Brachytherapy for Pediatric Patients at Gustave Roussy Cancer Campus: A Model of International Cooperation for Highly Specialized Treatments.

PURPOSE: Childhood cancer is rare, and treatment is frequently associated with long-term morbidity. Disparities in survival and long-term side effects encourage the establishment of networks to increase access to complex organ-conservative strategies, such as brachytherapy. We report our experience of an international cooperation model in childhood cancers. METHODS AND MATERIALS: We examined the outcome of all children referred to our center from national or international networks to be treated according to a multimodal organ-conservative approach, including brachytherapy. RESULTS: We identified 305 patients whose median age at diagnosis was 2.2 years (range, 1.4 months to 17.2 years). Among these patients, 99 (32.4%) were treated between 2015 and 2020; 172 (56.4%) were referred from national centers; and 133 (43.6%) were international patients from 31 countries (mainly Europe). Also, 263 patients were referred for primary treatment and 42 patients were referred for salvage treatment. Genitourinary tumors were the most frequent sites, with 56.4% bladder/prostate rhabdomyosarcoma and 28.5% gynecologic tumors. In addition to brachytherapy, local treatment consisted of partial tumor resection in 207 patients (67.9%), and 39 patients (13%) had additional external radiation therapy. Median follow-up was 58 months (range, 1 month to 48 years), 93 months for national patients, and 37 months for international patients (P < .0001). Five-year local control, disease-free survival, and overall survival rates were 90.8% (95% confidence interval [CI], 87.3%-94.4%), 84.4% (95% CI, 80.1%-89.0%), and 93.3% (95% CI, 90.1%-96.5%), respectively. Patients referred for salvage treatment had poorer disease-free survival (P < .01). Implementation of image guided pulse-dose-rate brachytherapy was associated with better local control among patients with rhabdomyosarcoma referred for primary treatment (hazard ratio, 9.72; 95% CI, 1.24-71.0). At last follow-up, 16.7% patients had long-term severe treatment-related complications, and 2 patients (0.7%) had developed second malignancy. CONCLUSIONS: This retrospective series shows the feasibility of a multinational referral network for brachytherapy allowing high patient numbers in rare pediatric cancers. High local control probability and acceptable late severe complication probability could be achieved despite very challenging situations. This cooperation model could serve as a basis for generating international reference networks for high-tech radiation such as brachytherapy to increase treatment care opportunities and cure probability.

Dermatofibrosarcoma protuberans in children and adolescents: The European Paediatric Soft Tissue Sarcoma Study Group prospective trial (EpSSG NRSTS 2005).

BACKGROUND: As dermatofibrosarcoma protuberans (DFSP) are rare with no prospective series within paediatric sarcoma trials, the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) examined the clinical data and outcomes of DFSP enrolled in a multinational study of non-rhabdomyosarcoma soft tissue sarcomas (NRSTS). PATIENTS AND METHODS: Forty-six patients with confirmed DFSP were enrolled into the EpSSG NRSTS 2005 study. All had surgical resection and none had any further therapy at diagnosis. RESULTS: The median age at diagnosis was 6.9 years (range 0.4-17.5). All patients had localised disease, and the majority had small <5 cm tumours (93%), and 76% had Intergroup Rhabdomyosarcoma Study (IRS) I tumours. All patients had up front surgery, 32 requiring two operations. There were 11 patients with IRS II tumours, of which only two went on to have a local recurrence. After a median follow up of 49.0 months (range 4.2-130.9), all patients were alive at the time of this report, with 5-year event-free survival of 92.6% (CI 78.8-97.6) with a 100% overall survival. CONCLUSION: This report demonstrates the ability to run prospective paediatric studies in NRSTS in multiple European countries, with reasonable numbers of DFSP patients, with few events and no deaths, and hence excellent outcomes.

Prospective, real-time monitoring of pegylated Escherichia coli and Erwinia asparaginase therapy in childhood acute lymphoblastic leukaemia and non-Hodgkin lymphoma in Belgium.

Asparaginase (ASNase) is an important anti-leukaemic drug in the treatment of childhood acute lymphoblastic leukaemia (ALL) and non-Hodgkin lymphoma (NHL). A substantial proportion of patients develop hypersensitivity reactions with anti-ASNase neutralising antibodies, resulting in allergic reactions or silent inactivation (SI), and characterised by inactivation and rapid clearance of ASNase. We report results of a prospective, real-time therapeutic drug monitoring of pegylated Escherichia coli (PEG-)ASNase and Erwinia ASNase in children treated for ALL and NHL in Belgium. Erwinia ASNase was given as second-line after hypersensitivity to PEG-ASNase. In total, 286 children were enrolled in the PEG-ASNase cohort. Allergy was seen in 11·2% and SI in 5·2% of patients. Of the 42 patients treated with Erwinia ASNase, 7·1% experienced allergy and 2·4% SI. The median trough PEG-ASNase activity was high in all patients without hypersensitivity. After Erwinia administration significantly more day 3 samples had activities <100 IU/l (62·5% vs. 10% at day 2 (D2)). The median D2 activity was significantly higher for intramuscular (IM; 347 IU/l) than for intravenous Erwinia administrations (159 IU/l). This prospective, multicentre study shows that monitoring of ASNase activity during treatment of children with ALL and NHL is feasible and informative. Treatment with Erwinia ASNase warrants close monitoring and optimally adherence to a 2-day interval of IM administrations.

Outcome and prognostic factors in pediatric malignant peripheral nerve sheath tumors: An analysis of the European Pediatric Soft Tissue Sarcoma Group (EpSSG) NRSTS-2005 prospective study.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are rare tumors of childhood. The role of standard chemotherapy in unresectable MPNST is still unclear. We report the outcome and prognostic factors in the EpSSG risk-adapted prospective study for localized pediatric MPNST. METHODS: Patients were stratified into four treatment groups defined by surgical resection, tumor size, and tumor grade (G): (a) surgery-only group-resected tumors G1; (b) adjuvant radiotherapy group-R0/R1, G2 tumors; (c) adjuvant chemotherapy group-R0/R1, G3 tumors; and (d) neoadjuvant chemotherapy group-R2 resected tumors and/or nodal involvement. Chemotherapy consisted of four courses of ifosfamide-doxorubicin and two courses of ifosfamide concomitant with radiotherapy (50.4-54 Gy). RESULTS: Overall, the study included 51 patients. The 5-year event-free survival (EFS) and overall survival (OS) were 52.9% (95% confidence interval, 38.1-65.8) and 62.1% (46.7-74.3), respectively. The 5-year EFS was 92% (56.6-98.9) for treatment group 1 (N = 13), 33% (0.9-77.4) for treatment group 2 (N = 4), 29% (4.1-61.2) for treatment group 3 (N = 7), and 42% (23.1-60.1) for treatment group 4 (N = 27). Response rate to chemotherapy (partial response + complete response) in patients with measurable disease was 46%. The presence of neurofibromatosis type 1 (NF1; 51% of patients) was an independent poor prognostic factor for OS and EFS. CONCLUSION: The outcome for patients with resectable MPNST was excellent. Standard ifosfamide-doxorubicin for unresectable MPNST rendered the best reported outcome. Children with NF1 disease seem to have worse prognosis.

Clinical features and outcomes of young patients with epithelioid sarcoma: an analysis from the Children's Oncology Group and the European paediatric soft tissue Sarcoma Study Group prospective clinical trials.

BACKGROUND: Data on the clinical features, optimal treatment and outcomes of paediatric patients with epithelioid sarcoma (ES) are limited and mostly retrospective. METHODS: A subset analysis of ES patients < 30 years of age enrolled on two international prospective clinical trials conducted between 7/2005 and 11/2015 was performed. Risk-adapted therapy was based on tumour diameter, histologic grade, extent of surgery and presence/absence of metastases and included surgery ± radiotherapy for all patients with the addition of ifosfamide/doxorubicin chemotherapy for intermediate-/high-risk patients. Response to therapy, event-free and overall survival and pattern and predictors of treatment failure were evaluated. RESULTS: Sixty-three ES patients (median age 13.1 years, 52% male) were eligible. Clinical features included the following: 68% extremity, median tumour diameter 3.5 cm, 56% high histologic grade, 14% nodal metastases, 14% distant metastases. Thirty-four low-risk patients underwent surgery (n = 30) or surgery/radiotherapy (n = 4); 16 intermediate-risk and 13 high-risk patients received chemotherapy ± surgery ± radiotherapy. Partial response was observed in 11/22 (50%) patients receiving neoadjuvant therapy. Events were local recurrence (n = 10) and distant recurrence (n = 15); estimated 5-year survival was 86.4%, 63.5% and 0%, respectively, for low-, intermediate- and high-risk patients. Locoregional nodal involvement, invasive tumour, high grade and lesser extent of resection predicted event-free survival in patients without metastases. CONCLUSIONS: Most low-risk ES patients who have undergone an adequate resection fare well without adjuvant therapy. Large tumour size, high histologic grade, tumour invasiveness, inadequate tumour resection and metastatic disease predict poorer outcomes in higher risk ES patients, for whom more effective therapies are needed. CLINICAL TRIAL REGISTRATION: COG ARST0332: ClinicalTrials.gov Identifier NCT00346164, EpSSG NRSTS 2005: European Union Drug Regulating Authorities Clinical Trials No. 2005-001139-31.

Microscopic Infiltration of Cryopreserved Ovarian Tissue in 2 Patients With Ewing Sarcoma.

We report the clinical history of 2 female patients with Ewing sarcoma and microscopic ovarian infiltration. In both cases, the initial workup found no metastasis. However, the examination of cryopreserved ovarian tissues revealed the presence of CD99 positive tumor cells with rearrangement of EWS gene confirmed by FISH. Both children were treated as patients with localized tumor and are currently in remission. These reports underline that, in Ewing sarcoma patients, retransplantation of cryopreserved ovarian tissue is not harmless and could lead to cancer relapse. These observations question also on the significance of ovarian dissemination on Ewing sarcoma prognosis and therapy.

Alveolar soft part sarcoma in children and adolescents: The European Paediatric Soft Tissue Sarcoma study group prospective trial (EpSSG NRSTS 2005).

BACKGROUND: As alveolar soft part sarcomas (ASPS) are rare with no prospective series within pediatric sarcoma trials, the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) examined the clinical data and outcomes of ASPS enrolled in a multinational study of nonrhabdomyosarcoma soft tissue sarcomas (NRSTS). PATIENTS AND METHODS: Twenty-two patients with ASPS were enrolled into the EpSSG NRSTS 2005 study. After surgical resection, subsequent treatment depended on the stratification of patients for completeness of resection and Intergroup Rhabdomyosarcoma Study (IRS) stage, size, and French Federation of Cancer Centres Sarcoma Group (FNCLCC) grade. Chemotherapy using ifosfamide and doxorubicin was performed in IRS group III. Radiotherapy was performed in IRS groups II and III, and FNCLCC grades 2 and 3 tumors. RESULTS: The median age at diagnosis was 11.5 years (range 2.7-17.5 years). The majority in the series had localized disease (20), with small IRS I tumors (12), and in total 19 had surgical resection upfront. Of the four patients who received conventional chemotherapy, there were no responses. Three of 20 patients with localized tumors and all metastatic patients developed metastases. The median follow up of patients with localized disease is 61.7 months (range 25.7-135.5 months) from diagnosis. The 5-year event-free survival is 94.7% (95% confidence interval: 68.1-99.2), and therefore the overall survival (OS) is 100%. CONCLUSION: This report demonstrates the ability to run prospective pediatric studies in NRSTS in multiple European countries, despite the small numbers of ASPS patients. We can conclude that for the majority with small resected tumors, there were few events and no deaths.

The EpSSG NRSTS 2005 treatment protocol for desmoid-type fibromatosis in children: an international prospective case series.

BACKGROUND: In 2005, the European Pediatric Soft Tissue Sarcoma Study Group (EpSSG) proposed a conservative treatment algorithm-consisting of an initial wait-and-see strategy, non-mutilating surgery, and minimal-morbidity chemotherapy (in the case of tumour progression)-for paediatric patients with desmoid-type fibromatosis. We aimed to investigate the outcomes of this algorithm. METHODS: In this case series, patients (<25 years) with desmoid-type fibromatosis from 57 centres in eight countries were prospectively registered through a web-based system. Diagnosis was based on histological analysis of the tumour specimen after biopsy or surgery, and we classified patients by tumour site, clinical stage (TNM system), and post-surgical stage (Intergroup Rhabdomyosarcoma Study system). Progression-free survival was defined as the time from diagnosis until disease progression (clinical or radiological progressive disease, relapse, or death from any cause). FINDINGS: From Oct 1, 2005, to July 31, 2016, 173 patients (median age 11·4 years [IQR 4·0-14·1], 88 [51%] male patients) were registered. After excluding patients with missing data, 54 (35%) patients had no immediate therapy (wait-and-see strategy), 47 (31%) had immediate surgery, and 53 (34%) had immediate chemotherapy after diagnosis. 5-year progression-free survival was 36·5% (95% CI 27·8-45·2) overall, 26·7% (14·2-41·0) in the wait-and-see group, 41·2% (25·8-55·9) in the surgery group, and 42·8% (27·2-57·6) in the chemotherapy group (overall log-rank p=0·17; wait-and-see vs surgery p=0·12; wait-and-see vs chemotherapy p=0·13). In multivariable analysis, large tumour size (>5 cm) was associated with worse progression-free survival (hazard ratio 2·25, 95% CI 1·34-3·76; p=0·0021). Apart from one patient in the chemotherapy group who died from a secondary tumour (head and neck anaplastic embryonal rhabdomyosarcoma), all patients were alive at the time of analysis. 13 (8%) patients had biopsy only (no further treatment), 65 (42%) had chemotherapy only, 31 (20%) had surgery only, 36 (23%) had both chemotherapy and surgery, and nine (6%) had radiotherapy in addition to other therapies. INTERPRETATION: In paediatric patients with desmoid-type fibromatosis, the EpSSG conservative strategy did not compromise outcomes and could be adopted to reduce treatment burden. FUNDING: S Wisnia and la Città della Speranza Foundation.

Outcome of extracranial malignant rhabdoid tumours in children registered in the European Paediatric Soft Tissue Sarcoma Study Group Non-Rhabdomyosarcoma Soft Tissue Sarcoma 2005 Study-EpSSG NRSTS 2005.

BACKGROUND: Extracranial malignant rhabdoid tumours (MRT) are rare lethal childhood cancers that often occur in infants and have a characteristic genetic mutation in the SMARCB1 gene. The European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) conducted a multinational prospective study of registered cases of extracranial MRT to test an intensive multimodal approach of treatment for children with newly diagnosed extracranial MRT. METHODS: Between December 2005 and June 2014, we prospectively registered 100 patients from 12 countries with a diagnosis of MRT tumour at an extracranial site on the EpSSG Non-Rhabdomyosarcoma Soft Tissue Sarcoma 2005 Study (NRSTS 2005). They were all treated on a standard multimodal protocol of surgery, radiotherapy, and chemotherapy over 30 weeks as follows: vincristine, cyclophosphamide, and doxorubicin (VDCy) at weeks 1, 10, 13, 22, and 28; vincristine was also given alone on weeks 2, 3, 11, 12, 14, 15, 23, 24, 29, and 30. Cyclophosphamide, carboplatin, and etoposide (Cy*CE) was given at weeks 4, 7, 16, 19, and 25. Radiotherapy was recommended for all primary tumour sites and all sites of metastatic disease. RESULTS: Forty-three patients completed the protocol treatment. Median follow-up for alive patients of the complete cohort was 44.6 months (range 11.5-84.6). For the whole cohort, the 3-year event-free survival (EFS) was 32.3% (95% confidence interval [CI] 23.2-41.6%) with a 3-year overall survival (OS) of 38.4% (95% CI 28.8-47.9%). For localised disease, the 4-year EFS was 39.3% (95% CI 28.2-50.1%) with a 4-year OS of 40.1% (95% CI 28.4-51.5%). For metastatic disease, the 2-year EFS was 8.7% (95% CI 1.5-24.2%) with a 2-year OS of 13.0% (95% CI 3.3-29.7%). Multivariable analysis disclosed that all patients ≤1 year of age were associated with at higher risk of death (hazard ratio [HR]: 2.6; 95% CI 1.0-6.8; p-value = 0.0094). Risk of death was also related with gender in metastatic patients (HR for males: 2.9, 95% CI 1.0-8.0; p-value = 0.0077). CONCLUSIONS: The EpSSG NRSTS 2005 protocol of intensive therapy can be delivered to extracranial MRT patients, with a possible improvement in outcome. The outcome, however, remains poor for patients who progress or with metastatic disease.

Conservative strategy in infantile fibrosarcoma is possible: The European paediatric Soft tissue sarcoma Study Group experience.

BACKGROUND: Infantile fibrosarcoma (IFS) is a very rare disease occurring in young infants characterised by a high local aggressiveness but overall with a favourable survival. To try to reduce the total burden of therapy, the European pediatric Soft tissue sarcoma Study Group has developed conservative therapeutic recommendations according to initial resectability. MATERIAL AND METHODS: Between 2005 and 2012, children with localised IFS were prospectively registered. Initial surgery was suggested only if possible without mutilation. Patients with initial complete (IRS-group I/R0) or microscopic incomplete (group II/R1) resection had no further therapy. Patients with initial inoperable tumour (group III/R2) received first-line vincristine-actinomycin-D chemotherapy (VA). Delayed conservative surgery was planned after tumour reduction. Aggressive local therapy (mutilating surgery or external radiotherapy) was discouraged. RESULTS: A total of 50 infants (median age 1.4 months), were included in the study. ETV6-NTRK3 transcript was present in 87.2% of patients where investigation was performed. According to initial surgery, 11 patients were classified as group I, 8 as group II and 31 as group III. VA chemotherapy was first delivered to 25 children with IRS-III/R2 and one with IRS-II/R1 disease. Response rate to VA was 68.0%. Mutilating surgery was only performed in three cases. After a median follow-up of 4.7 years (range 1.9-9.0), 3-year event-free survival and overall survival were respectively 84.0% (95% confidence interval [CI] 70.5-91.7) and 94.0% (95% CI 82.5-98.0). CONCLUSIONS: Conservative therapy is possible in IFS as only three children required mutilating surgery, and alkylating or anthracycline based chemotherapy was avoided in 71.0% of patients needing chemotherapy. VA regimen should be first line therapy in order to reduce long term effects.

CASP8 SNP D302H (rs1045485) is associated with worse survival in MYCN-amplified neuroblastoma patients.

BACKGROUND: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis. PATIENTS AND METHODS: We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays. RESULTS AND CONCLUSION: We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors.

Identification of 2 putative critical segments of 17q gain in neuroblastoma through integrative genomics.

Partial gain of chromosome arm 17q is the most frequent genetic change in neuroblastoma (NB) and constitutes the strongest independent genetic factor for adverse prognosis. It is assumed that 1 or more genes on 17q contribute to NB pathogenesis by a gene dosage effect. In the present study, we applied chromosome 17 tiling path BAC arrays on a panel of 69 primary tumors and 28 NB cell lines in order to reduce the current smallest region of gain and facilitate identification of candidate dosage sensitive genes. In all tumors and cell lines with 17q gain, large distal segments were consistently present in extra copies and no interstitial gains were observed. In addition to these large regions of distal gain with breakpoints proximal to coordinate 44.3 Mb (17q21.32), smaller regions of gain (distal to coordinate 60 Mb at 17q24.1) were found superimposed on the larger region in a minority of cases. Positional gene enrichment analysis for 17q genes overexpressed in NB showed that dosage sensitive NB oncogenes are most likely located in the gained region immediately distal to the most distal breakpoint of the 2 breakpoint regions. Interestingly, comparison of gene expression profiles between primary tumors and normal fetal adrenal neuroblasts revealed 2 gene clusters on chromosome 17q that are overexpressed in NB, i.e. a region on 17q21.32 immediately distal to the most distal breakpoint (in cases with single regions of gain) and 17q24.1, a region coinciding with breakpoints leading to superimposed gain.

F-18 FDG PET in infectious diseases in children.

PURPOSE: The aim is to report our observations regarding the role of F-18 FDG PET in children's infectious processes. MATERIAL AND METHODS: A presentation is made of 3 neonatal infections unresponsive to treatment, 2 invasive infections in immunocompromised children, and 1 discitis in an immunocompetent child. PET or PET/CT was performed to address a specific question pertaining to the management of the diseases. Results were correlated with the clinical outcome. The impact on patient management is discussed. RESULTS: In 1 neonate, PET localized the infection in a bone which allowed surgical curettage. In another one, it localized the infection in recently renewed exogenous material and led to subsequent removal. It was negative in the third one, whose evolution was rapidly favorable. In the immunocompromised children, treatment of invasive infection was adapted according to the metabolic inflammatory activity of the disease. In a limping child with slight abnormalities on bone scintigraphy but major misleading involvement on MRI, PET/CT demonstrated hypermetabolism limited to a disc, thus avoiding further invasive procedures. CONCLUSIONS: Although not meant as a first choice examination, F-18 FDG PET should be considered in difficult cases of neonatal infection or in challenging diagnoses like discitis in the young child. It provides more accurate staging and treatment monitoring of the inflammatory process in invasive infections of immunocompromised children.

Contribution of NMR spectroscopy to the differential diagnosis of a recurrent cranial mass 7 years after irradiation for a pediatric ependymoma.

CASE REPORT: We describe the case of a 5-year-old-boy who underwent surgery and focal radiotherapy for an anaplastic ependymoma of the fourth ventricle. One year later, a spinal metastasis was treated the same way. Six years later, a 16-mm lesion was found on a control MRI in the posterior fossa. To help the differential diagnosis between a relapse, a radio-induced modification, and a new tumor, magnetic resonance spectroscopy was performed. The main findings were a peak at the expected resonance frequency of reduced glutathione, a prominent peak of glutamate/glutamine compounds, a low N-acetylaspartate, and the absence of elevated choline. These findings were suggestive of a meningioma, although the latency between irradiation and development of the lesion was quite short. The diagnosis was confirmed by the pathological examination. CONCLUSION: This case exemplifies the fact that magnetic resonance spectroscopy provides useful biochemical information in such a clinical setting.

Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay.

Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for time-consuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C(T) method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.