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Detection of minimal residual disease (MRD) in follow-up samples from patients with ALL is essential for evaluation of treatment response. We applied multicolor flow cytometry and real-time quantitative PCR (RQ-PCR) to compare MRD results in 71 follow-up samples from 22 children treated for ALL. When results obtained by flow cytometry and RQ-PCR were grouped into positive-negative categories, a significant level of agreement was found in 72% of samples (P<0.001). However, if a cutoff level of 0.01% was applied, the concordance was 89%. MRD could be quantified in 19 samples by both methods, showing a strong correlation (P<0.01). Nevertheless, MRD levels differed more than five-fold between both methods in 4/19 samples. In 20 (28%) samples, the two techniques showed discordant results. Most discordant results (17/20) were due to the limited sensitivity of flow cytometry analysis within the range 0.01-0.001%; remaining discordant results were due to the instable or subclonal IG/TCR gene rearrangements or a limited quantitative range of the applied RQ-PCR targets. Although concordant results could be obtained by flow cytometry and RQ-PCR analysis, MRD levels may differ. Therefore, MRD data obtained by these two techniques are not yet easily exchangeable.
Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.
PURPOSE: To report the results of a prospective Phase III trial for patients with newly diagnosed glioblastoma multiforme (GBM), treated with either accelerated hyperfractionated irradiation with or without difluromethylornithine (DFMO) or standard fractionated irradiation with or without DFMO. METHODS AND MATERIALS: Adult patients with newly diagnosed GBM were registered and randomized following surgery to one of 4 treatment arms: Arm A, accelerated hyperfractionation alone using 2 fractions a day of 1.6 Gy to a total dose of 70.4 Gy in 44 fractions; Arm B, accelerated hyperfractionation as above plus DFMO 1.8 gm/m2 by mouth every 8 h beginning one week before radiation until the last fraction was given; Arm C, single-fraction irradiation of 1.8 Gy/day to 59.4 Gy; Arm D, single-fraction irradiation as in Arm C plus DFMO given as in Arm B. Patients were followed for progression-free survival (PFS) and overall survival (OS), as well as for toxicity. Eligibility required histologically proven GBM, age > or =18, Karnofsky performance status (KPS) > or =60, and no prior chemotherapy or radiotherapy. Adjuvant chemotherapy was not used in this protocol. RESULTS: A total of 231 eligible patients were enrolled. There were 95 men and 136 women with a median age of 57 years, and median KPS of 90. Extent of resection was total in 23, subtotal in 152, and biopsy only in 56 patients. The 4 arms were balanced with respect to age, KPS, and extent of resection. Times to event measurements are from date of diagnosis. Median OS and PFS were 40 and 19 weeks for Arm A; 42 and 22 weeks for Arm B; 37 and 16 weeks for Arm C; and 44 and 19 weeks for Arm D (p = 0.48 for survival; p = 0.32 for PFS). Comparison of the 2 arms treated with DFMO to the 2 arms without DFMO revealed no difference in OS (37 weeks vs. 42 weeks, p = 0.12) or PFS and thus no benefit to the use of DFMO. Comparison of the 2 standard fractionation arms to the 2 accelerated hyperfractionation arms also resulted in no difference in OS (42 weeks vs. 41 weeks, p = 0.75) or PFS, showing no benefit to accelerated hyperfractionated irradiation. CONCLUSION: In this prospective Phase III study, no survival or PFS benefit was seen with accelerated hyperfractionated irradiation to 70.4 Gy, nor was any benefit seen with DFMO as a radiosensitizer. Standard fractionated irradiation to 59.4 Gy remains the treatment of choice for newly diagnosed patients with glioblastoma multiforme.
There is recent evidence that the perfusion of the choroid changes during dark-light transitions. We set out to investigate this response in more detail and to elucidate possible mechanisms involved in this process. For this purpose, the effect of dark-light transitions on choroidal perfusion was studied in healthy subjects. Choroidal blood flow and ocular fundus pulsation amplitude were measured as indices of choroidal perfusion during dark-light transitions using laser Doppler flowmetry and laser interferometry, respectively. In the first experiment, subjects were first kept in room light for 20 min, then light conditions were changed to darkness for 20 min, and thereafter, subjects were exposed to room light again. Both choroidal parameters decreased (-12% to -14%) during darkness but returned to baseline after the final room light period. In the second experiment, the index eye underwent the same procedure, whereas the contralateral eye was kept in light throughout the experiment. Choroidal haemodynamic parameters in the index eye reacted in a way comparable to that seen in the first experiment. The eye that was kept in light also reacted, but the effect tended to be less pronounced than that seen in the index eye (-8% to -10%). The observation that choroidal blood flow in both eyes reacts during unilateral light-dark transitions indicates that choroidal perfusion rate is adapted to retinal illumination conditions by neural control mechanisms.
The analysis of minimal residual disease (MRD) has assumed a growing role in the follow-up of patients with acute lymphoblastic leukemia (ALL). We have applied multiparameter flow cytometry (FC) with 'live-gate' analysis and allele-specific oligonucleotide (ASO)-PCR detecting leukemia-specific T cell receptor gamma and delta gene rearrangements for MRD follow-up in 30 ALL patients. The comparison of results obtained in 89 follow-up samples from 23 patients showed significantly consistent results in 70 samples (78%); (P < 0.001). Bone marrow samples taken during the first phase of treatment (during or immediately after induction) showed a lower level of consistency when compared to samples taken during later phases of treatment (69% vs 85% consistent results, respectively). Some of the discrepant results were due to low cellularity of the samples obtained for FC and some due to the presence of PCR inhibitors. Of 29 patients evaluated at the end of the induction treatment, 18 (62%) had detectable levels of MRD and six of these patients suffered relapse. In all these patients MRD levels by FC increased preceding relapse. Our results suggest that FC offers a MRD detection tool that can be easily applied in clinical practice and is as informative as molecular methods.
We conducted a single-arm phase II study to evaluate the efficacy and safety of radiotherapy combined with 6-thioguanine, procarbazine, dibromodulcitol, lomustine, and vincristine (TPDCV) chemotherapy for treating malignant astrocytoma in children and anaplastic ependymoma in patients of all ages. Between 1984 and 1992, 42 patients who had malignant astrocytomas (glioblastomas multiforme, anaplastic astrocytomas, or mixed anaplastic oligoastrocytomas) were treated with TPDCV chemotherapy and radiation therapy. Of these patients, 40 were younger than 18 years, but 2 were older (22 and 23 years) when treated. Cranial radiation averaged 58 Gy. TPDCV chemotherapy was given for 1 year or until progression. Between 1989 and 1991, 17 patients with malignant ependymoma were treated with TPDCV chemotherapy and craniospinal radiation. Radiation was given at an average dose of 54 Gy to the tumor, 28 Gy to the whole brain, and 31 Gy to the spinal axis. TPDCV chemotherapy was given for 1 year or until tumor progressed. Of the patients with glioblastoma multiforme, 13 of 17 died; the median time to progression was 49 weeks, and median survival was 85 weeks. The four patients surviving at this writing were followed a median 537 weeks (range 364-635 weeks). Of the patients with nonglioblastoma malignant astrocytoma, 14 of 25 died; the median time to progression was 224 weeks. Median survival was not reached in this group. The median follow-up for those surviving was 494 weeks. For the patients with ependymoma, 11 of 17 died with a median time to progression of 141 weeks. The median follow-up for the eight who survive was 469 weeks. Nine patients died with a median survival of 183 weeks. The combination of TPDCV and radiotherapy has activity against childhood anaplastic astrocytoma, glioblastoma multiforme, and anaplastic ependymoma. The results of this study for children with glioblastoma were comparable to results in the literature, while the results for children with anaplastic astrocytoma appeared better than most reports. The combination of TPDCV chemotherapy and radiation therapy for anaplastic ependymomas appears to be active and at least as good as published reports using radiation therapy alone.
Primary fallopian tube carcinoma is a rare, aggressive gynecological cancer; little is known about its cause. Previous studies have indicated that p53 immunopositivity is correlated with short-term survival in primary fallopian tube carcinoma. We examined p53 and p21/WAF1 immunostaining and TP53 mutation in exons 5 to 8 by single-stranded conformation polymorphism and constant denaturant gel electrophoresis in nine cases of primary fallopian tube carcinoma and their metastases/recurrences from patients who survived for between a few months and more than 20 years after diagnosis. We found that 1.) p53 immunopositivity without detectable p21/WAF1 immunostaining did not correlate with TP53 mutations in the conserved domains; 2.) mutations in TP53 occurred in two metastases/recurrences but not in their corresponding primary tumors; 3.) in two cancers, a TP53 mutation was observed in the primary tumor but not in the metastases/recurrences; 4.) constant denaturant gel electrophoresis seems to be more sensitive than single-stranded conformation polymorphism in detecting TP53 mutations; and 5.) in the nine cases studied, p53 immunoreactivity and/or TP53 mutation analysis did not correlate with tumor progression, survival, or response to treatment.
Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A <--> B) and nested PCR (internal primers C <--> D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A <--> B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.
PURPOSE: This study was designed to evaluate a combined modality treatment for malignant gliomas using radiation therapy with a radiosensitizer and an adjuvant chemotherapy regimen designed to modify resistance to BNCU. METHODS AND MATERIALS: Patients were eligible if they were 15 years of age or older, and had newly diagnosed glioblastoma multiforme (GBM), or anaplastic glioma (AG). Treatment consisted of external beam radiotherapy given to a dose of 60 Gy using a single daily fraction Monday to Friday. Concurrent hydroxyurea at a dose of 300 mg/m2 every 6 h every other day was given during radiation. Following radiotherapy, patients were then treated with BCNU and 6-Thioguanine (6TG). The 6-TG was given by mouth every 6 h for 12 doses prior to BCNU. Patients were initially treated with 60 mg/m2/dose of 6TG, with escalation to a maximum dose of 100 mg/m2/dose. The primary study end points were time to tumor progression and survival. RESULTS: A total of 245 eligible patients were enrolled from 1/18/88 to 12/26/91. The histologic subtypes included 135 GBM, and 110 with AG (103 with anaplastic astrocytoma, 7 with high-grade mixed oligoastrocytoma). For the GBM group, the median time to tumor progression (TTP) and median survival were 33 (95% CI 26, 39) and 56 (95% CI 49, 69) weeks, respectively. For the AG group the median TTP was 282 weeks (95% lower confidence bound = 155 weeks). Median survival for this group has not been reached (95% lower confidence bound = 284 weeks) with a median follow-up for surviving patients of 298 weeks. A proportional hazards model was used to look at potential prognostic factors for survival, including initial Karnofsky Performance Scale (KPS), age, and extent of surgery, as well as dose of 6TG. Higher KPS, and lower age, predicted for longer survival (p < 0.01, < 0.001) in GBM patients; lower age was significant (p = 0.05) for AG cases. A higher (greater than 95 mg/m2) or lower dose of 6TG was not statistically significant in this model. CONCLUSIONS: This therapy was no more effective in patients with GBM than other reported series. In patients with malignant gliomas other than GBM, prolonged progression-free and overall survival is noted, without a median survival reached at the time of this report. In this subset of AG patients, survival is comparable to recent studies using halogenated prymidines during radiation and Procarbazine, CCNU, and Vincristine (PCV) as adjuvant chemotherapy.
PURPOSE: To determine the maximum-tolerated dose (MTD) of paclitaxel administered as a 3-hour infusion in patients with recurrent malignant glioma. PATIENTS AND METHODS: Patients were stratified by starting dose of paclitaxel and concurrent anticonvulsant (AC) use and were treated in cohorts of three patients. The starting dose was 240 mg/m2 administered intravenously with escalations of 30 mg/m2 until the MTD was established. Pharmacokinetic data were obtained for each patient for the first infusion. Tumor response was assessed at 6-week intervals and treatment was continued until documented tumor progression, unacceptable toxicity, or a total of 12 paclitaxel infusions. RESULTS: From April 1995 to December 1996, 34 patients were treated; 27 patients in the AC group and seven patients in the non-AC group. The MTD for patients who received ACs was established at 360 mg/m2 and the dose-limiting toxicity (DLT) was central neurotoxicity, characterized as transient encephalopathy and seizures. In contrast, the MTD for patients who did not receive ACs was 240 mg/m2, and myelosuppression, gastrointestinal toxicity, and fatigue were the DLTs. Pharmacokinetic data confirmed that the plasma drug levels and clearance rates were similar for patients in both groups at the respective dose levels that produced DLTs. CONCLUSION: The pharmacokinetics of paclitaxel are altered by ACs, and significantly larger doses of the drug can be administered to patients with brain tumors on AC therapy. The toxicity profile is different for patients on AC therapy treated at these higher doses. A phase II study has been initiated that uses a dose of 330 mg/m2 for patients on AC therapy and 210 mg/m2 for patients not on AC therapy.
The paper presents 43 patients treated between 1971 and 1995 in the Otolaryngology Clinic of the Pomeranian Medical Academy in Szczecin because of deep neck space infections. The authors analyse their own clinical material paying particular attention to the diagnosis, microbiology, patients prognosis and basic therapeutic procedures. Out of the 43 cases, deaths occurred in 3, which denotes 7% mortality rate. The reason for failure was discussed. It was revealed that needle aspiration and sonography were for valuable to identify early abscess formation. The analyses have confirmed that the prognosis of the deep neck space infections is influenced by early diagnosis and prompt, extensive surgical treatment.
The paper presents a 26-year experience of the ENT Clinic of the Pomeranian Medical Academy in Szczecin with 20 patients who underwent reconstruction of the hypopharynx after resection for locally advanced carcinoma. The procedures were carried out in years 1970-1995. The reconstructions included 7 musculomucosal pedicle flaps obtained from the base of the tongue, 5 epiglottis pedicle flaps, 4 tubed pectoralis major musculocutaneous flaps, 1 vascular pedicle submandibular gland flap, 1 deltopectoral flap, 1 skin flap and 1 island pectoralis major musculocutaneous flap. An analysis of the procedures with emphasis on the oncological and functional results indicates that each techniques has advantages in specific circumstances. Guidelines for the application of the techniques are described.
One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor.
In a Phase II trial, 63 evaluable patients with recurrent glioma received i.v. infusions of carboplatin every 3 weeks beginning at a dose of 400 mg/m2. The dose was increased by 50 mg/m2 at each subsequent infusion until the maximum tolerated dose reached, as defined by a platelet count < 25,000/mm3 or an absolute neutrophil count (ANC) < 500/mm3. Treatment was then resumed at the previous dose level and continued until tumor progression occurred. There were 43 men and 20 women studied (mean age, 41 years; range, 6 months to 70.6 years). The combined response and stabilization rate was 29% for 31 patients with glioblastoma and 71.9% for 32 patients with other tumors; median time to tumor progression was 8.2 and 20.3 weeks and median survival was 25.9 and 58.3 weeks, respectively. Twenty patients had level 4 platelet toxicity and nine had level 4 ANC toxicity. Most tumors progressed before the maximum tolerated dose was reached. These results were not better than those from a previous trial of carboplatin at an initial dose of 350 mg/m2, which was escalated by 25 mg/ m2 after every two infusions. Therefore, an optimal dosing schedule was not achieved in this trial.
Two cases of total subglottic stenosis of the larynx and of the upper segment of the trachea following an automobile accident and a prolonged endotracheal intubation are presented. Both were observed in ENT Department of Pomeranien Medical Academy in Szczecin during the years 1991-1992. In both cases the ring of the cricoid cartilage and upper segment of trachea was excised and the primary thyrotracheal anastomosis was performed. The functional results of the treatment in either case was satisfactory.
Six cases of the rarely encountered, advanced tumours of the larynx treated in ENT Clinic of Pomeranian Medical Academy in Szczecin are presented: chondroma, rhabdomyosarcoma, neurinoma, cylindroma and 2 cases of granular cell tumour. There were certain diagnostic difficulties in rhabdomyosarcoma and chondroma cases. The immunocytochemical investigations were of decisive significance in differential diagnosis of the larynx sarcoma. In the case of chondroma there were problems with biopsy taking the tense tissue of the tumour was compressing the subglottic space. The radiological examinations turned out to be very helpful in this case. All patients were treated by surgery, two of them with rhabdomyosarcoma and cylindroma were treated by irradiation too. Observation periods were from 0.5 to 10 years. The oncological and functional results in 4 cases after partial laryngectomies turned out to be satisfactory. All larynx activities were preserved. The patient with sarcoma of the larynx died 6 years after diagnosis of neoplasm. Finally author discuss diagnostic problems and methods of treatment.
The authors present a retrospective study of 72 classic frontolateral partial laryngectomy and 63 extended frontolateral resection performed in the years 1973-1992 in the ENT Clinic of Pomeranian Medical Academy in Szczecin. The operation was performed in selected glottic carcinomas. For reconstruction of the larynx after laryngectomy, the skin, vascular pedicled flap of the thyroid gland and the epiglottis were used. The functional results in regard to respiration, deglutition and phonation were satisfactory. 88% of patients survived 3 years and 86% of patients 5 years.