Genomic profiling by whole-genome single nucleotide polymorphism arrays in Wilms tumor and association with relapse.

Despite the excellent survival rate of Wilms tumor (WT) patients, only approximately one-half of children who suffer tumor recurrence reach second durable remission. This underlines the need for novel markers to optimize initial treatment. We investigated 77 tumors using Illumina 370CNV-QUAD genotyping BeadChip arrays and compared their genomic profiles to detect copy number (CN) abnormalities and allelic ratio anomalies associated with the following clinicopathological variables: relapse (yes vs. no), age at diagnosis (≤ 24 months vs. >24 months), and disease stage (low stage, I and II, vs. high stage, III and IV). We found that CN gains at chromosome region 1q21.1-q31.3 were significantly associated with relapse. Additional genetic events, including allelic imbalances at chromosome arms 1p, 1q, 3p, 3q, and 14q were also found to occur at higher frequency in relapsing tumors. Interestingly, allelic imbalances at 1p and 14q also showed a borderline association with higher tumor stages. No genetic events were found to be associated with age at diagnosis. This is the first genome wide analysis with single nucleotide polymorphism (SNP) arrays specifically investigating the role of genetic anomalies in predicting WT relapse on cases prospectively enrolled in the same clinical trial. Our study, besides confirming the role of 1q gains, identified a number of additional candidate genetic markers, warranting further molecular investigations.

Complementary and alternative medical therapies used by children with cancer treated at an Italian pediatric oncology unit.

BACKGROUND: Research has identified a growing use of complementary and alternative medicines (CAM) in the pediatric oncology setting and health care professionals should consider how they might interact with and/or be used in lieu of conventional treatment. The present study was designed to establish the prevalence of CAM usage at an Italian pediatric oncology department, and the reasons why patients used these unconventional therapies. PROCEDURE: This was an observational study involving parents whose children were treated for tumors at the pediatric oncology unit of the Istituto Nazionale Tumori in Milano. Data were collected on their sociodemographic variables and their use of CAM by means of a self-administered questionnaire. RESULTS: We distributed 145 questionnaires and 97 of them (67%) were returned. Judging from this survey, 12.4% of the children used at least one type of CAM and homoeopathy was the most often used. Benefits were reported by 83% of parents. The most common reasons for using CAM were to reduce the side-effects of conventional therapies. The oncologists taking care of the patients were notified of the child's use of CAM in only one case. CONCLUSION: CAM were used not as a substitute but in addition to conventional treatments. In almost all cases, oncologists were not informed that a child was using CAM, posing a risk of any interaction with pharmacological treatments being inadequately understood.

A novel WT1 mutation in a 46,XY boy with congenital bilateral cryptorchidism, nystagmus and Wilms tumor.

The WT1 gene plays a crucial role in urogenital and gonadal development. Germline WT1 alterations have been described in a wide spectrum of pathological conditions, including kidney diseases, genital abnormalities and Wilms tumor (WT), frequently occurring in combination. We report on a novel WT1 nonsense mutation (c.1105C>T), introducing a premature stop codon in exon 8 (p.Q369X), in a young XY male patient who presented with bilateral cryptorchidism, nystagmus, mild proteinuria and WT, but no sign of severe nephropathy. Although the majority of congenital urogenital abnormalities are not due to constitutional defects of the WT1 gene, our findings provide a rational for considering WT1 mutational analysis as one of the screening options in newborns with congenital defects of the urogenital tract due to the associated high risk of WT.

Brain magnetic resonance imaging after high-dose chemotherapy and radiotherapy for childhood brain tumors.

PURPOSE: Brain necrosis or other subacute iatrogenic reactions has been recognized as a potential complication of radiotherapy (RT), although the possible synergistic effects of high-dose chemotherapy and RT might have been underestimated. METHODS AND MATERIALS: We reviewed the clinical and radiologic data of 49 consecutive children with malignant brain tumors treated with high-dose thiotepa and autologous hematopoietic stem cell rescue, preceded or followed by RT. The patients were assessed for neurocognitive tests to identify any correlation with magnetic resonance imaging (MRI) anomalies. RESULTS: Of the 49 children, 18 (6 of 25 with high-grade gliomas and 12 of 24 with primitive neuroectodermal tumors) had abnormal brain MRI findings occurring a median of 8 months (range, 2-39 months) after RT and beginning to regress a median of 13 months (range, 2-26 months) after onset. The most common lesion pattern involved multiple pseudonodular, millimeter-size, T1-weighted unevenly enhancing, and T2-weighted hyperintense foci. Four patients with primitive neuroectodermal tumors also had subdural fluid leaks, with meningeal enhancement over the effusion. One-half of the patients had symptoms relating to the new radiographic findings. The MRI lesion-free survival rate was 74%+/-6% at 1 year and 57%+/-8% at 2 years. The number of marrow ablative courses correlated significantly to the incidence of radiographic anomalies. No significant difference was found in intelligent quotient scores between children with and without radiographic changes. CONCLUSION: Multiple enhancing cerebral lesions were frequently seen on MRI scans soon after high-dose chemotherapy and RT. Such findings pose a major diagnostic challenge in terms of their differential diagnosis vis-à-vis recurrent tumor. Their correlation with neurocognitive results deserves further investigation.

Radiation effects on development of HER2-positive breast carcinomas.

PURPOSE: Neither hormone-related nor genetics risk factors have been associated with the development of highly proliferative HER2-positive breast carcinomas. Because the majority of HER2-positive tumors present the amplification of the oncogene, we asked whether genomic instability triggered by irradiation might be involved in the induction of HER2-overexpressing breast carcinomas. EXPERIMENTAL DESIGN: Sixty-six infiltrating breast carcinomas from patients treated with radiation therapy for Hodgkin's lymphoma or other pediatric solid tumors and a control series of 61 consecutive sporadic breast tumors were analyzed by immunohistochemistry for HER2 expression with HercepTest. A panel of antibodies against estrogen receptor, progesterone receptor, c-kit, cytokeratin 5/6, p53, and ki67 antigen was also used to identify differentiation subsets and molecular characteristics of the analyzed breast carcinomas. RESULTS: Although no differences between the two tumor series were found with respect to HER2 expression scored 2+ and 3+, the percentage of 3+ HER2-positive tumors was significantly higher in patients irradiated during breast maturation compared with patients irradiated after breast maturation (35.3% versus 12.5%, P = 0.046). In the latter group, 52.5% of the breast carcinomas showed basal-like differentiation (estrogen receptor, progesterone receptor, and HER2 negative) versus only 5.9% in the group irradiated during breast development (P < 0.0001). Analysis adjusted for age confirmed the significant increase in basal-like tumor development in patients irradiated within 4 years of menarche, but also showed that the differences between patients irradiated before and after puberty in HER2 3+ tumor frequencies are due to age-related differences in HER2 3+ tumor onset. CONCLUSION: Together, our data indicate that the development of HER2-positive tumors correlates with timing rather than type of carcinogenic hits and provide clear evidence that radiation is a risk factor for breast carcinomas showing basal-like differentiation.

Psychological assessment of women on an early breast screening program after radiotherapy to the chest wall for childhood cancer.

AIMS AND BACKGROUND: This study investigates the psychological status in a population of female patients who received chest irradiation for a childhood cancer and were screened for second primary breast cancer. METHODS: Sixty-eight consecutive such young women were included. Compilation of the Crown-Crisp Index questionnaire was requested and 49/68 patients accepted to fill it in; 14 women in the sample had children (28%). RESULTS: Twenty-seven of 49 patients achieved a normal score, whereas in 22 the score was slightly above the normal range in at least one scale. Pathological scores were more frequent among the women without children. CONCLUSION: Quality of life in this series of long-term survivors does not seem to be severely affected by previous treatment for cancer nor by the concern for the onset of a second primary malignancy.

Thyroid-stimulating hormone suppression for protection against hypothyroidism due to craniospinal irradiation for childhood medulloblastoma/primitive neuroectodermal tumor.

PURPOSE: Hypothyroidism is one of the earliest endocrine effects of craniospinal irradiation (CSI). The effects of radiation also depend on circulating thyroid-stimulating hormone (TSH), which acts as an indicator of thyrocyte function and is the most sensitive marker of thyroid damage. Hence, our study was launched in 1998 to evaluate the protective effect of TSH suppression during CSI for medulloblastoma/primitive neuroectodermal tumor. PATIENTS AND METHODS: From Jan 1998 to Feb 2001, a total of 37 euthyroid children scheduled for CSI for medulloblastoma/primitive neuroectodermal tumor underwent thyroid ultrasound and free triiodothyronine (FT3), free thyroxine (FT4), and TSH evaluation at the beginning and end of CSI. From 14 days before and up to the end of CSI, patients were administered l-thyroxine at suppressive doses; every 3 days, TSH suppression was checked to ensure a value <0.3 mum/ml. During follow-up, blood tests and ultrasound were repeated after 1 year; primary hypothyroidism was considered an increased TSH level greater than normal range. CSI was done using a hyperfractionated accelerated technique with total doses ranging from 20.8-39 Gy; models were used to evaluate doses received by the thyroid bed. RESULTS: Of 37 patients, 25 were alive a median 7 years after CSI. They were well matched for all clinical features, except that eight children underwent adequate TSH suppression during CSI, whereas 17 did not. Hypothyroidism-free survival rates were 70% for the "adequately TSH-suppressed" group and 20% for the "inadequately TSH-suppressed" group (p = 0.02). CONCLUSIONS: Thyroid-stimulating hormone suppression with l-thyroxine had a protective effect on thyroid function at long-term follow-up. This is the first demonstration that transient endocrine suppression of thyroid activity may protect against radiation-induced functional damage.

Adult-type soft tissue sarcomas in pediatric-age patients: experience at the Istituto Nazionale Tumori in Milan.

PURPOSE: Nonrhabdomyosarcoma soft tissue sarcomas are a heterogeneous group of tumors for which optimal treatment remains controversial. We report on a large group of 182 patients younger than 18 years old treated at a single institution over a 25-year period. PATIENTS AND METHODS: In this relatively homogeneous subgroup of adult-type histotypes, surgery was the mainstay of treatment; radiotherapy was administered to 73 patients, and chemotherapy was administered to 114 patients (70 received chemotherapy as adjuvant therapy). RESULTS: Overall survival at 5 years was 89% in patients who underwent complete resection at diagnosis, 79% in patients who had marginal resection, 52% in initially unresected patients, and 17% in patients with metastases at onset. Outcome was unsatisfactory in patients with large and high-grade tumors, even after gross resection; adjuvant chemotherapy seemed to improve the results in this group. Initially unresected patients who responded well to chemotherapy and subsequently underwent complete resection had an event-free survival rate of approximately 70%. The rate of response to chemotherapy was 58%. CONCLUSION: The identification of prognostic variables should enable risk-adapted therapies to be planned. Patients with initially unresectable disease and patients with resected large and high-grade tumors are at high risk of metastases and treatment failure. Although the limits of this retrospective analysis are self-evident, our data would suggest that intensive chemotherapy (with an ifosfamide-doxorubicin regimen) might have a more significant role in these patients than what is generally assumed.

Supratentorial primitive neuroectodermal tumors (S-PNET) in children: A prospective experience with adjuvant intensive chemotherapy and hyperfractionated accelerated radiotherapy.

PURPOSE: Supratentorial primitive neuroectodermal tumors (S-PNET) are rare and have a grim prognosis, frequently taking an aggressive course with local relapse and metastatic spread. We report the results of a mono-institutional therapeutic trial. METHODS AND MATERIALS: We enrolled 15 consecutive patients to preradiation chemotherapy (CT) consisting of high-dose methotrexate, high-dose etoposide, high-dose cyclophosphamide, and high-dose carboplatin, craniospinal irradiation (CSI) with hyperfractionated accelerated radiotherapy (HART) plus focal boost, maintenance with vincristine/lomustine or consolidation with high-dose thiotepa followed by autologous stem-cell rescue. RESULTS: Median age was 9 years; 7 were male, 8 female. Site of disease was pineal in 3, elsewhere in 12. Six patients were had no evidence of disease after surgery (NED). Of those with evidence of disease after surgery (ED), 2 had central nervous system spread. Of the 9 ED patients, 2 had complete response (CR) and 2 partial response (PR) after CT, 4 stable disease, and 1 progressive disease. Of the 7 ED patients before radiotherapy, 1 had CR, 4 PR, and 2 minor response, thus obtaining a 44% CR + PR after CT and 71% after HART. Because of rapid progression in 2 of the first 5 patients, high-dose thiotepa was systematically adopted after HART in the subsequent 10 patients. Six of 15 patients relapsed (4 locally, 1 locally with dissemination, 1 with dissemination) a mean of 6 months after starting CT, 2 developed second tumors; 5 of 6 relapsers died at a median of 13 months. Three-year progression-free survival, event-free survival, and overall survival were 54%, 34%, and 61%, respectively. CONCLUSION: Hyperfractionated accelerated RT was the main tool in obtaining responses in S-PNET; introducing the myeloablative phase improved the prognosis (3/10 vs. 3/5 relapses), though the outcome remained unsatisfactory despite the adoption of this intensive treatment.

Detecting CD56+/NB84+/CD45- immunophenotype in the bone marrow of patients with metastatic neuroblastoma using flow cytometry.

BACKGROUND: In order to identify neuroblastoma cells infiltrating the bone marrow, a triple-color flow-cytometric assay was developed combining CD56 and CD45 with the intracellular anti-NB84 specific antibody. MATERIALS AND METHODS: The bilateral aspirates obtained from 27 consecutive children over the age of one year with stage 4 neuroblastoma were evaluated. RESULTS: Neuroblastoma cells were detected in the bone marrow of 17/27 (63%) and 19/27 (70%) cases using cytomorphology and triple-color flow-cytometry, respectively. Using cytometry, the percentage of CD56+/NB84+/CD45-cells infiltrating the bone marrow ranged from 0.02% to 65%. Five out of eight patients without bone marrow involvement according to cytometry are in continuous complete remission, while only 3 out of 19 patients whose bone marrow gave positive results are still alive. CONCLUSION: By combining CD45 and CD56 with the specific antibody, NB84, directed against neuroblastoma cells, we developed a rapid and reliable cytometric assay that can be associated with conventional cytomorphological bone marrow evaluation to detect infiltrating neuroblastoma cells, especially in cases of dubious positivity.

Assistance to parents who have lost their child with cancer.

AIMS AND BACKGROUND: To assess the psychological needs of parents after the death of their child from cancer. METHODS: The study comprises a preliminary retrospective phase to identify parents who spontaneously contacted the medical staff, followed by a prospective phase in which families were contacted by telephone and were invited to a meeting. RESULTS: The retrospective study demonstrated that more than 50% of the families spontaneously sought contact with the department. In the prospective study, 17 families were contacted and the majority of them subsequently decided to come to the department for a talk. CONCLUSIONS: Our experience shows that parents have a strong need to have further contact with the team that took care of their children for months. In the process of coping with bereavement, anxiety and depression are common and not necessarily pathological, though there may be psychopathological reactions that can interfere with the parents' quality of life.

Antigen-specific immunity in neuroblastoma patients: antibody and T-cell recognition of NY-ESO-1 tumor antigen.

Neuroblastoma cells have been shown to express molecularly defined tumor-associated antigens, which could represent potential targets of T and/or B cell-mediated immunity. However, the existence of a spontaneous immune response to such tumor antigens in neuroblastoma patients has yet to be investigated. In the present work we addressed the issue of whether NY-ESO-1, a germ cell antigen aberrantly expressed in different tumor types, is expressed by neuroblastoma cells and may represent a target for humoral and/or cellular immune responses in neuroblastoma patients. We found that a large fraction of neuroblastoma biopsies, independently from the clinical stage and degree of tumor cell differentiation, expressed significant levels of NY-ESO-1 as assessed by reverse transcription-PCR and immunohistochemistry. NY-ESO-1-specific IgG antibodies were detected in the sera of 10% of neuroblastoma patients with stage III or IV disease, but not in patients in clinical remission or with earlier stages. This suggests that antibody production occurred as a late event in the course of disease. NY-ESO-1-specific immune responses were observed for CD4(+) and CD8(+) T cells from peripheral blood lymphocytes in 4 of 8 neuroblastoma patients, as detected by IFN-gamma enzyme-linked immunospot assay after in vitro stimulation either with the NY-ESO-1 recombinant protein or with the HLA-A2-restricted peptide NY-ESO-1(157-167). NY-ESO-1-specific CD4(+) and CD8(+) T cells were also able to recognize NY-ESO-1 expressing neuroblastoma cells. The presence of T cells specific for NY-ESO-1 antigen was not associated with the stage of disease, or to the presence or absence of NY-ESO-1 specific antibodies. We conclude that NY-ESO-1 is an immunogenic antigen in neuroblastoma patients and represents a candidate target for immune-based therapy.

Intensive, very short-term chemotherapy for advanced Burkitt's lymphoma in children.

PURPOSE: To improve the 63% event-free survival (EFS) achieved before 1986 in Murphy's stage III to IV Burkitt's lymphoma (BL), both chemotherapy and supportive care were intensified. PATIENTS AND METHODS: From May 1987 to February 2001, 60 children, median age 9 years (range, 2.1 to 17 years), with advanced BL were enrolled onto two sequential institutional studies. From 1987 to 1992, 30 patients were stratified according to the absence (regimen IA, n = 19) or presence (regimen IB, n = 11) of bone marrow (BM) or CNS involvement. After 5-week cytoreductive chemotherapy consisting of vincristine, cyclophosphamide, doxorubicin, high-dose (HD) methotrexate (MTX), and intrathecal MTX or cytarabine, HD cytarabine and cisplatin were provided as a 4-day continuous infusion. Regimen IB was intensified by adding etoposide and HD ifosfamide and escalating MTX doses. Since 1992, regardless of BM or CNS status, 30 patients have been placed on regimen II, which is identical to IB but without ifosfamide. The scheduled duration of regimen II was 45 days. RESULTS: EFS and disease-free survival at 5 years are 81% +/- 5% and 87% +/- 5%, respectively, for 59 assessable patients (73% +/- 8% and 85% +/- 7% for regimen IA + IB, 89% +/- 6%, EFS and disease-free survival, for regimen II; median follow-up, 6.7 years; range, 0.6 to 13.5 years). Six patients, two of whom were receiving regimen II, died as a result of initial treatment failure or relapse, and five patients, none receiving regimen II, died as a result of treatment-related complications. CONCLUSION: This 45-day intensive chemotherapy program is the shortest schedule for disseminated BL and overcomes previously recognized risk factors such as BM and CNS infiltration.

High risk of subsequent neoplasms continues with extended follow-up of childhood Hodgkin's disease: report from the Late Effects Study Group.

PURPOSE: We present an update of a previously reported Late Effects Study Group cohort of 1,380 children with Hodgkin's disease (HD) diagnosed between 1955 and 1986 in patients aged 16 years or younger. We describe the pattern and incidence of subsequent neoplasms (SNs) occurring with extended follow-up. PATIENTS AND METHODS: Median age at diagnosis of HD was 11.7 years (range, 0.3 to 16.9 years) and at last follow-up was 27.8 years. Median length of follow-up was 17.0 years. RESULTS: An additional 103 SNs were ascertained (total SNs = 212). The cohort was at an 18.5-fold increased risk of developing SNs compared with the general population (standardized incidence ratio [SIR], 18.5, 95% CI, 15.6 to 21.7). The cumulative incidence of any second malignancy was 10.6% at 20 years, increasing to 26.3% at 30 years; and of solid malignancies was 7.3% at 20 years, increasing to 23.5% at 30 years. Breast cancer was the most common solid malignancy (SIR, 56.7). Other commonly occurring solid malignancies included thyroid cancer (SIR, 36.4), bone tumors (SIR, 37.1), and colorectal (SIR, 36.4), lung (SIR, 27.3), and gastric cancers (SIR, 63.9). Risk factors for solid tumors included young age at HD and radiation-based therapy. Thirty-two patients developed third neoplasms, with the cumulative incidence approaching 21% at 10 years from diagnosis of second malignancy. CONCLUSION: Additional follow-up of this large cohort of HD survivors documents an increasing occurrence of known radiation-associated solid tumors, (breast and thyroid cancers), as well as emergence of epithelial neoplasms common in adults, (colon and lung cancers) at a younger age than expected in the general population, necessitating ongoing surveillance of this high risk population.

High response rate to cisplatin/etoposide regimen in childhood low-grade glioma.

PURPOSE: The aim of this study was to avoid radiotherapy and to induce an objective response in children with low-grade glioma (LGG) using a simple chemotherapy regimen based on cisplatin and etoposide. PATIENTS AND METHODS: Thirty-four children (median age, 45 months) with unresectable LGG were treated with 10 monthly cycles of cisplatin (30 mg/m(2)/d on days 1 to 3) and etoposide (150 mg/m(2)/d on days 1 to 3). Tumor originated in the visual pathway in 29 patients, in the temporal lobe in two, in the frontal lobe in two, and in the spine in one. Eight children were affected by neurofibromatosis type 1. Objective tumor response and toxicity were evaluated by magnetic resonance imaging and neurologic and functional tests at 3-month intervals. RESULTS: An objective response was obtained in 24 (70%) of 34 patients, whereas the others had stable disease. None of the children were electively irradiated. In 31 previously untreated children, overall survival was 100% and progression-free survival was 78% at 3 years, with a median follow-up of 44 months. Acute toxicity was unremarkable; 28% patients evaluated for acoustic neurotoxicity revealed a loss of perception of high frequencies. CONCLUSION: Cisplatin and etoposide combined treatment is one of the most active regimens for LGG in children and allows avoidance of radiotherapy in the vast majority of patients.

Sequential chemotherapy, high-dose thiotepa, circulating progenitor cell rescue, and radiotherapy for childhood high-grade glioma.

Childhood malignant gliomas are rare, but their clinical behavior is almost as aggressive as in adults, with resistance to therapy, rapid progression, and not uncommonly, dissemination. Our study protocol incorporated sequential chemotherapy and high-dose thiotepa in the preradiant phase, followed by focal radiotherapy and maintenance with vincristine and lomustine for a total duration of one year. The induction treatment consisted of two courses of cisplatin (30 mg/m2) plus etoposide (150 mg/m2) x 3 days and of vincristine (1.4 mg/m2) plus cyclophosphamide (1.5 g/m2) plus high-dose methotrexate (8 g/m2), followed by high-dose thiotepa (300 mg/m2 x 3 doses), with harvesting of peripheral blood progenitor cells after the first cisplatin/etoposide course. From August 1996 to March 2003, 21 children, 14 females and 7 males, with a median age of 10 years were enrolled, 18 presenting with residual disease after surgery. Histologies were glioblastoma multiforme in 10, anaplastic astrocytoma in nine, and anaplastic oligodendroglioma in two; sites of origin were supratentorial areas in 17, spine in two, and posterior fossa in two. Of the 21 patients, 12 have died (10 after relapse, with a median time to progression for the whole series of 14 months; one with intratumoral bleeding at 40 months after diagnosis; and one affected by Turcot syndrome for duodenal cancer relapse). Four of 12 relapsed children had tumor dissemination. At a median follow-up of 57 months, overall survival and progression-free survival at four years were 43% and 46%, respectively. Sequential and high-dose chemotherapy can be afforded in front-line therapy of childhood malignant glioma without excessive morbidity and rather encouraging results.

Survival of adults treated for medulloblastoma using paediatric protocols.

We retrospectively studied 26 consecutive adults treated for medulloblastoma using paediatric protocols. Between 1987 and 2003, patients 18 years old were given adjuvant chemotherapy consisting of one of two 'paediatric' regimens (depending on the time of presentation) and craniospinal local-boost radiotherapy: regimen A (n = 12), vincristine (VCR), intrathecal and/or intravenous methotrexate and conventional radiotherapy; or regimen B (n = 11) sequencing intensive doses of multiple agents followed by hyperfractionated accelerated radiotherapy (HART). A VCR-lomustine-based maintenance followed both regimens. Three additional patients received a tailored treatment due to their impaired neurological status after surgery. The median age at diagnosis was 26 years (range 18-41 years). With a median follow-up of 46 months, 5-year disease-free and overall survival rates were 65+/-11% and 73+/-10%, respectively, for the series as a whole. All patients who received regimen B (5 of whom had metastatic Chang M2-M3 disease) are alive with no evidence of disease at 39 months. Although the number of patients is limited, our data suggest that the sandwich sequential, moderately intensive chemotherapy in combination with HART is an effective treatment for medulloblastoma in adults, and this approach seems to overcome previously-recognised risk factors.

Germline mutations of the POU6F2 gene in Wilms tumors with loss of heterozygosity on chromosome 7p14.

Wilms tumor (WT) is a kidney malignancy of childhood characterized by highly heterogeneous genetic alterations. We previously reported the molecular and cytogenetic characterization of a WT (Case 30) carrying an interstitial deletion in chromosome 7p14 between markers D7S555 and D7S668. Loss of heterozygosity (LOH) analyses had revealed that this same region was lost in 8 out of 38 examined WTs, suggesting that the identified interval contains a putative tumor suppressor gene. To confirm this hypothesis, in this work, we analyzed an additional 35 WTs, four of which showed LOH in the region of interest. Furthermore, we were able to more accurately define the extension of the deletion in Case 30, mapping it within an interval not exceeding 390 kb, proximally to D7S555. To date, only a single expressed gene, POU6F2 (the POU domain, class 6, transcription factor 2; also known as RPF1), has been recognized in this interval. Sequencing of the gene in the 12 WTs showing LOH and in a corresponding numbers of WT cases without LOH, led to the identification of two germline nucleotide substitutions. The first occurred in the 5'-untranslated region, while the second caused an amino acid change in a glutamine repeat domain. These mutations, whose occurrence was not observed in more than 100 control subjects, were detected in two patients showing the loss of the constitutionally wild-type allele in tumor DNA. Together with the finding of the expression of the POU6F2 mouse homolog in both fetal and adult kidney, our observations suggest that the gene is a tumor suppressor and is involved in hereditary predisposition to WT.

Molecular detection of dopamine decarboxylase expression by means of reverse transcriptase and polymerase chain reaction in bone marrow and peripheral blood: utility as a tumor marker for neuroblastoma.

A highly sensitive molecular method was used to evaluate the presence of dopamine decarboxylase (DDC) mRNA in the bone marrow and peripheral blood of patients with neuroblastoma (NB). DDC, like tyrosine hydroxylase (TH), is an enzyme involved in the catecholamine synthesis pathway and has recently been proposed as a specific marker of NB among pediatric malignancies. DDC transcript was detected in five of five NB cell lines, 10 of 10 NB primary tumors, 17 of 18 (94%) bone marrow samples, and 12 of 18 (66%) blood samples drawn at diagnosis in 18 patients affected by disseminated NB. In contrast, no PCR signal was found in 20 bone marrow samples obtained from patients with other malignancies or in eight of nine marrow and blood samples drawn from patients with localized NB (two stage 2 and seven stage 3). In addition, all marrow and blood samples obtained from NB patients at relapse revealed DDC mRNA. Furthermore, the percentage of DDC-positive samples was lower among the samples drawn from these patients during treatment. By comparison with conventional methods for disease evaluation, DDC transcript research can increase the sensitivity of NB cell detection in marrow and blood samples at diagnosis and during the treatment and follow-up of NB patients. These results suggest that finding DDC mRNA in NB patients could be a potential marker for minimal residual disease study.

Immunomodulation in a treatment program including pre- and post-operative interleukin-2 and chemotherapy for childhood osteosarcoma.

AIMS AND BACKGROUND: The treatment applied in our Institution to children with localized osteosarcoma between 1991 and 1999 consisted of four interleukin 2 (IL-2) courses (9 x 10(6) IU/mL/daily x 4), alternated with pre- and post-operative polichemotherapy. The aims of the present study were to quantify the modifications of some immunological parameters induced by IL-2 and to verify whether polychemotherapy could reduce them. An additional aim was to assess whether any correlation between the immune modifications and the clinical outcome could be found. PATIENTS AND METHODS: We evaluated in 18 consecutive patients the following changes, induced in blood by each IL-2 course: number of lymphocyte subpopulations and natural killer (NK) cells, lymphokine activated killer (LAK) and NK activities. RESULTS: Chemotherapy did not influence the modifications of the number of NK and CD4+ cells and of the LAK and NK activities, induced by each of the four courses of IL-2. The magnitudo of the NK activity and the peak of the NK absolute counts significantly correlated with the clinical outcome. CONCLUSIONS: The results show that the use of IL-2 permitted a repeated immune activation despite the intensive chemotherapy. Furthermore, although the limited number of cases precludes any definitive conclusion, the results suggest a possible role of the NK cells in the control of osteosarcoma.