Second brain tumors following central nervous system radiotherapy in childhood.

OBJECTIVE: The second tumour (ST) occurrence is a relatively uncommon late complication of radiotherapy but represents one of the most significant issues, especially in childhood oncology. We describe our experience with patients who developed second brain neoplasm following cranial irradiation in childhood. METHODS: We identified nine patients who received radiotherapy owing to central nervous system tumour in childhood and subsequently developed the second brain tumour. The full clinical and radiological documentation and histopathological reports were reviewed. Risk factors such as age at irradiation, latency period to ST diagnosis, radiotherapy doses and volumes and other therapy methods were evaluated. We correlated the ST location with the three levels of irradiation dose (high, >40 Gy; medium, 25-40 Gy; and low <25 Gy). RESULTS: Five meningiomas and four gliomas occurred as the ST after the mean time of 11.7 years after radiotherapy. The average age of children during irradiation was 4.6 years. The shorter latency time to the ST induction was found in children treated with chemotherapy (9 years vs 17.2 years). Seven STs developed in the area of high and moderate dose (>25 Gy), only two low-grade gliomas appeared in the low-dose region. CONCLUSION: Our data suggest that the STs usually develop in the brain tissues that received doses >25 Gy in patients irradiated at a young age. ADVANCES IN KNOWLEDGE: The low-dose volume seems not to be so significant for second brain neoplasm induction. Therefore, the modern intensity-modulated radiotherapy technique could be safely applied in paediatric patients.

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

Radiotherapy of central nervous system tumors in young children: benefits and pitfalls.

BACKGROUND: The prognosis for young children suffering from brain tumors is, as many authors report, generally poor. The probability of late complications in young children is also high, and their quality of life is significantly worse than that of older children. This study presents the experience of one center in management of central nervous system tumors in children in aged 1-3 years with radiation therapy. PROCEDURE: From 1981 to 1990, 52 children (aged 1-3 years) with CNS tumors were treated at the First Radiotherapy Department in the Center of Oncology in Warsaw, Poland. These patients represented 18% of the total number of 293 children with brain and spine tumors treated in this period. The radiation treatment policy for young children was similar to that applied to older children, but total doses and doses per fraction were lower and did not exceed 50 Gy to the tumor site and 30 Gy to the CNS axis. RESULTS: Overall 5-year survival was 52%, and disease-free survival was 50%. These results were similar to those obtained in older children (53% and 50%, respectively). Serious mental retardation (IQ < 70) was observed in 30% of young children in comparison to 7% of older patients, but in 62% these symptoms were noted even before the start of radiotherapy. CONCLUSIONS: The results obtained in this series did not confirm the hypothesis of more aggressive biology of CNS tumors in younger children. The proportion of children with serious psychological disturbances in this group is apparently high, although in most cases these symptoms were observed before the start of radiotherapy and were a result of tumor mass effects.

Human thyroid peroxidase (TPO) isoforms, TPO-1 and TPO-2: analysis of protein expression in Graves' thyroid tissue.

Thyroid peroxidase (TPO) is the key enzyme involved in the biosynthesis of thyroid hormones and is an important autoantigen in autoimmune thyroid disease. Different messenger RNA species coding for TPO are present in thyroid tissue, including the species coding for a 933-amino acid protein (termed TPO-1) and a second in which exon 10 is deleted and which is 57 residues shorter (termed TPO-2). However, it is not known whether the smaller, TPO-2 isoform is expressed as a protein in thyroid cells. In SDS-PAGE under reducing conditions, TPO appears in the thyroid microsome and purified protein preparations as a closely migrating double band of approximately 105 (larger form) and 100 kilodaltons (smaller form). We investigated the presence of the isoform TPO-2 polypeptide in Graves' thyroid tissue using rabbit antisera to three different synthetic peptides from exon 10 (specific for TPO-1) and a polyclonal rabbit and monoclonal anti-TPO antibody (both of which are specific for the two forms of TPO). The larger and smaller forms of TPO were purified by electroelution after gel electrophoresis of highly purified natural TPO from Graves' thyroid microsomes. Both of the purified forms of TPO react with all three anti-exon 10 peptide antibodies, the polyclonal anti-TPO and the monoclonal antibody anti-TPO. This shows that both forms of TPO contain exon 10-encoded polypeptide of TPO-1. Interestingly, the proportion of the larger and smaller forms of TPO varied in different Graves' thyroid microsome preparations. To investigate the presence of the smaller TPO-2 isoform in the purified natural TPO preparation, affinity depletion of TPO-1 using the anti-exon 10 peptide antibodies was carried out. The binding of anti-exon 10 peptide antibodies to the immunodepleted TPO-1 fraction was considerably diminished in comparison to binding of polyclonal anti-TPO, suggesting the presence of small amounts (< 10%) of TPO-2 expressed as a protein in thyroid cells. Our results extend previous observations by showing that the alternatively spliced form of TPO, in which exon 10 is excised, is expressed at low levels in Graves' thyroid tissue. Furthermore, we confirm that both the larger and smaller forms of TPO observed on gel electrophoresis contain TPO-1, suggesting that the difference is caused by posttranslational modifications. The presence of small amounts of TPO-2 in Graves' thyroid glands argues for its role in thyroid function, which remains to be clarified.

Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis.

Antibodies (Ab) to thyroid peroxidase (TPO) are common in patients with autoimmune thyroid disease and may play a role in disease pathogenesis. We have prepared immunoglobulin G kappa (IgG kappa) and IgG lambda phage display combinatorial libraries from the cervical (thyroid-draining) lymph nodes of 2 Hashimoto's thyroiditis patients and from the thyroid of 1 patient. After selection with purified recombinant human TPO, up to 10 high affinity IgG kappa clones from each tissue source were analyzed further. No IgG lambda Fab were detected in the patient with the highest TPO Ab titer. Sequence analysis of the clones showed restricted heavy and light chain usage, similar to that in previously published TPO-reactive Fabs. This was despite the substantially larger sizes of the initial libraries, the use of lymph node tissue to generate libraries, and the analysis of the repertoire in patients with Hashimoto's thyroiditis rather than Graves' disease. There was overall similarity in sequences obtained from lymph node and thyroid libraries, with higher levels of somatic hypermutation in the former. The Fab inhibited binding of serum TPO Ab from five patients by 55-95%. These data together with those from previous reports indicate that although there is no unique Ab gene usage, there is the recurrent presence of certain variable regions in the high affinity TPO Ab response.

Characterisation of the antibody response to the extracellular region of recombinant thyrotropin receptor.

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.

Majority of thyroid peroxidase autoantibodies in patients with autoimmune thyroid disease are directed to a single TPO domain.

Murine monoclonal antibodies (mAb) produced against native human thyroid peroxidase (TPO) are powerful tools for analyzing the autoantibody (Aab) epitopes on TPO. Binding sites of thirteen mAbs cover all or most antigenic regions on TPO. We determined the competition between Aabs from 75 AITD patients and 13 mAbs in binding to TPO. Autoantibodies recognize predominantly the TPO area close or identical to mAb#9 epitope. All sera tested inhibited this mAb binding by 92.9 +/- 14.8 (mean +/- SD), range from 69-100%. AITD patients' sera with low Aabs titer up to 1/2,000 inhibited mAb#9 binding to TP0 by 85 +/- 11.5% (mean +/- SD) and did not influence remaining mAbs binding to TPO. With elevated Aab levels the inhibition of other mAbs binding was higher, but never exceeded 35%. The amount of Aabs yielding 50% inhibition of mAbs binding was lowest for mAb#9. In order to obtain this degree of inhibition for other mAbs 5 to 25 times more Aabs were needed. Our results demonstrate that the majority of autoantibodies in sera of patients with AITD recognize a single immunodominant region on the TPO mapped by mAb#9. They account for about 80-90% of serum TPO autoantibodies. The autoimmune response to other regions on TPO molecule is directed to several other epitopes, but represents quantitatively a minority of autoantibodies. This response intensifies with increasing Aabs level in the serum.

Hyperfractionated radiotherapy for brain stem tumours in children.

From 1988 to 1992 in the Centre of Oncology, Warsaw, 42 children with brain stem tumours were treated with hyperfractionated radiotherapy (HFRT). Two-year survival in nine (27%) patients was obtained. The HFRT treated group was compared with the historical, conventionally irradiated group with the same diagnosis. The hyperfractionated radiotherapy was well tolerated, but did not improve survival rate in comparison with conventionally treated group.

Iodine induced splitting of peptide bonds in human thyroglobulin.

Human thyroglobulin pretreated with iodine (1mM) at alkaline pH is split to small molecular weight fragments after reduction with dithiothreitol. Iodine pretreatment alone did not induce any changes in the thyroglobulin molecular weight.

Radiotherapy of midbrain and brainstem tumours in children.

From 1984 to 1987 in the First Radiotherapy Department in the Centre of Oncology in Warsaw, 11 children with midbrain (group I) and 14 with brainstem (group II) tumours were treated. In 4 cases diagnostic biopsy was performed and in 21 diagnosis was established by CT scan. All children were treated with megavoltage radiotherapy with a Co-60 unit. The initial radiotherapy treatment volume was determined from CT scan and was subsequently adapted to include whole brain or whole cranio-spinal axis, depending on the response to treatment. Improvement or stabilization of disease in 23/25 (92%) of cases was observed. Total survival, longer than 3 years in 14/25 (56%) was observed, while 9/11 (82%) survived greater than 3 years (NED) in group I, and in 4/14 (28%) in group II. Ninety two percent of living children have normal school life, with minimal or no neurological defects.

[Immunoenzymatic method for determining thyroglobulin levels in human blood serum]. / Immunoenzymatyczna metoda oznaczania stezenia tyreoglobuliny w surowicy ludzkiej.

An inexpensive enzyme immunoassay method was designed for the determination of thyroglobulin concentration in human blood serum. The range of concentrations of thyroglobulin which can be measured by the method is between 6 and 800 ng/ml. The sensitivity of the method is comparable to that of the commercial test kits. The values of thyroglobulin concentration obtained with the use of the described method are strongly correlated (r = 0.946) with those obtained by using the reference method (IRMA kit of Byk, Sweden). The intraassay coefficient of variation ranged from 5.5 to 10.2% and interassay coefficient of variation from 9.5 to 13.2% depending on the thyroglobulin concentration. The upper limit of blood serum thyroglobulin concentration in healthy subjects was 70 ng/ml. The results of thyroglobulin determination obtained with the described method are falsely lowered in the presence of antithyroglobulin antibodies; simultaneous determination of these antibodies is thus necessary in such a case. It seems that the described method may be used for monitoring the patients after surgical treatment of differentiated thyroid cancer aimed at early detection of metastases.

[Preliminary evaluation of the methods of radiotherapy in the complex treatment of children with soft tissue sarcomas]. / Wstepna analiza metod radioterapii w kompleksowym leczeniu dzieci chorych na miesaki tkanek miekkich.

44 children with the diagnosis of soft tissue sarcoma have been irradiated in the 1st Radiotherapy Department of the Institute of Oncology in Warsaw in the 1978-1986 period. Radiotherapy has been applied in cases previously non radically operated (32), not operated (9), and preoperatively irradiated (3). Most numerous group consisted of rhabdomyosarcomas (77%). As localization is concerned most frequent have been neoplasms of the head and neck area and the orbita (68%), retroperitoneal space and urinary bladder. Megavoltage Co60 gamma radiation has been applied and electron beam irradiation in part of the cases. Methods of irradiation were individualized. Special protective shields and cellon masques applied allowed precise reproduction of positioning and immobilization of children during irradiation. The tolerance of treatment has been in general good. Exaggerated early postradiation reactions appeared in cases receiving synchronously cytostatics. Over 2 year survival without symptoms of neoplasms has been obtained in 20 children (45%) and with symptoms of recurrence in 7 (16%); 17 (39%) children died.

[Preliminary results of radiotherapy of children with neoplasms of the central nervous system]. / Wstepne wyniki leczenia napromienianiem dzieci chorych na nowotwory centralnego ukladu nerwowego.

42 children with diagnosis of neoplasm of the central nervous system have been treated in the Ist Teleradiotherapy Unit of the Oncology Center--Maria Sklodowska-Curie Institute in Warsaw in the years 1980-1985. The age of children has been within 16 months--16 years limits. In 38 cases brain tumors have been irradiated and in 4 spinal ones. The treatment consisted of megavoltage Co60 irradiation, and in part of the spinal tumors the irradiation with electrons of 13-17 MeV energy. The tolerance of the treatment has been in general very good. 25 children (60%) survived 3-8 years, without symptoms of the disease, including 15 (60%) in very good psychosomatic condition, 17 children died.

The effect of dithiotreitol on thyroid peroxidase and microsomal antigen epitopes recognized by auto and monoclonal antibodies.

The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).

Comparison of autoantibodies level in thyroid autoimmune diseases estimated by different methods.

Autoantibodies to different thyroid cell components in serum of patients with thyroid disease have been estimated by several methods. The TSH receptor antibodies (TRAb) have been detected in 77% of untreated Graves-Basedow, in 70% of thionamide drugs treated Graves-Basedow patients up to four months of the treatment and in 48% of patients with Hashimoto's thyroiditis. Microsomal antibodies (MAb) and thyroid plasma membrane antibodies (ATMA) have been detected in 84% and 68% respectively, in thionamide drugs treated Graves-Basedow and in 91% and 72% in Hashimoto's thyroiditis. The thyroglobulin antibodies have been detected in 42% of untreated Graves-Basedow, in 47% of thionamide drugs treated Graves-Basedow and in 77% of Hashimoto's thyroiditis. In 19% to 21% of patients with toxic nodular goiter underlying autoimmunological process have been established by the detection of high levels of serum autoantibodies (ATMA and MAb). In the non-toxic nodular goiter, non-thyroid autoimmune diseases and healthy blood donors only thyroglobulin antibodies were detected in a significant number of patients. Other types of antibodies (ATMA or MAb) were detected only in sporadic cases of non-thyroid autoimmune diseases.

The presence of autoantibodies directed to thyroid plasma membrane antigens in sera of patients with thyroid disorders, estimated by the reaction with labelled protein A.

The presence of antithyroid plasma membrane antibodies (ATMA) has been detected in 97% of patients with untreated hyperthyroid Graves' disease, 85% of methimazole treated hyperthyroid Graves' disease, 25% of Hashimoto's thyroiditis and 6.9% of patients with toxic nodular goitre. The ATMA index was negative in all healthy blood donors, in patients with non-toxic nodular goitre, with the thyrocardiac syndrome and with simple obesity. Studies of patients with non-thyroid autoimmune diseases revealed that ATMA is positive in 11% of patients with scleroderma, 17.6% of systemic lupus erythematosus and 16% of rheumatoid arthritis. The amount of immunoglobulin bound to thyroid plasma membranes after pre-incubation with serum from patients with Graves' disease varied from 4.2 to 25.2 pmoles per mg of membrane protein; these values are several times higher than the maximal binding capacity for thyrotrophin which is 1.28 pmole/mg protein. In the majority of the cases studied TSH did not significantly inhibit IgG bound from thyroid plasma membranes. Significant amounts of IgG were displaced by an excess of TSH only in three cases with untreated hyperthyroid Graves' disease.