.

Loss of A, B, and H antigens from the surface of red blood cells has been a recurrent observation in patients with hematologic malignancy, particularly those malignancies in which the myeloid lineage is involved. To better understand this phenomenon, a 2-color flow cytometric method was developed to determine quantitative and qualitative alterations of A, B, and H antigens in patients with myeloid malignancies. Characteristic patterns, dependent on the genotype, were seen for healthy individuals from each of the blood groups. Fifty-five percent (16/29) of patients of blood group A, B, or AB had a proportion of red cells with decreased expression of A or B antigens compared with no changes in 127 healthy A, B, and AB individuals. In most cases, the changes were not detected by routine serologic typing. The loss of A or B antigens was the primary change in 28% (8/29) of patients. In 17% (5/29) of patients, loss of A or B antigens was an indirect consequence of loss of the precursor H antigen. Alterations involving both the H and the A or B antigens were seen in 10% (3/29) of patients. Loss of H was also detected in 21% (6/28) of group O patients whereas none of 51 healthy O individuals showed changes. Alterations of ABO antigens can now be considered a common event in myeloid malignancy. (Blood. 2001;97:3633-3639)

Molecular detection of blood-borne epithelial cells in colorectal cancer patients and in patients with benign bowel disease.

In colorectal cancer (CRC), a proportion of patients with early stage disease still die of metastatic or recurrent disease within 5 years of "curative" resection. Detection of carcinoma cells in the peripheral circulation at presentation may identify a subgroup of patients with micro-metastatic disease who may benefit from adjuvant chemotherapy or radiotherapy. Our aim was to determine the presence and clinical significance of colon carcinoma cells in peripheral blood at the time of surgery. Preoperative peripheral blood samples were collected from 94 patients with CRC and 64 patients undergoing bowel resection for benign conditions (adenoma, diverticular disease or Crohn's colitis). Blood was also obtained from 20 normal donors not undergoing bowel surgery. Immunomagnetic beads were used to isolate epithelial cells followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression of cytokeratin (CK) 19, CK 20, mucin (MUC) 1 and MUC 2. Nineteen of 94 (20%) CRC patients were positive for epithelial cells in preoperative blood, including 6 with early stage disease. Kaplan-Meier survival analysis showed that detection of epithelial cells in preoperative blood was associated with reduced disease-free and overall survival (log-rank test, p = 0.0001). Surprisingly, circulating epithelial cells were detected in 3/30 (10%) patients resected for adenoma, and in 4/34 (12%) patients resected for benign inflammatory conditions, suggesting that cells from nonmalignant colonic epithelium may also gain entry into the bloodstream in the presence of bowel pathology. All 20 normal control bloods were negative for epithelial cells.

Methadone-maintenance outcomes for Hispanic and African-American men and women.

Six-month methadone-maintenance response and outcome were examined for African-American and Hispanic men and women in a large urban sample. A consistent pattern of improvement was indicated for both races and genders on the addiction severity index (ASI). There were virtually no statistically significant differences in ASI outcomes between Hispanics and African-Americans and men and women using conventional analysis of variance (ANOVA) procedures. Results from an additional equivalence analysis, however, indicated that baseline to 6-month changes for the different groups were generally not similar enough to consider them equivalent. Urine toxicologies obtained during the 6-month treatment period were also not statistically equivalent by race and gender. Evaluating outcomes by gender and race are discussed, as are the implications of using equivalence tests when examining group differences.

Treatment of metastatic breast cancer with continuous infusional 5 fluorouracil.

BACKGROUND: Single agent continuous infusional 5 fluorouracil (CI-5FU) via a central venous catheter (CVC) is usually reserved for breast cancer patients who have previously failed one or more chemotherapy regimens. The patients are usually heavily pre-treated with later stage disease. Previously published studies of CI-5FU have reported response rates as high as 54%. It is considered an approach with an acceptable side effect profile in such patients. AIMS: To evaluate the efficacy and toxicity of CI-5FU in previously treated metastatic breast cancer. METHODS: A retrospective review of advanced breast cancer patients treated with CI-5FU between October 1992 and October 1996 was performed. Response to treatment, toxicity, CVC complications and patient survival were analysed. RESULTS: Twenty-four patients with metastatic breast cancer were treated with CI-5FU. All had received previous chemotherapy, including 19 patients (79%) with prior 5FU exposure and eight patients (33%) who had previous high dose chemotherapy with autologous stem cell transplantation. The median duration of CI-5FU treatment was 3.1 months. Nineteen patients had evaluable disease, three (16%) of whom demonstrated a partial response and four patients had stable disease. There were no complete responses. All responses occurred in soft tissue sites with no objective evidence of response in liver or bone metastases. The survival rate at one year was 21% (five of 24) and the median survival of all patients was 6.1 months. Five patients (21%) stopped treatment due to treatment related morbidity (two CVC complications and three CI-5FU side effects). Diarrhoea, nausea, and palmar-plantar erythrodysaesthesia were the major side effects of chemotherapy. CVC complications requiring intervention, the most notable of which were infection and thrombosis, occurred in 11 patients (46%). There were no treatment related deaths. CONCLUSIONS: Single agent CI-5FU has modest activity in women with previously treated advanced breast cancer. The efficacy is lower than in previously published series. This may reflect patient selection factors. The toxicity was mainly related to CVC complications. Important issues relating to quality of life need to be objectively measured in future studies of CI-5FU.

Somatic mutations, acetylator status, and prognosis in colorectal cancer.

BACKGROUND: Somatic mutations in K-ras and TP53 may be associated with both acetylator status and prognosis in colorectal cancer. AIMS: To determine whether cancers with somatic mutations are more frequent in fast acetylators and whether mutations or acetylator status influence prognosis after colorectal surgery. PATIENTS: One hundred consecutive subjects undergoing elective surgery for colorectal cancer. METHODS: Acetylator status was determined by polymerase chain reaction (PCR) genotyping for polymorphism in the N-acetyltransferase 2 (NAT2) gene. Mutations in K-ras (codon 12) and TP53 were determined by PCR analysis using restriction enzyme digestion and single strand conformation polymorphism respectively. Survival from colorectal cancer for up to five years after diagnosis was analysed using the Kaplan-Meier product limit estimator. Cox proportional hazards regression was used to compare survival rates after adjusting for tumour stage. RESULTS: Mutations in K-ras and TP53 were independent of acetylator status. By log rank test, survival was significantly reduced in subjects with TP53 mutations (p = 0.003) but was not significantly related to acetylator status or the presence of K-ras mutations. After adjustment for tumour stage, subjects with both TP53 and K-ras mutations had a 4.2-fold case fatality (95% confidence interval 1.5 to 11.6) when compared with that of a TP53 negative reference group. CONCLUSION: The presence of both TP53 and K-ras mutations in colorectal tumours is an adverse prognostic marker which is independent of tumour stage.

Effect of high-dose chemotherapy on intestinal permeability in humans.

1. Mucositis is a common side-effect of chemotherapy which is difficult to assess except by invasive means such as upper gastrointestinal endoscopy. Differential absorption of mono- and di-saccharides, such as rhamnose and lactulose, is a non-invasive measure of intestinal damage. 2. The purpose of the study was to assess the duration and severity of intestinal damage in patients undergoing high-dose chemotherapy and autologous blood stem-cell transplantation for malignant disease. 3. Thirty-five patients were studied before treatment and at 7, 28, 60 and 90 days after treatment. 4. The median lactulose/rhamnose ratios before treatment and at 7 and 90 days post-treatment were 0.09, 0.62 and 0.06 respectively. Altered permeability was due to both increased lactulose permeation and decreased rhamnose absorption. These abnormalities suggest a defect in tight-junction integrity as well as a decrease in surface area of small bowel. 5. We conclude that chemotherapy given for malignant disease is associated with a transient abnormality in intestinal sugar permeability, which peaks at 7 days after treatment and is composed of both mono- and di-saccharide absorption abnormalities.

Mobilization of predominantly Philadelphia chromosome-negative blood progenitors using cyclophosphamide and rHUG-CSF in early chronic-phase chronic myeloid leukaemia: correlation with Sokal prognostic index and haematological control.

Mobilization of Philadelphia chromosome (Ph) negative blood progenitors was attempted in 23 newly diagnosed chronic myeloid leukaemia (CML) patients using a regimen of cyclophosphamide (CY) 5 g/m2 and rHUG-CSF 150 microg/m2 daily. This regimen was well tolerated with no major adverse events reported. More than 2 x 10(6)/kg CD34+ cells were collected in 21 patients (91%). Predominantly Ph-negative mobilization (0-25% Ph-positive) was seen in 30% of cases overall and was confined to patients with a Sokal prognostic score < 1 (7/11 with Sokal score <1; 0/12 with Sokal score > or = 1). Within the low Sokal index group, a low WBC count pre-mobilization and a low WBC nadir both correlated strongly with Ph-negative mobilization (P = 0.006 and 0.02 respectively). Five of 19 patients receiving at least 6 months of Roferon A therapy post mobilization achieved a major cytogenetic response; all five patients were Ph-negative mobilizers. Therefore CML patients can be divided into a good-prognosis group in whom predominantly Ph-negative progenitors can be mobilized using a regimen of moderate intensity if haematological control is achieved pre-mobilization, and a poor-prognosis group for whom predominantly Ph-positive cells are mobilized with this regimen regardless of haematological control.

Detection of circulating tumor cells in colorectal cancer by immunobead-PCR is a sensitive prognostic marker for relapse of disease.

BACKGROUND: Recurrent and metastatic carcinoma of the colorectum remains a major problem, with survival at 5 years post curative resection still only about 50%. Moreover, up to 30% of patients who present with early stage disease also relapse and die within 5 years, suggesting the presence of micrometastatic disease at diagnosis. One route of metastatic spread is via the blood stream, hence the detection of tumor cells in blood is likely to provide an important predictive tool with respect to relapse of disease. We have developed a sensitive molecular technique to identify tumor cells in blood using mutations in codon 12 of the K-ras gene as a marker. MATERIALS AND METHODS: Twenty-seven patients whose tumor carried a mutation in codon 12 of K-ras were studied for the presence of tumor cells in perioperative peripheral blood samples. Immunomagnetic beads, labeled with an epithelial-specific antibody, were used to harvest epithelial cells from blood. K-ras mutations were identified in this selected population using a polymerase chain reaction (PCR)-based analysis (immunobead-PCR). RESULTS: Circulating K-ras mutant cells were detected in 9 or 27 patients; seven of these nine patients have since died due to recurrent or metastatic disease. Mutant cells were not detected in 18 patients, and 16 or 18 have remained disease free (median follow-up: 16 months; range: 7-42 months). Kaplan-Meier analysis showed that detection of K-ras mutant cells in bloods was associated with significantly reduced disease-free survival (p = 0.0001). CONCLUSION: This study indicates that detection of circulating tumor cells perioperatively by immunobead-PCR provides a sensitive prognostic marker for recurrent and metastatic colorectal cancer.

Impaired responsiveness of platelets from patients with stable angina pectoris to antiaggregating and cyclicAMP-elevating effects of prostaglandin E1.

The antiplatelet effects of prostacyclin (PGI2) and prostaglandin E1 (PGE1) are mediated by the same receptor and are secondary to intraplatelet cyclicAMP formation. Therefore, any dysfunction in PGI2/PGE1-stimulated cyclicAMP generation might lead to pathologically increased platelet aggregation. This possible consequence has not yet been studied. We examined antiaggregating effects of PGE1 in comparison with its cyclicAMP-elevating potency in platelets obtained from normal subjects and patients with stable angina pectoris; platelet hyperaggregability in such patients has been documented by us previously. ADP-induced aggregation was measured in platelet-rich plasma (PRP); PGE1 was added to platelets 0.5 min after ADP for assessment of reversal of incipient aggregation. Concentrations of PGE1 associated with 50% reversal of aggregation (C50) were 2.4 +/- 0.3 x 10(-8) M in normal subjects and 6.3 +/- 1.6 x 10(-7) M in patients (p < 0.01). PGE1 produced a concentration-dependent increase in intraplatelet cyclicAMP, and there was a strong correlation between cyclicAMP-stimulating and antiaggregating effects of PGE1. Maximal increases in cyclicAMP with PGE1 10(-4) M were 330 +/- 10% for normal subjects and 220 +/- 20% for patients (p < 0.01). Thus, the observed decrease in PGE1-induced reversal of platelet aggregation in patients can be attributed to a suppressed cyclicAMP response to PGE1. These results are likely also to imply reduced platelet sensitivity in vivo to endogenous PGE1 and PGI2, which in turn might contribute to platelet hyperaggregability observed in cardiovascular diseases.

Significance of molecular marker-positive cells after autologous peripheral-blood stem-cell transplantation for non-Hodgkin's lymphoma.

PURPOSE: To evaluate the significance of molecular marker-positive cells in a cohort of non-Hodgkin's lymphoma (NHL) patients undergoing high-dose chemotherapy and autologous peripheral-blood stem-cell transplantation (PBSCT). PATIENTS AND METHODS: Twenty-eight PBSC transplants have been performed in 24 patients with poor-prognosis NHL. Molecular analysis of the t(14;18) (q32;q21) translocation (bcl-2/immunoglobulin [Ig] heavy-chain joining locus [JH] fusion) or antigen receptor gene rearrangements was performed to determine the presence of lymphoma cells at presentation, in PBSC harvests, and before and after autologous PBSCT. Kaplan-Meier estimates of survival and Cox regression analyses were used to test the effect of bone marrow involvement, tumor-cell contamination of PBSCs, disease stage, and chemotherapy sensitivity at transplantation, and presence of marker-positive cells post-PBSCT on disease-free and overall survival. RESULTS: Thirteen of 24 patients (54%) are alive following PBSCT at a median follow-up time of 654 days (range, 193 to 1,908). Nine patients are in complete remission (CR) at day 216 to 1,799 (median, 805) and four are alive following relapse (day 440, 573, 1,188, and 1,908). Eleven patients (46%) have died: three of transplant-related complications at day 0, 1, and 13, and eight of recurrent disease (day 132 to 1,330; median, 451). Longitudinal marker studies post-PBSCT showed that of 16 relapse events, 13 (81%) were positive for the lymphoma marker at or before clinically documented relapse. Marker studies became negative post-PBSCT in nine of nine patients who entered and remained in CR. Disease-free survival (DFS) was significantly shortened in patients in whom marker-positive cells were detected in serial samples posttransplantation (P = .006). Cox regression analysis showed that patients in this group had a 24 times higher risk of relapse (P = .03). CONCLUSION: The results show that the reappearance or persistence of marker-positive cells after autologous PBSCT is strongly associated with relapse.

Dose intensive therapy with autologous blood stem cell transplantation in breast cancer.

BACKGROUND: Breast cancer is the commonest form of cancer in Australian women. Although approximately 50% of women with breast cancer achieve long term survival by current management methods, recurrent or metastatic disease is generally incurable. In addition, women with Stage II disease with > 10 positive axillary lymph nodes and also women with locally advanced disease (Stage III) have a poor survival even with adjuvant therapy. AIMS: To assess the toxicity and efficacy of high-dose chemotherapy with autologous peripheral blood stem cell (PBSC) transplantation in women with both metastatic and poor prognosis primary breast cancer. METHODS: Twenty-eight women with either metastatic (15) or poor prognosis (13) primary breast cancer were enrolled in the study between November 1988 to January 1993. PBSC were harvested using high-dose cyclophosphamide (Cy) with or without granulocyte-colony stimulating factor (G-CSF) and a myeloablative regimen of Cy, melphalan and carboplatin (CMCp) was used in the transplantation phase. RESULTS: Optimum numbers of stem cells were harvested in 85% of patients. The use of five G/m2 Cy plus G-CSF resulted in better PBSC yields and a significant reduction in haematologic morbidity when compared to mobilisation with Cy alone. Twenty-two women underwent 23 PBSC transplants (PBSCT). There have been two early deaths due to sepsis. The predominant morbidities observed following high dose chemotherapy and transplantation have been nausea, mucositis and diarrhoea. The median number of days to discharge following infusion of PBSC was 15 (range 11-21). At a median follow up time of 1.1 years (range 0 months-3.6 years), 8/22 (36%) evaluable patients remain alive and disease free while 14/22 (64%) have relapsed or progressed or died. CONCLUSION: High-dose chemotherapy and autologous PBSCT is a potentially highly effective treatment of women with metastatic and poor prognosis primary breast cancer. Randomised studies are required to compare this form of therapy to more standard forms of treatment in breast cancer.

Immunobead-PCR: a technique for the detection of circulating tumor cells using immunomagnetic beads and the polymerase chain reaction.

The presence of tumor cells in the circulation may predict disease recurrence and metastases. We have developed a sensitive technique for the detection of carcinoma cells in blood, using immunomagnetic beads to enrich for epithelial cells and the polymerase chain reaction to identify a tumor marker. The colon carcinoma cell line SW480, homozygous for a K-ras codon 12 mutation, was used to establish optimal conditions. The SW480 cells were serially diluted in normal blood and incubated with immunomagnetic beads labeled with a monoclonal antibody specific for epithelial cells. Cells bound to the beads were retrieved using a magnetic field and the presence of K-ras codon 12 mutations determined by a polymerase chain reaction based analysis. SW480 cells could be detected in dilutions up to 1 SW480 cell/10(5) leukocytes in whole blood.

High dose hydroxyurea in collection of Philadelphia chromosome-negative stem cells in chronic myeloid leukaemia.

High dose hydroxyurea (HU) was used in a pilot study to assess its efficacy in mobilizing Philadelphia (Ph) chromosome negative progenitor cells in chronic myeloid leukaemia (CML). Five patients received 12 g/M2 oral HU in divided doses. Side effects were minimal, allowing outpatient administration. Nine to fourteen days later 4 patients achieved a mean leukocyte nadir of 3.5 x 10(9)/L (Range 2.4-4.9) and a mean platelet nadir of 99 x 10(9)/L (Range 95-108). Peripheral blood mononuclear cells (PB-MNC) sampled prior to the HU priming were 100% Ph positive. Between 10 and 18 days post HU, 3 patients achieved a marked reduction (80-100%) in the number of Ph positive metaphases in PB-MNC collected by apheresis. One patient failed to achieve any Ph suppression. Polymerase chain reaction analysis (PCR) for the bcr-abl fusion product remained positive in all samples. Rapid rises in CFU-GM numbers were associated with a return to 100% Ph positive metaphases however slower rises represented recovery with predominantly Ph negative cells, allowing apheresis collection of these cells. We conclude that HU induces a reproducible leukocyte nadir with sufficient stem cell mobilization for potential autologous transplantation. Higher doses of HU together with early and intensive apheresis is required to maximize Ph negative progenitor cell harvests.

Antiplatelet effects of nitroglycerin in healthy subjects and in patients with stable angina pectoris.

The effects of nitroglycerin (NTG) on platelet aggregation are controversial. Most in vitro investigations suggest that NTG suppresses platelet aggregation only at suprapharmacologic concentrations. We investigated various aspects of the antiaggregating effects of NTG in both normal individuals and in patients with stable angina pectoris not treated with nitrates. Platelets from patients exhibited hyperresponsiveness to ADP as an inductor of aggregation. Sublingual administration to patients of NTG (300 micrograms) decreased platelet aggregability; ADP concentrations inducing 50% aggregation were 3.3 +/- 0.3 microM after NTG versus 2.1 +/- 0.1 microM before NTG (p < 0.01). Consistent with previous findings, NTG was a weak inhibitor of platelet aggregation in vitro when added before induction of aggregation. When added after the beginning of aggregation, however, NTG induced both inhibition of developing aggregation and marked disaggregation at concentrations > or = 10(-8) M NTG; concentration associated with 50% reversal of aggregation was 1.4 +/- 0.3 x 10(-6) M. Therefore, antiplatelet effects of NTG in vitro are demonstrable in low, clinically achievable concentrations; previously reported effects of NTG have been underestimated owing to suboptimum experimental conditions. Platelets from patients with angina pectoris were 100-fold less responsive to the cyclic GMP-increasing and disaggregating effects of NTG in vitro, which, together with increased aggregability, could imply reduced platelet sensitivity to endogenous sources of nitric oxide (NO) in vivo. The observed antiplatelet effects of NTG raise the question of its potential utility to reduce the risk of thrombotic complications in patients with ischemic heart disease.

Molecular detection of residual lymphoma cells in peripheral blood stem cell harvests and following autologous transplantation.

Twenty-seven patients with non-Hodgkin's lymphoma (NHL) have undergone peripheral blood stem cell (PBSC) harvesting for autologous transplantation (Tx). A molecular marker was found at presentation in 23/27 patients. Immunoglobulin heavy chain (IgH) or T cell receptor beta (TCR beta) rearrangements were detected by Southern blotting or the polymerase chain reaction (PCR) in 13 patients; PCR detected the bcl-2/JH fusion in 10 patients. Fifteen autologous PBSC transplants have been performed in 11 patients. In 5/11 patients, the marker was present in at least one PBSC collection (in four patients, every PBSC collection was positive). Survival data are available for nine patients (two early deaths); three patients relapsed and died (221 - 930 d), one is alive and in relapse (354 + d) and five are alive and in complete remission (330 - 1290 + d). These findings suggest that tumour cell contamination of PBSC harvests is not uncommon. Whether these cells are clonogenic and contribute to disease relapse remains to be elucidated. The presence of residual disease at the time of transplantation and the reappearance (or persistence) of marker positive cells post-transplantation both appear to be poor prognostic factors for disease-free survival.

Reversal of human platelet aggregation by low concentrations of nitroglycerin in vitro in normal subjects.

The potential reversal of platelet aggregation in vitro by nitroglycerin in low concentrations was explored using both optical aggregometry and electron microscopy. Venous blood was collected from a cohort of normal volunteers (20 men and 10 women) aged 21 to 65 years. Aggregation in platelet-rich plasma was induced by adenosine diphosphate in concentrations just sufficient to maintain a steady state of aggregation, without a spontaneous disaggregation phase (3.5 to 5 microM). Administration of nitroglycerin after the induction of aggregation caused both inhibition of the primary wave of developing aggregation and marked disaggregation. This combined effect was maximal when nitroglycerin was added at 0.5 minute after the beginning of aggregation. The observed reversal of platelet aggregation by nitroglycerin was concentration-dependent. Significant effects occurred with nitroglycerin concentrations greater than or equal to 10(-8) M. Concentration associated with 50% reversal of aggregation was 1.52 +/- 0.24 (SEM) x 10(-6) M. Electron microscopy revealed that 10(-6) M nitroglycerin induced a significant reduction in both platelet clumping and morphologic changes associated with aggregation. The results of the current study suggest a beneficial antiplatelet effect of nitroglycerin in restoring homeostasis in the face of incipient platelet aggregation. The clinical use of nitroglycerin in patients with acute ischemic syndromes may rest on this action.