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Anti-cancer treatment in children can deteriorate gonadal function and affect future fertility. We analyzed the hormonal markers of gonadal function in adolescent leukemia survivors, treated in childhood with different levels of aggressiveness. We analyzed hormone levels in 69 adolescents and young adults, leukemia survivors stratified into standard (SR), intermediate (IR), and high (HR) risk groups, and in 80 healthy controls (38 men) at a similar age. We assessed follicular stimulating hormone (FSH), luteinizing hormone (LH), and inhibin B in the whole group, testosterone in males, and E2 and anti-Mullerian hormone (AMH) in females. Males classified into HR group presented, in comparison to control, higher levels of FSH, LH, lower inhibin B, and normal testosterone, whereas in SR and IR group, the hormonal values were comparable to the control. In females, in all risk groups, the levels of FSH, LH, E2, and inhibin B were comparable with the control, but the mean AMH levels were slightly lowered. We did not observe the effect of prophylactic cranial irradiation (12 or 18 Gy) or the time of treatment (before vs. during puberty) on hormone levels. In females, a positive correlation was found between the time interval after the end of treatment and AMH levels. Male leukemia survivors having undergone more intensive chemotherapy show the symptoms of disturbed spermatogenesis and need to be followed-up in the future. Women, irrespective of the risk group, can develop the signs of preterm ovarian insufficiency. They should be informed about the impact of the treatment on gonadal function.

Anticancer treatment can disturb gonadal function and deplete the primordial follicle pool, leading to premature menopause. We made a prospective analysis of serum hormone levels in young female cancer survivors who had been treated during childhood and adolescence. Serum anti-Müllerian hormone (AMH) as a marker of ovarian reserve, FSH, LH, and estradiol were measured in 33 women treated previously (6-11 years earlier) for Hodgkin Lymphoma, solid tumours, and after bone marrow transplantation, and in 34 healthy controls. The group of survivors was divided according to the risk of gonadotoxicity into the low risk and median risk group (LR+MR), and into the high risk (HR) group. The measurements were repeated after 5 years. In the HR group, AMH levels were significantly lower than in controls (p=0.001) and in the LR+MR group (p=0.006) at the time of the first examination fell progressively after 5 years (p=0.03), whereas elevated FSH values (p=0.053) increased (p=0.001). Unchanged LH values in the first measurement rose in the second one (p=0.001). In the LR+MR group, the levels of AMH and FSH were normal (compared to the control) at baseline, but after 5 years serum AMH decreased (p=0.027) and FSH increased (p=0.008). Our findings indicate that anticancer treatment during childhood and adolescence is associated with a serious, progressive risk of ovarian failure. It is necessary to inform female cancer survivors, especially the high risk patients, about the risk of premature menopause.

Children with severe aplastic anemia (AA) require multiple transfusions of the red blood cells during the immunosuppressive therapy. This leads to iron overload and manifests as elevated levels of ferritin in blood. The aim of this study was a retrospective analysis of the influence of the elevated serum ferritin on the overall survival, event-free survival, the risk of relapse, and response to treatment in children with AA during immunosuppressive therapy. We analyzed 38 children with AA (19 girls, 19 boys, aged 2–17 years) treated according to the obligatory protocol for AA in Poland. The response rate was assessed on days 84, 112, and 360. Patients were divided into three groups: group I consisted of children with ferritin below 285 ng/mL (6 children), group II with ferritin between 286 and 1,000 ng/mL (13 children), and group III ferritin >1,000 ng/mL (19 children). Kaplan-Meier plot was used to estimate the overall survival and event-free survival. We found the overall survival did not differ between the three groups. Event-free survival was significantly shorter (p = 0.03) in patients with ferritin levels >1,000 ng/mL compared with the groups with ferritin bellow 1,000 ng/mL. The time to relapse was significantly shorter in group III than in the other two groups (p = 0.02). We also found the differences in the treatment response at day 84 (p = 0.03) and day 112 (p < 0.0001) of immunosuppressive therapy. These findings confirm a negative influence of iron overload in children with AA on the effect of treatment and the risk of relapse.

Immunosuppressive therapy is the treatment of choice in children with acquired severe aplastic anemia (AA) and no HLA-matched family donor. The paper presents results of a multicenter study of 63 children with AA treated with rabbit antithymocyte globulin (r-ATG) and cyclosporine A as the first line treatment in the years 1996–2012. Therapeutic effects were evaluated at Days 112, 180, and 360. At Day 112, remission was achieved in 28 out of the 63 patients (44.4 %), complete remission in 10 patients (15.9 %), and partial remission in 18 (28.5 %). At Day 180, 31 patients (49.2 %) were in remission including 15 cases in complete (23.8 %), and 16 cases in partial remission (25.4 %). One year after therapy onset, 34 patients (64.9 %) were in remission including 24 patients (38.0 %) in complete and 10 (15.9 %) in partial remission. Relapse occurred in 4 patients, from 8 months up to 2 years and 2 months after remission. One child, 5 years after remission, was diagnosed with paroxysmal nocturnal hemoglobinuria. The estimated 10-year overall survival rate and 10-year event-free survival rate were 67 % and 57 %, respectively.

We evaluated ovarian function by measuring the levels of anti-Müllerian hormone (AMH), estradiol, and gonadotropins in 83 young women treated for cancer during childhood and adolescence, and classified according to post-treatment gonadal toxicity versus 38 healthy females.Results. The mean AMH values were lower in the entire cohort independently of the risk group as compared to the control, whereas FSH was elevated only in the high risk group. The lowest AMH values were noted in patients after bone marrow transplantation (BMT) and those treated for Hodgkin lymphoma (HL). Nineteen patients (22.9%) had elevated FSH. They all had low AMH values. Lowered AMH values (but with normal FSH and LH) were observed in 43 patients (51.8%). There was no effect of age at the time of treatment (before puberty, during or after puberty) on AMH levels.Conclusion. Our results show the utility of AMH measurement as a sensitive marker of a reduced ovarian reserve in young cancer survivors. Patients after BMT and patients treated for HL, independently of age at treatment (prepuberty or puberty), are at the highest risk of gonadal damage and early menopause.

The proteasome chymotrypsin-like (Cht-L) activity was determined in plasma of children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) in correlation to baseline leukocytosis, immunophenotype, LDH at the time of diagnosis and after remission induction.The activity was assayed using the fluorogenic peptide substrate in the presence of an artificial activator sodium dodecyl sulfate (SDS) in the plasma of healthy donors (n=25), acute lymphoblastic leukemia, ALL (n=95) and AML (n=17) patients.As compared to healthy subjects, the plasma proteasome ChT-L activity was significantly increased (p=0.001) in ALL patients prior to treatment, especially in those with T-ALL immunophenotype and with high LDH activity. Similarly, in AML patients the plasma proteasome ChT-L activity was elevated (p=0.001). Following remission-inducing chemotherapy, the activity of the ChT-L proteasome was significantly reduced both in ALL and AML patients.Plasma proteasome ChT-L activity may serve as an additional marker in monitoring anticancer therapy.

Introduction Acquired aplastic anemia is very rare disease characterized by and pancytopenia and hypoplastic bone marrow. In 70% of cases the etiology is idiopathic but some cases can be caused by drugs, chemical substances and viruses. One of the main pathophysiological mechanism of aplastic anemia is immune-mediated destruction of hematopoietic cells. As a result of that the immunosuppressive therapy (IST) persists the treatment of choice in patients with severe acquired aplastic anemia (SAA), who don’t have matched sibling donor. Since few last years there is a discussion if it is still the best way of treatment. We wanted to show the side effects of IST during the therapy of SAA using the rabbit antithymocyte globulin(r-ATG) Methods The group of 55 children ( 32 boys, 23 girls) with SAA and vSAA (very severe aplastic anemia) treated with r-ATG , steroids, CSA, and/or GSF-CS was retrospectively analyzed. The median age of the children was 10 years (range 6mth-17,5 years). Response rate was assessed on days 84, 112,180, 360. Kaplan–Meier estimator, univariate analysis and multivariate Cox regression models were used to estimate the influence of side effects for PFS (progression free survival) and EFS( event free survival). Results Infectious complications (fever of unknown origin-FUO-26,92%, stomatitis -19,23% and sepsis -17,03% ) were the most common side effects of IST. The most common etiology were : E. coli, P. aureginosa, S. aureus, Kl. pneumonie, E. fecium, E. cloace. There was a significant association between EFS and infectious side effects (p=0,00093- log rank). Among side effects of r-ATG fever (30,30%) and dyspnoe (24,24%) were the most common.After CSA mainly gingival hyperplasia, hirsutysm and hypertension were observed. The most common hemorrhagic side effects of IST were skin petechiae. The other were central nervous system bleeding, gastrointestinal tract bleeding. There was significant correlation between the hemorrhagic side effects and PFS(p= 0,0144 log rank).After the G-CSF therapy muscular pains, and capillary leak syndrome, was observed but it didn’t influence survival. Among all patients 15 deaths was observed (27,27%)-5 early and 10 late deaths. Infections were the main causes of death. The relapse rate was 1,81%. The 2-year probability of not having relapse was 87,68%. Relapse free survival was 75,16% at 5 and 10 years. There was one transformation to paroxysmal nocturnal hemoglobinuria (PNH). There were no transformation to MDS or AML.Discussion. Infections are the most common side effects during the IST with r- ATG. Hemorrhagic side effects and infectious complications are very important factors which can affect the survival rate in children with SAA. Disclosures: Szczepanski: Octapharma AG: Investigator Other.

Abstract 4375 Antithymocyte horse globulin (hATG) or rabbit (rATG) is used to treat children with severe aplastic anemia (SAA) in the absence of a compatible bone marrow family donor’s. We presenting (viewing) retrospective evaluation of the effects of SAA therapy using hATG and rATG. 49 children (20 girls and 29 boys, aged 1.2–17) were treated between 1993 and 2000 - hATG (15mg/kg/day for 5 days with cyclosporine 5mg/kg/day for 12–24 mths). On day 180 day of treatment there was a complete response (CR) in 22 of 49 children (44.5%) and a partial response (PR) in 12 children (24.4%). 34 of 49 children (69,4%) responded to treatment. There was no remission (NR) in 15 of 49 children (30.6%). In the whole group there were 7 deaths during the first 6 months. There were no relapses within 5 years of observation. The 5-year probability of survival in this group was 84%. 55 children (22 girls and 33 boys, aged 3,5–17,7 years) were treated in the years 1996–2009 using rATG (3.75 mg/kg/day for 5 days and cyclosporine 5mg/kg/day for 12–24 mths). On day 180 of treatment 5 patients (9%) achieved a CR, 23 children (41.8%) had a PR. 28 of 55 children (50.9%) responded to treatment. NR was found in 27 children (49%).In the rATG group 15 deaths (27%) were recorded, 4 patients (7%) had relapses. In one child paroxysmal nocturnal hemoglobinuria was noted.The 5-year survival in the rATG.group was 63.54%. Our results show the greater effectiveness of treatment with horse globulin than rabbit globulin in terms of hematological response and overall survival. Disclosures: No relevant conflicts of interest to declare.

Abstract 4220 Bone marrow transplantation (BMT) is a therapy of choice in children with severe aplastic anemia (SAA), but it is possible only in about 15-20% patients who have family donors, so the majority of them received the immunosuppressive therapy (IST). We want to present the results of IST obtained in 116 children treated between 1993-2008 years at 11 pediatric hematology centers of the Polish Pediatric Hematology Group (PPHG),, 55 patients (22 girls and 33 boys aged 0,6-17,5 years) received antithymocyte rabbit globulin (ATG) as a first course of therapy. ATG was administered according to PPHG and Working Party SAA Group of EBMT protocol: ATG 3.75mg/kg iv for 5 days, cyclosporin A (CSA) 5mg/kg orally from day 1 to day 180 and granulocyte-stimulating factor during deep neutropenia. Remission of the disease was assessed on days 112, 180 and 360 from start of therapy on the basis of blood and bone marrow examination. The results on 112 and 180 were similar. The remission was achieved in 28 of 55 children (51%), complete remission (CR) in 5 children (9%), and partial remission (PR) in 23 children (42%). On day 360 remission was obtained in 30 of 55 patients (42 %), CR in 10 children (18%) and PR in 20 children (36%) There was no response to treatment (NR) in 25 patients (45%). In the group of 55 patients, 15 died (27%). 5 of them died early (day 52 and day 72 of therapy) due to septicemia and central nervous system (CNS) hemorrhage. The other 10 died late (day 99 and day 360 of therapy) due to CNS hemorrhage. Observation time of patients ranged from 1 to 14 years. During this time we noted four relapse. PNH was observed in one patient. The probability of 14 year survival in our group of patients treated initially with rabbit ATG is 72,7%. The results in the last follow up (Jun 2009) are better then earlier. We observed CR in 23/55 children (42%), PR in 10/55 (18%), NR 18/55 (33%). o information about 4 patients. We conclude that initial treatment with rabbit ATG is safe and effective in children. Further studies are needed, to assess long-term effectiveness of rabbit ATG in IST. Disclosures: No relevant conflicts of interest to declare.

Abstract 3435 Objective: One of the main patophysiological mechanism of aplastic anemia (AA) is immune-mediated destruction of hematopoietic cells. In children with severe acquired aplastic anemia (SAA) allogenic bone marrow transplantation is the first choice. If there is no matched sibling donor the first line of treatment is immunosuppressive therapy (IST) which consists of ATG combined with cyclosporine A. Aim. The aim of this study was to analyze the results of IST containing rabbit anti-thymocyte globulin (r-ATG) performed on children with SAA and vSAA treated between 1996–2009 yr. We wanted to assess the influence of such factors as response to treatment, etiology, sex, age, type of AA, for: event free survival (EFS), and progression free survival (PFS) We analyzed deaths rate, relapses rate, clonality and side effects of IST. Materials and methods. We retrospectively analyzed group of 55 children with SAA and vSAA. The group consisted of 23 girls and 32 boys, the median age was 10 years (range 6mth-17,5 years). IST contained antithymocyte globulin,given intravenous at a dose of 3,75 mg/kg/day for 5 days, and cyclosporine A(CSA) at 5 mg/kg/day by mouth in divided doses started on day 1 and continued for at least 6 months(till 24 months). Response rate was assessed on 112, 180 and 360 day and defined as CR, PR, NR. Kaplan–Meier estimator, univariate analysis and multivariate Cox regression models were used to estimate the influence of such factors as response to treatment, etiology, sex, age for EFS, PFS, toxicity and other side effects. Results. The 2- year probability of event free survival (EFS) was 71,52%. The 5 and 10 –year EFS was 57,7%. Progression free survival (PFS) was 66,53%, at 2 years and 59,13% at 5- and 10 year Patients with CR and PR had significantly longer EFS than children with no response(NR) (p 0,05 Log rank). In all group we observed 15 deaths (27,27%), 5 early and 10 late deaths. Infections were the main causes of death. The relapse rate was 1,81%. The 2-year probability of not having relapse was 87,68%. Relapse free survival was 75,16% at 5 and 10 years. There was one transformation to paroxysmal nocturnal hemoglobinuria (PNH).Non responders (4 patients) received second IST and 11 children underwent MUD-BMT. Infections were the most often side effects of IST (FUO-26,92% of infectious side effects). There was significant association found between EFS and infectious side effects. (p=0,00093 log rank) The most common adverse effects after rabbit immunoglobulin were fever (30,30%) and dyspnoe (24,24%). After CSA treatment we observed mainly gingival hyperplasia (29,16% of side effects of CSA). Most common hemorrhagic side effects were skin petechie. There was significant correlation between hemorrhagic side effects and PFS (p= 0,0144 log rank) Conclusions. 1. Rabbit-ATG is an effective frontline therapy for children with SAA without familiar bone marrow donor.2.Remission achieved on day 112 seems to be constant. 3.5-year EFS seems to be stable within next 5 years.4.5-year relapse free survival seems to be constant during the next 5 years.5.Etiology, type of SAA, age and sex have no influence on the survival.)6.Infectious and hemorrhagic side effects are a very important factors affecting the survival rate in children with SAA. Disclosures: No relevant conflicts of interest to declare.

In children with severe aplastic anemia (SAA) bone marrow transplantation is the chosen therapy. However, it is only possible in about 15–20% patients who have family donors. Immunosuppressive therapy (IST) is administered in the remaining cases. 105 children in 11 hematology centers of the Polish Pediatric Hematology Group (PPHG) were given IST treatment. 27 patients (12 girls and 15 boys aged 6–16 years) received antithymocyte rabbit globulin (ATG) in the first course of therapy. ATG was administered according to PPHG and Working Party SAA Group of EBMT protocol: ATG 3.75mg/kg iv for 5 days, cyclosporin A (CSA) 5mg/kg orally from day 1 to day 180 and granulocyte-stimulating factor during deep neutropenia. Remission of the disease was assessed on days 84, 112 and 180 from start of therapy on the basis of blood and bone marrow examination. On day 180 remission was obtained in 17 of 27 patients (62.9%), complete remission (CR) in 11 children (40.7%) and partial remission in 6 children (22.2%) There was no response to treatment (NR) in 10 patients (37%). In the no response group, 1 child died and 4 had bone marrow transplants from unrelated donors. The remaining 5 patients received a second course of IST therapy, and 2 of them had CR. In the group of 27 patients, 4 died (14.8%). 2 of them died early (day 52 and day 72 of therapy) due to septicemia and central nervous system (CNS) hemorrhage. The other 2 died late (day 99 and day 365of therapy) due to CNS hemorrhage. Observation time of patients ranged from 1 to 13 years. During this time we noted one relapse. MDS/AML or PNH was not observed in any of the patients. The probability of 13 year survival is 85.1% in our group. Our data suggest that initial SAA treatment with ATG is safe and effective in children. Further studies are needed, however, to assess long-term effectiveness of ATG in IST.

Introduction: Bone marrow transplantation (BMT) from HLA identical family donors is the treatment chosen, according indication of Polish Pediatric Hematology Group, for children with severe aplastic anemia (SAA). When no donor is available, combined immunosuppressive therapy (IST) is given. Material and method: SAA was recognized in 105 children (42 girls, 73 boys) aged 2–18 years in eleven pediatric hematological center in Poland between 1993–2006 years. All patients received IST according Polish Pediatric Hematology and Working Party SAA of the EBMT protocol: antithymocyte globulin horse or rabbit, cyclosporine A (CSA), prenisolon and in 79 patients G-CSF or GM-CSF was additionally administered. Hematological response was evaluated on day 84 and 180 of therapy. Results and conclusion: On day 180 remission was obtained in 80 from 105 patients (76.1%), complete remission (CR) in 53 patients (51.5%), partial remission (PR) in 27 (24.7%), There was no response to treatment (NR) in 25 patients (23.8%). Period of observation was from 10 months to 13 years. During this time relapse occurred in 28 patients (25.7%). Patients with NR and relapse were treated second course of IST or BMT from matched bone marrow donors. Unrelated BMT was performed in 15 patients with NR after IST. We observed 22 deaths (8 early during the first 3 months of IST) in all group. During the 13 year of observation we notice one case with late clonally complication (PNH), one case of transformation to AML and one to MDS. Probably of 13 year survival was 75.2% in our group. Our results are effective and compared to other hematological centers.