Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6-10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.

Sepsis, low platelet nadir at mobilization and previous IFN use predict stem cell mobilization failure in patients with multiple myeloma.

BACKGROUND: Successful stem cell mobilization is a prerequisite for autologous blood cell transplantation. We analyzed factors that may predict the success of stem cell mobilization in patients with multiple myeloma (MM). METHODS: We analyzed 124 consecutive patients and compared those who failed to mobilize a sufficient amount of CD34(+) cells (peak blood CD34(+) cell count <20x10(6)/L) (n=20) with those with successful mobilization (n=104). The peak blood CD34(+) cell count after mobilization was used as the marker of mobilization success against which the various predictive factors were tested. RESULTS: In univariate analysis the best predictive factors for mobilization failure were the number of different chemotherapy regimens (P<0.001), number of chemotherapy cycles (P<0.001), time from diagnosis to mobilization (P<0.001) and previous use of IFN (P<0.001). The distributions of treatment responses at mobilization were similar in the groups with successful and unsuccessful mobilization, and were CR or VGPR in 10% of all patients, PR in 54% and stable or progressive disease in 36%. Regarding the mobilization-related factors, lower leukocyte nadir (P<0.001), longer duration of leukocyte counts <1x10(9)/L (P<0.001), lower platelet nadir (P=0.001), longer duration of platelet counts <20x10(9)/L (P<0.001) and the occurrence of sepsis after the mobilization therapy (P=0.001) were significantly associated with mobilization failure. In multivariate analysis, the amount of earlier chemotherapy cycles (P=0.002), low platelet nadir (P=0.020), occurrence of sepsis at mobilization (P=0.040) and previous use of IFN (P=0.052) remained as significant predictive factors for mobilization failure. DISCUSSION: Predicting the success of stem cell mobilization beforehand may have important practical consequences. By identifying those patients who will fail to mobilize stem cells, unnecessary mobilization and collection attempts can be avoided.

Oral treatment of acute myeloid leukaemia with etoposide, thioguanine, and idarubicin (ETI) in elderly patients: a prospective randomised comparison with intravenous cytarabine, idarubicin, and thioguanine in the second and third treatment cycle.

A randomised multicentre study was conducted among patients over 65 yr of age with newly diagnosed acute myeloid leukaemia (AML) to compare oral treatment with etoposide 80 mg/m(2) and thioguanine 100 mg/m(2) twice daily on 5 d and idarubicin 15 mg/m(2) on 3 d (ETI) to a mainly i.v. combination of cytarabine 100 mg/m(2) twice daily on 5 d, idarubicin 12 mg/m(2) x 1, and thioguanine (TAI). Ninety-two patients were enrolled. Their median age was 72 yr, range 65-84 yr. Sixty-five patients had de novo AML, 21 AML subsequent to myelodysplastic syndrome, and six treatment-related AML. They received at first a 6-d i.v. treatment with cytarabine and idarubicin. After the first treatment, 68 patients were randomised to receive two cycles of ETI (n = 36) or TAI (n = 32) and thereafter maintenance with mercaptopurine and methotrexate. Of the 92 patients, 52 (57%) achieved remission at some stage. The median survival was 10 months. There were no significant differences between the patients randomised to ETI or TAI in the remission rate (67% vs. 72%), survival (12 months from randomisation in both arms), event-free survival or relapse rate. The patients randomised to receive ETI spent significantly fewer days at hospital during the two randomised cycles (20 vs. 41 d, P = 0.010), and they had fewer days with infusions, shorter neutropenias and thrombocytopenias and fewer and less severe infections. In conclusion, treatment with oral ETI resulted in a similar antileukaemic effect as obtained with mainly i.v. TAI, with less toxicity and reduced need for hospitalisation.

Low-dose or intermediate-dose cyclophosphamide plus granulocyte colony-stimulating factor for progenitor cell mobilisation in patients with multiple myeloma.

Cyclophosphamide (CY) combined with granulocyte colony-stimulating factor (G-CSF) is commonly used to mobilise blood progenitor cells to support high-dose therapy in patients with multiple myeloma (MM). The optimal dose of CY in this setting is unknown. We have retrospectively analysed mobilisation efficiency and need for supportive care in 57 patients with newly diagnosed myeloma previously treated with VAD+/-local radiotherapy. The patients were mobilised either with low-dose CY (LD-CY, 1.2-2 g/m(2)) (n=25) or intermediate-dose CY (ID-CY, 4 g/m(2)) (n=32) plus G-CSF. Both regimens proved to be effective in the progenitor cell mobilisation. At least 2 x 10(6)/kg CD34+ cells were collected from 88% and 84% of the patients with a single apheresis, respectively. Only one patient in the LD-CY group (4%) failed to mobilise vs none in the ID-CY group. Patients mobilised with LD-CY plus G-CSF had less toxicity: fewer hospital days during the mobilisation and apheresis procedures (5 vs 9 days, P<0.001), lower frequency of fever (20 vs 73%, P<0.001) and less need for supportive care including platelet transfusions (0 vs 24%, P=0.004) and days on parenteral antibiotics (0 vs 4 days, P<0.001). While these regimens seem to be equally effective in terms of progenitor cell mobilisation in newly diagnosed patients with MM, LD-CY+G-CSF is preferential because of more optimal resource utilisation and more favourable toxicity profile.

Autologous blood cell transplantation in multiple myeloma: impact of CD34+ cell selection with long follow-up.

Positive CD34(+) selection to purge blood cell harvests is one way to attempt to reduce the high relapse risk after high-dose chemotherapy (HDT) supported by autologous blood cell transplantation (ABCT) in patients with multiple myeloma (MM). Until recently, however, the impact of CD34(+) selection, if any, on long-term clinical outcome in MM has remained obscure. We have analyzed engraftment kinetics, response to HDT, progression-free survival (PFS), and overall survival (OS) for 64 consecutive MM patients who have been treated with up-front HDT plus ABCT at our institution between 1993 and 1998. Nonrandomized comparisons were made between transplants with unselected (39 patients) and CD34(+)-selected (25 patients) grafts. The engraftment kinetics, need of blood product support, discharge time from hospital, and response to HDT were similar for both unselected and selected transplants. The median PFS was also similar (26 and 30 months, respectively) for the both groups. With a median follow-up time for the survivors of 67.5 months, the median OS (78 and 75 months, respectively) did not differ between transplants with unselected and selected grafts. In conclusion, this nonrandomized study suggests that positive CD34(+) selection has no beneficial impact on long-term outcome of patients with MM.

Autocrine production and synergistic growth-promoting activity of interleukin-6 and oncostatin M in a new human myeloma cell line TU-1.

Human myeloma cell lines are difficult to establish, and they usually originate from patients with extramedullary disease. We describe a new human myeloma cell line, TU-1, which was established from the bone marrow of a patient without extramedullary myeloma. The myeloma cells were initially maintained in a conditioned medium derived from another well-known myeloma cell line U-266. This conditioned medium contained interleukin-6 (IL-6) and oncostatin M (OSM), and possibly other unknown growth factors as well. In 3 months the TU-1 cell line proliferated autonomously and secreted IL-6 and OSM with a synergistic growth response. As we have previously shown the cell line acquired a p53 mutation in vitro, which may be an important factor causing autonomous proliferation. In patients with multiple myeloma OSM is frequently found in the serum and OSM has been associated with serum IL-6 and progressive disease. Our study demonstrates the close relationship of OSM and IL-6 also in vitro.

Feasibility of prolonged use of interferon-alpha in metastatic kidney carcinoma: a phase II study.

BACKGROUND: Interferon-alpha has proven effective in the treatment of metastatic renal cell carcinoma. However, the optimal schedule has not yet been determined. The authors have studied the efficacy and toxicity of prolonged use interferon-alpha2a (IFN-alpha) in metastatic renal cell carcinoma (RCC). Interferon-alpha was administered intermittently for outpatients. METHODS: Seventy-five patients with metastatic RCC without prior biochemotherapy were treated. During the first month, the IFN-alpha dose was increased from 4.5 to 18 million units (MU) 3 times a week to define the individual maximal tolerated dose for each patient. The treatment was continued at the maximal tolerated dose with a 1-week pause each month until either progression or intolerable toxicity was observed or up to 2 years. RESULTS: The overall response rate (5 complete response [CRs] and 8 partial responses [PRs]) was 17% (95% confidence interval, 10-28%). Stable disease was observed in 32 patients (43%). Three late objective responses (4%) occurred after 12 months treatment. The median progression free time of all patients was 12.3 months, and median survival time was 19.3 months. The median duration of response in CR/PR patients was 16 months. In multivariate analysis independent prognostic factors were poor performance status (P = 0.004), presence of bone metastases (P = 0.001), and time to metastases less than 24 months (P = 0.003), which predicted poor survival. Six patients (8%) discontinued the treatment because of fatigue, elevation of liver enzymes, or cardiac arrhythmias. No life-threatening side effects were observed. CONCLUSIONS: Prolonged and intermittently administered IFN-alpha2a three times per week in 3 weekly cycles in metastatic RCC is a feasible and effective therapy. A prolonged treatment duration of more than 12 months for stable and responding patients is beneficial and may improve the outcome of patients with RCC.

Serum oncostatin M in multiple myeloma: impact on disease severity and prognosis.

Since high levels of serum IL-6 predict a poor prognosis of patients with multiple myeloma (MM), we investigated if a related cytokine, oncostatin M (OSM), correlates with clinical or biochemical findings or has prognostic significance in patients with MM. Among 82 newly diagnosed MM patients, OSM was detected in the sera in 45 (55%). Serum OSM had a borderline statistical correlation with serum IL-6 (r = 0.198, p = 0.074) and C-reactive protein (r = 0.199, p = 0.074) concentrations. However, OSM did not have prognostic significance alone or in combination with other factors. The median survival of patients with detectable serum OSM concentration was 41 months (range 2-124 months) and of OSM negative patients 35 months (1-75 months). Serum OSM concentration was not associated with clinical factors or severity of bone disease at diagnosis. We conclude that serum OSM concentration is not a prognostic factor in MM patients.

Comparison between four and eight cycles of intensive chemotherapy in adult acute myeloid leukemia: a randomized trial of the Finnish Leukemia Group.

In acute myelogenous leukemia (AML) intensive postremission treatment is needed for an optimal result. However, it is not known how long the treatment should last and how many courses are necessary. The object of this prospective study was to compare four and eight intensive chemotherapy cycles in the treatment of adult de novo AML. In a multicenter study, 248 consecutive patients, aged from 16 to 65 years, were treated with intensive induction treatment. The patients in remission after two courses were randomized to receive either two (short arm) or six (long arm) additional intensive cycles of chemotherapy. The median follow-up time of the living patients is 68 months. Of the patients, 77% achieved complete remission, and 36% of all patients survived for 5 years. Seventy-three patients were randomized to the short arm and 66 to the long arm. There was no significant difference in the relapse-free survival (median 21 months vs 17 months) or overall survival (43 months vs 39 months) between the short and long arms, respectively. Treatment-related deaths occurred in 31 patients (13%), 11 of them in first remission. More than one-third of the patients survived for 5 years. It seems probable that the first few months after diagnosis are decisive for the prognosis if the chemotherapy is intensive, and further treatment cannot markedly influence the outcome.

Plasma cell leukemia 3 months after autologous blood cell transplantation for multiple myeloma.

We describe a patient with multiple myeloma who was treated with intensive therapy and autologous blood cell transplantation as her first-line treatment. The disease relapsed 3 months after the transplant as plasma cell leukemia and the patient succumbed in 4 weeks. We suggest that an aggressive plasma cell clone may be selected during the course of intensive treatment. Complex karyotypic findings are also presented.

Long-term treatment with GM-CSF in patients with chronic lymphocytic leukemia and recurrent neutropenic infections.

In this prospective study we evaluated the multiple effects of long-term GM-CSF therapy on blood counts, granulocyte functions and disease progression in patients with chronic lymphocytic leukemia (CLL) with chronic neutropenia and recurrent bacterial infections. The treatment duration varied from 2 to 12 weeks. The neutrophil count was raised in all patients, by the median of 6.6-fold. The neutrophil level of 1.0 x 10(9)/l was usually reached after two weeks. The initial dose of GM-CSF was 5 microg/kg/day, and 1-7 microg/kg/day was required to maintain the neutrophil level above 1.0 x 10(9)/l. Granulocyte functions, i.e. chemiluminescence (CL), random migration, and fMLP-stimulated chemotaxis were initially depressed in all patients when compared to healthy controls. GM-CSF enhanced significantly CL even when given at small doses (less than 1 microg/kg/day), even lower than the dose required to promote granulopoiesis. We conclude that GM-CSF is effective in improving CLL associated chronic neutropenia and also enhances impaired granulocyte chemiluminescence. Thus, GM-CSF could be helpful for giving chemotherapy without neutropenic delays and for prophylaxis of infectious complications in CLL patients.

p53 gene mutations are rare in patients but common in patient-originating cell lines in multiple myeloma.

We have shown previously that detectable serum concentration of p53 protein is associated with poor prognosis in multiple myeloma (MM). In this study, we studied p53 gene mutations in 29 bone marrow samples of MM patients using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. No p53 mutations were found in these patients although 41% of the patients had an elevated serum p53 protein concentration. This result indicates that the detectable serum level of p53 protein is not associated with p53 mutations. In addition, we have also analysed three MM cell lines established from bone marrow samples. All the cell lines contained p53 mutations in exon 5. However, bone marrow samples of the patients associated with the cell lines did not have these mutations at the time of diagnosis, nor did the original samples which were used to establish the cell lines. This indicates that p53 mutations can arise during the cell passages.

Autologous blood cell transplantation in B-CLL: response to chemotherapy prior to mobilization predicts the stem cell yield.

Successful stem cell mobilization and collection is possible in B-CLL after a favorable response to preceding chemotherapy, and also after treatment with the new nucleoside analogues fludarabine and cladribine. A poor response, on the other hand, seemed to predict a mobilization failure. CD34+ cell selection resulted in a 2- to 3-log reduction of the CLL cells in the harvests. Engraftment with both unselected and selected progenitor cells was rapid, and need for hospitalization was short. High-dose therapy with stem cell rescue appears to be capable of inducing CRs of high quality, but so far the follow-up is too short to define whether this will be translated into prolonged disease-free survival.

High-dose chemotherapy with autologous stem cell support for the treatment of advanced ovarian cancer--initial experience in Uppsala and Turku.

BACKGROUND: With current standard-dose chemotherapy ovarian cancer is a chemosensitive but not chemocurable disease in the majority of cases. The widely used first-line chemotherapy including a platinum analogue combined with cyclophosphamide results in response rates of 60-80%. However, only 10-20% of patients with advanced disease are alive 5 years after the diagnosis. The efficacy of high-dose chemotherapy supported by autologous stem cell transplantation (ASCT) is currently under intensive investigation. METHODS: We report here our initial experiences of the use of high-dose chemotherapy supported by ASCT for patients with high-risk ovarian cancer. Two patients were treated at Uppsala University Hospital in 1992 and four patients at Turku University Central Hospital in 1994. RESULTS: The first four patients treated either after heavy previous chemotherapy or recurrent disease relapsed within 5-10 months. Two patients received high-dose therapy as part of first-line treatment. One of them had a relapse 18 months after therapy, the other one has been disease free for 28 months. No toxic deaths occurred, but the patients had neutropenic febrile episodes and moderate to severe gastrointestinal toxicity. CONCLUSIONS: Coordinated efforts in Nordic countries are indicated to evaluate the usefulness of high-dose therapy supported by ASCT in the treatment of advanced ovarian cancer.

Daily measurements of blood CD34+ cells after stem cell mobilization predict stem cell yield and posttransplant hematopoietic recovery.

The value of daily monitoring of the blood CD34+ cell concentration as a guide to the optimal timing of stem cell harvests was studied in 60 patients who underwent 66 stem cell mobilizations and 189 leukaphereses. There was a highly significant correlation between the blood CD34+ count and the CD34+ cell content in the apheresis product of the same day (r = 0.904, p < 0.01). Thus, the target yield of 4 x 10(6) CD34+ cells/kg can be harvested in one or two leukaphereses when the blood CD34+ cell count exceeds 50 x 10(6)/L. However, an insufficient harvest is to be expected when the blood CD34+ cell count is below 20 x 10(6)/L. The data from 35 autologous blood cell transplantations with a minimum CD34+ cell yield of 1.5 x 10(6)/kg showed that the recovery of blood neutrophil counts to 1.0 x 10(9)/L occurred in all patients within 9-14 days, but the time to recovery of the platelet counts to 20 x 10(9)/L may exceed 14 days, especially if the CD34+ cell content is below 4 x 10(6)/kg. Daily monitoring of blood CD34+ cell counts is a rapid and reliable means to guide the timing of stem cell collections. The count predicts well the CD34+ cell content of the harvests, the number of leukaphereses needed, and the speed of hematopoietic recovery.

Intensive chemotherapy of poor prognosis myelodysplastic syndromes (MDS) and acute myeloid leukemia following MDS with idarubicin and cytarabine.

Forty patients with high risk myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) preceded by MDS were treated with intensive induction and consolidation chemotherapy in a prospective multicenter pilot study. They were given two cycles of cytarabine 100 mg/m with 12-h intervals on days 1-7 and idarubicin 12 mg/m2 on days 5-7, both intravenously. Patients who were in remission after these two cycles were given two further cycles of cytarabine on days 1-5 and idarubicin on day 5. No maintenance treatment was given. Eleven out of 19 MDS patients (58%) and 10 out of 21 AML patients (48%), in total 21 out of 40 patients (53%), entered remission. Eight patients underwent allogeneic bone marrow transplantation. The follow-up time was 13-48 (median 33) months. At the time of the analysis, seven patients survived, four patients with MDS all of whom had been treated with bone marrow transplantation (three in continuous remission), and three patients with AML treated with chemotherapy only (two in continuous remission). The median survival of the patients treated with chemotherapy only was 12 months, with the median progression-free survival being 8 months. In view of the poor prognostic factors of the patients, the remission rate was satisfactory, but the responses as well as the survival were short. The post-remission treatment needs to be improved.

Type I diabetic pregnancy and subclinical human papillomavirus infection.

It has been suggested that diabetic pregnancy is an immunosuppressive state. To determine whether the possible immunosuppression in pregnant diabetics might result in an increased risk for human papillomavirus (HPV) infection, we studied exfoliated cells from the uterine cervix, vagina, and posterior commissure of the vulva by means of dot blot hybridization with use of a probe cocktail of HPV types 11, 16, and 18 under low stringency and by means of consensus primer-mediated polymerase chain reaction (PCR) targeted to the HPV L1 and E1 regions. For this study, samples from 31 pregnant diabetics whose glucose levels had been reasonably well controlled were analyzed during the first trimester; samples from 27 of these patients were analyzed again during the third trimester. Fifty-one healthy pregnant women were included as controls. Only one of the pregnant diabetics was positive for HPV DNA. The L1 PCR products of the first and third trimester samples from this patient were sequenced, and both were found to represent HPV 61. Three of the pregnant controls were positive for HPV: two were positive for HPV 16, and one was positive for HPV 6. The 95% confidence limits for the prevalence of HPV were calculated to be 0.1%-16.7% for the diabetics and 1.2%-16.2% for the controls. The 95% confidence limits for the difference between the groups were -11.6%-6.3%. These results suggest that pregnant diabetics do not have an increased risk of developing HPV infection, at least when their glucose levels remain well controlled.

Febrile infection changes the expression of IgG Fc receptors and complement receptors in human neutrophils in vivo.

We have examined the expression of the IgG Fc receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3) in neutrophils from 42 patients with febrile bacterial infection, 20 patients with febrile viral infection and 69 non-febrile healthy individuals. Using receptor-specific MoAbs and immunofluorescence flow cytometry the relative fluorescence intensity (as a measure of receptor number) and the proportion of receptor-positive cells were determined in peripheral blood neutrophils exposed to minimal processing, consisting only of erythrolysis. Both the percentage of FcRI-positive neutrophils and FcRI number per neutrophil were significantly (P < 0.001 and P < 0.0001) increased in bacterial infected patients compared with controls, whereas in viral infected patients only the FcRI percentage was markedly elevated (P < 0.05). In addition, both FcRII and CR1 levels were significantly higher in the bacterial infection group than in the viral infection and control groups (bacterial versus control P < 0.001, bacterial versus viral P < 0.0001). No changes in expression of FcRIII or CR3 were found in the patient groups. The kinetic analysis of receptor expression in bacterial infection patients revealed a shift in the percentage of FcRI-bearing neutrophils towards normal values already on day 2 after the first analysis. On the other hand, the levels of FcRI, FcRII and CR1 remained clearly elevated in these patients during 1 week's follow-up period. We conclude that febrile infection may cause systemic activation of the entire pool of circulating neutrophils, resulting in alterations in cell surface receptor expression, some of which are characteristic of the nature of the infectious agent.

Serum oncostatin M in multiple myeloma: association with prognostic factors.

The serum concentration of oncostatin M (OSM) was measured in 40 multiple myeloma patients at diagnosis. Serum OSM level exceeded the sensitivity limit of the ELISA assay in eight (20%) of these patients (OSM+ patients). The serum levels of IL-6, another member of the gp180 cytokine family and C-reactive protein (CRP) as a surrogate of IL-6 were significantly higher in OSM+ patients. There was a trend towards higher serum beta 2M concentration in OSM+ patients, whereas there was no difference in the serum sIL-6R level or clinical data (age, gender, myeloma protein or stage) between the two groups. Two human myeloma cell lines secreted OSM and IL-6, but not IL-11 or leukaemia inhibitory factor (LIF), which suggests an important role for OSM and IL-6 in supporting growth of myeloma cells.