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Absence of epstein-barr and cytomegalovirus infection in neuroblastoma cells by standard detection methodologies

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Absence of epstein-barr and cytomegalovirus infection in neuroblastoma cells by standard detection methodologies
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  Pediatr Blood Cancer Acute Onset of Ovarian Dysfunction in Young Females After Start of Cancer Treatment Helena Mo ¨rse,  MD , 1 Maria Elfving,  MD ,  PhD , 2 Anna Lindgren,  MSc ,  PhD , 3 Pa˚l Wo ¨lner-Hanssen,  MD ,  PhD , 4 Claus Yding Andersen,  MSc ,  PhD , 5 and Ingrid Øra,  MD ,  PhD 1,6 *INTRODUCTION Several studies of late complications established years aftercancer treatment have shown that female childhood cancer survi-vors and women treated with high cumulative doses of alkylatingagents and/or pelvic radiotherapy are at risk of acute ovarianfailure and premature ovarian insufficiency (POI) [1–3]. Acuteovarian failure is classified as loss of ovarian function during orshortly after treatment with gonadotoxic agents, and POI is de-fined as menopause at the age of 40 years or younger [4]. Fur-thermore, reduced ovarian function is also associated with loss of bone mass and an increased risk of cardiovascular disease [5,6].Anti-Mu¨llerian hormone (AMH) is produced by the granulosacells of immature ovarian follicles before the preovulatory stage[7]. Compared to inhibin B, AMH is a better marker for ovarianreserve, because it acts independently throughout the menstrualcycle [8]. AMH is present in human females at birth, and levelsincrease during the first years of life, peaks at age 24.5 years, andis no longer detectable after menopause [9,10]. Hence, in contrastto inhibin B and follicle-stimulating hormone (FSH), AMH can beused to study ovarian reserve, even in very young and premenar-chal females.A limited number of long-term follow-up studies of femalesurvivors of childhood cancer have demonstrated that AMH is auseful marker for ovarian reserve and POI. In one investigation,14 young female cancer survivors had significantly reduced levelsof AMH compared to controls [11]. Others observed that among32 women treated for Hodgkin lymphoma during adolescence,those who had had more than six cycles of MOPP showed de-creased AMH [12]. Also, in a large study of 182 female childhoodcancer survivors, it was found that AMH levels were reduced inadult life in those who had had (1) Hodgkin lymphoma, (2)irradiation below the diaphragm, and/or (3) higher cumulativedoses of alkylating agents [13]. In another investigation, 32females who had undergone stem cell transplantation (SCT) dur-ing prepuberty were examined after the onset of puberty, and allwere found to have low levels of AMH as a sign of an attenuatedovarian reserve [14]. Similarly, data on 7/22 young females whohad been treated for brain tumors showed that low AMH levelspredicted ovarian failure [15]. Equivalent results have beenobtained in studies of female survivors of adult cancer [16–18].Four prospective studies have evaluated adult female cancerpatients regarding AMH and other ovarian markers during andafter treatment for breast cancer [19,20], lymphoma [21], or lym-phoma/breast cancer/Ewing sarcoma [22], and the results showeda marked reduction in AMH levels after start of treatment. Insome patient subgroups, AMH levels rose again after conclusionof treatment, although they never reached the initial levels or wereat lower levels compared to controls 1–3 years after cessation of treatment. The patients with lymphoma given high cumulative Background.  Female childhood cancer survivors are at risk of ovarian failure and premature ovarian insufficiency. We herebypresent an interim analysis of a prospective observational study of ovarian function during cancer treatment of young females in rela-tion to clinical factors.  Procedure.  Thirty-four consecutive femalecancer patients aged 0–18 year were included after informed con-sent. Serum/Plasma levels of anti-Mu¨llerian hormone (AMH), inhib-in B, FSH, LH, and oestradiol (E2) were measured at diagnosis andevery 3–4 months during and after treatment.  Results.  All patientshad detectable AMH levels at diagnosis. Eleven patients hadreached menarche (mean age 14½ years) and the remainingpatients had a mean age of 6½ years. They all showed a rapiddecline in AMH after 3 months of treatment, regardless of AMHat diagnosis, age, menarche, or treatment given. Those givenradiotherapy below the diaphragm and/or stem cell transplantation(SCT) (n  ¼  9) had no ovarian recovery during or 1½-year aftertreatment. However, recovery was observed in those given standardtreatment for acute lymphatic leukemia (n  ¼  7) already duringmaintenance chemotherapy. For the remaining patients, longer fol-low-up is required for analysis of ovarian recovery after treatment. Conclusions.  Rapid ovarian dysfunction is observed in all femalesafter initiation of cancer treatment as measured by AMH and inhib-in B. Our data regarding those who require abdominal radiotherapyand/or SCT confirms the recommendations in the Nordic countrieswhere these patients are eligible for cryopreservation of ovariancortical tissue before start of cancer treatment. Pediatr BloodCancer    2012 Wiley Periodicals, Inc. Key words:  anti-Mu¨llerian hormone; childhood cancer; female; prepuberty; puberty; ovarian reserve 1 Department of Pediatric Oncology and Hematology, Clinical Scien-ces, Lund University, Lund, Sweden;  2 Department of Pediatric Endo-crinology, Clinical Sciences, Lund University, Lund, Sweden;  3 Centrefor Mathematical Sciences, Lund University, Lund, Sweden;  4 Depart-ment of Reproductive Medicine, Clinical Sciences, Malmo¨ andCarema Specialist Care, Lund University, Lund, Sweden;  5 Laboratoryof Reproductive Biology, Rigshospitalet, University of Copenhagen,Copenhagen, Denmark;  6 Department of Oncogenomics, AcademicMedical Center, University of Amsterdam, Amsterdam, TheNetherlandsGrant sponsor: Swedish Childhood Cancer Foundation; Grant spon-sor: Ska˚ne University Hospital Donation Fund; Grant sponsor: DanishCancer Society; Grant number: DP05112/R2-A41-09-S2; Grant spon-sor: Danish Medical Research Council; Grant numbers: 271-07-0452,09-072265.Conflict of interest: Nothing to declare.The funders of the study had no role in study design, data collection,data analysis, data interpretation, or writing of the report. The corre-sponding author had full access to all the data in the study and hadfinal responsibility for the decision to submit for publication*Correspondence to: Ingrid Øra, MD, PhD, Department of PediatricOncology and Hematology, Clinical Sciences, Lund University,Getingeva¨gen, Lund 22185, Sweden. E-mail: ingrid.ora@med.lu.seReceived 8 May 2012; Accepted 22 August 2012  2012 Wiley Periodicals, Inc.DOI 10.1002/pbc.24327Published online in Wiley Online Library(wileyonlinelibrary.com).  doses of alkylating agents and those who received SCT developedPOI, which agrees with previous long-term follow-up studies.An increasing number of cancer treatment centers offer em-bryo- or oocyte cryopreservation to postpubertal females who arescheduled to receive highly gonadotoxic therapy that entails risk of ovarian failure. Preservation of gonadal tissue, particularlyprepubertal ovarian tissue, remains experimental [23]. Neverthe-less, during the last few years, autotransplantation of cryopre-served ovarian tissue has enabled female cancer survivors withchemotherapy-induced POI to give birth to 18 healthy babies[24,25].The first prospective study of ovarian response in 22 prepu-bertal and pubertal females during chemotherapy was very recent-ly published [26]. In this study the patients were grouped in low/ median or high risk based on cumulative dose of alkylating agentsand radiotherapy involving the ovaries. AMH decreased signifi-cantly during chemotherapy treatment in both prepubertal andpubertal patients with hormonal recovery in the low/median risk groups after completion of treatment. Here, we present the firstresults of a single-centre prospective investigation of ovarianmarkers in 34 young females during chemotherapy related toclinical features such as age, menarche, diagnosis, and treatment. PATIENTS AND METHODS Patients eligible for this study comprised females 0–18 yearsof age diagnosed with cancer or hematological disease at theDepartment of Pediatric Oncology and Hematology, Ska˚ne Uni-versity Hospital, Lund University, Sweden. Each subject wasrecruited after written informed consent from the patient herself and/or her parents.All participants had blood drawn from a central venous cathe-ter or a peripheral vein at diagnosis and approximately onceevery 3 months during and after treatment (up to 36 monthsafter end of treatment). The samples were centrifuged and batchedfor assessment of AMH, inhibin B, FSH, luteinizing hormone(LH), and 17 b -oestradiol (E2), and were stored at   80 8 C untilanalyzed.Serum levels of AMH and inhibin B were measured at theLaboratory of Reproductive Biology, Copenhagen, Denmark,using specific ELISA kits according to the instructions of themanufacturers. AMH was measured using DSL-10-14400 (Diag-nostic System Laboratories Inc., Webster, TX) and Gen II (Beck-man Coulter, Copenhagen, Denmark). The DSL AMH assaybecame unavailable during the project period, and thus it wasnot possible to apply the same assay to all samples. However,there was a strong significant linear correlation between theresults obtained using the two assays (Gen II assay  ¼  1,181DSL-assay  þ  0.067, n  ¼  66, r  ¼  0.98,  P  <  0.001). Inhibin Bwas measured using MCA1312KZZ (Oxford Bio-InnovationLtd., Oxfordshire, UK) with a detection limit of   < 20 pg/ml.Plasma/Serum levels of FSH (Roche 11775863) and LH(Roche 11732234) were measured at the Department of ClinicalChemistry, Ska˚ne University Hospital, Lund, Sweden, and thedetection limit was  < 0.2 IE/L in the LH assay. Plasma levels of E2 were measured at the Department of Clinical Chemistry,Ska˚ne University Hospital, Malmo¨, Sweden, and this assay hada detection limit of   < 10 pg/ml. Intraassay and interassay coeffi-cients of variation were less than 10%. All analyses were per-formed on thawed serum/plasma samples. The study wasapproved by the Regional Ethics Committee, Medical Faculty,Lund University, Sweden (approval no. 211/2006). Statistical Analysis All statistical analyses were performed using Statistical Pack-age for Social Sciences version 20 (SPPS Inc., Chicago, IL).Comparisons between AMH levels in different groups weredone using  t  -test. Change in AMH levels from diagnosis to after3 months of treatment was analyzed using paired samples  t  -test.Correlations between AMH levels and other continuous variableswere evaluated using Pearson’s correlation. All statistical testswere two-sided, and  P  <  0.05 was considered as statisticallysignificant. RESULTSPatient Characteristics A total of 51 consecutive patients were eligible for inclusion inthis interim analysis. Seventeen of those individuals were exclud-ed: seven who had brain tumors and treated only with surgerywere lost to follow-up; four who died shortly after diagnosis;three who declined participation; one who was on contraceptives;one who had ovarian cancer; and one with Down syndrome. Thus,a total of 34 patients were included in the current analysis.The clinical characteristics of the 34 patients are summarizedin Table I. The treatment regimen was selected according to thenational standard, which included alkylating agents for 27 out of all patients (79.4%). At the time of diagnosis, the youngest patientwas 4½ months and the oldest 16½ years, mean 9½ years. Elevenhad reached menarche (mean age 14½ years). One 14-year-oldgirl had pituitary insufficiency due to intracranial germinoma withhypogonadotropic hypogonadism. The remaining 22 patients hada mean age of 6½ years and menarche occurred in two of thosepatients during treatment and in an additional two during follow-up. We did not include clinical recovery of menarche into thisinterim analysis because of the large variance of follow-up intime.Distribution of the diagnoses was as follows (Table I): acutelymphatic leukemia (ALL) in eight patients, acute myeloblasticleukemia (AML) in three, Hodgkin lymphoma in four, Wilmstumor in three, Ewing sarcoma or osteosarcoma in seven, braintumors in four, and other diagnoses in the remaining five cases.In the present interim analysis, 32/34 patients were followedfor a minimum of 1-year after diagnosis. Seven patients were stillunder treatment and five had died of disease (Table I). The timeafter treatment varied between 0–36 months with a median of 5 months. Because of the variance in follow-up we focused on thehormonal status before and during treatment in the current analy-sis, except for the patients who received radiotherapy below thediaphragm and/or SCT with a median follow-up of 1½-year aftercompletion of treatment. Ovarian Hormones Before Treatment AMH was detectable in all patients at diagnosis. Before treat-ment, AMH was positively correlated with age ( P  ¼  0.018;Fig. 1A), inhibin B ( P  <  0.001), and LH ( P  ¼  0.005) but notto FSH, E2, or menarche at diagnosis. However, when the patientswith menarche were excluded there was no correlation between 2 Mo ¨rse et al. Pediatr Blood Cancer   DOI 10.1002/pbc  TABLE I. Patients’ Characteristics N Age year Menarche DiagnosisRT belowdiaphragm SCT Alkylating agentsMonths afterTRM/state1 2.3 No ALL No No No TRM2 2.7 No ALL No No Cyclophosphamide 3 g/m 2 63 3.3 No ALL No No Cyclophosphamide 2 g/m 2 114 4.7 No ALL No No No TRM5 8.3 No ALL No No Cyclophosphamide 2 g/m 2 TRM6 10.3 No ALL No No Cyclophosphamide 2 g/m 2 37 10.2 No ALL No No Cyclophosphamide 2 g/m 2 TRM8 14.9 Yes ALL 12 Gy TBI Yes Cyclophosphamide 4.4 g/m 2 179 0.4 No AML No No No 1810 8.6 No AML No No No 111 11.9 No AML No No No TRM12 13.1 Yes Hodgkin lymphoma 19.8 Gy No Cyclophosphamide 4 g/m 2 30Procarbazine 9 g/m 2 13 15.8 Yes Hodgkin lymphoma No No Cyclophosphamide 2 g/m 2 5Procarbazine 3 g/m 2 14 15.8 Yes Hodgkin lymphoma No No Cyclophosphamide 2 g/m 2 10Dacarbazine 1.5 g/m 2 15 16.8 Yes Hodgkin lymphoma 19.8 Gy No Cyclophosphamide 2 g/m 2 11Procarbazine 6 g/m 2 16 1.6 No Wilms tumor No No No 617 6.4 No Wilms tumor 21 Gy No No 1918 11.4 Yes Wilms tumor 25 Gy Yes Ifosfamide 27 g/m 2 19 DODCyclophosphamide 2.7 g/m 2 Carboplatin 3.6 g/m 2 Temozolomide 570 mg/m 2 Melphalan 120 mg/m 2 Thiotepa 10 mg/m 2 19 5.9 No Ewing sarcoma No No Ifosfamide 102 g/m 2 820 8.1 No Ewing sarcoma 54 Gy No Ifosfamide 102 g/m 2 1921 13.3 Yes Ewing sarcoma No No Ifosfamide 102 g/m 2 TRM22 13.4 No Ewing sarcoma No Yes Ifosfamide 60 g/m 2 8Melphalan 140 mg/m 2 Busulfan 12.8 mg/kg23 11 No Osteosarcoma No No Cisplatin 480 mg/m 2 324 14.2 Yes Osteosarcoma No No Cisplatin 480 mg/m 2 225 14.5 Yes Osteosarcoma No No Cisplatin 360 mg/m 2 15Ifosfamide 60 g/m 2 32 6.2 No Low-grade glioma No No Cyclophosphamide 4,5 g/m 2 TRMCarboplatin 4.4 g/m 2 Cisplatin 300 mg/m 2 33 14.2 No CNS germinoma No No Ifosfamide 18 g/m 2 36Carboplatin 1.2 g/m 2 30 3.6 No Medulloblastoma No No Cyclophosphamide 1.2 g/m 2 TRMCarboplatin 3 g/m 2 31 5.5 No Supratentorial PNET 23.4 Gy spinal No Cyklophosphamide 4.8 g/m 2 DODCisplatin 280 mg/m 2 Carboplatin 1.2 g/m 2 CCNU 300 mg/m 2 27 15.9 Yes Rhabdomyosarcoma No No Ifosfamide 36 g/m 2 DODCarboplatin 2.4 g/m 2 28 5 No Rhabdoid tumor No No Ifosfamide 33 g/m 2 DODCyclophosphamide 11.9 g/m 2 Carboplatin 1.2 g/m 2 29 14.3 Yes Midline carcinoma No No Cisplatin 500 mg/m 2 DOD26 3.5 No Neuroblastoma No No Cyclophosphamide 4.2 g/m 2 23Carboplatin 1.8 g/m 2 34 11 No Fanconi anemia No Yes Cyclophosphamide 1.2 g/m 2 36RT, radiotherapy; SCT, stem cell transplantation; ALL, acute lymphatic leukemia; AML, acute myeloid leukemia; CNS, central nervoussystem; PNET, primitive neuroectodermal tumor; TRM, under treatment; DOD, death of disease; Gy, gray; TBI, total body irradiation; CCNU,lomustine. Ovarian Response to Pediatric Cancer Therapy 3 Pediatr Blood Cancer   DOI 10.1002/pbc  AMH levels and age. Inhibin B at diagnosis was detectable in47% of the cases and correlated with age ( P  <  0.001) and men-arche ( P  ¼  0.001) at diagnosis as illustrated in Figure 1B, andeven to LH ( P  <  0.001), FSH ( P  ¼  0.003), and E2 ( P  <  0.001).LH was detectable in 44% of the patients at diagnosis, while FSHwas detected in all and E2 in all but two. Ovarian Hormones After 3 Months of Treatment All patients showed significantly decreased levels of AMH( P  <  0.001) and inhibin B ( P  ¼  0.045) 3 months after initiationof chemotherapy (Fig. 2, time point B). AMH was below thedetection limit in 22 patients (65%). For all patients, AMH after3 months did not depend on the AMH level ( P  ¼  0.142), age( P  ¼  0.220), or menarche ( P  ¼  0.843) recorded at the time of diagnosis (Fig. 1), nor diagnosis or type or dose of chemothera-peutic agents given.After 3 months of treatment inhibin B was below detectionlimit in 29 patients (85%) and the decrease was independent of the inhibin B level at diagnosis as illustrated in Figure 1B. LHincreased significantly ( P  ¼  0.045) as did FSH ( P  ¼  0.015), andfor LH the increase correlated with the LH value at diagnosis( P  ¼  0.019). The E2 levels showed a large variance in the totalgroup of patients and no significant change occurred during thefirst 3 months of treatment.The 12 patients with measurable AMH after 3 months includ-ed four of the eight who had ALL, and they were all older than8 years. Although not significant, the AMH levels at diagnosiswere higher in the four older than in the four younger ALLpatients (mean 1.70 and 0.41 ng/ml respectively,  P  ¼  0.145). 01530456001234  A B C D E F G H I J    i  n   h   i   b   i  n   B  p  g   /  m   L   A   M   H  n  g   /  m   L 040801201600255075100  A B C D E F G H I J    E   2  p  m  o   l   /   L   F   S   H  a  n   d   L   H   I   U   /   L FSH, LH, and E2 over time (A-J) AMH and inhibin B over time (A-J) 34 33 33 32 21 18 13 11 10 7 Fig. 2.  Mean levels of AMH (black), inhibin B (blue), LH (orange), FSH (red), and E2 (green) in 34 premenarchal and postmenarchal femalepatients before start of cancer treatment (time point A) and during and after treatment at intervals of 3–4 months (time points B–J). The numberof samples for each time point is shown in italic under the X-axis on the left panel. The error bars show standard deviation (SD; omitted for E2due to large variation). The lines in the upper left corner on the left panel illustrate the individual duration of treatment (dotted lines depictsmaintenance treatment). Fig. 1.  A : Levels of AMH in 34 young females before cancer treatment in relation to age at diagnosis and whether they had (red dots) or hadnot (black dots) reached menarche. The levels of AMH were significantly correlated with age ( P  ¼  0.018) but not with menarche. The filledand open dots represent cases that, respectively, did and did not have detectable AMH after 3 months of cancer treatment. The level of AMHafter 3 months was not correlated with age, menarche, AMH before diagnosis, or cancer treatment given.  B : Levels of inhibin B in 34 youngfemales before cancer treatment in relation to age at diagnosis and whether they had (red dots) or had not (black dots) reached menarche. Thefilled and open dots represent cases that, respectively, did and did not have detectable inhibin B after 3 months of cancer treatment. 4 Mo ¨rse et al. Pediatr Blood Cancer   DOI 10.1002/pbc  Two patients with Hodgkin lymphoma had detectable AMH levelsthat were not associated with other clinical parameters, which wasalso true for the remaining six patients. Ovarian Hormones During/After Treatment Levels of the ovarian hormones during treatment differed con-siderably between the patients treated with irradiation below thediaphragm and/or stem cell transplantation (n  ¼  9; Fig. 3A) andthose given ALL treatment (i.e., without SCT, n  ¼  7; Fig. 3B).Radiotherapy affecting the gonads led to depletion of AMH fol-lowed by depletion of inhibin B, whereas it was associated withincreased levels of FSH, LH, and E2 during the observation time(Fig. 3A). The nine irradiated/stem cell transplanted patients werefollowed between 5 and 36 months after treatment (median  ¼  18months) and could therefore be long-term evaluated. After amedian of 1½-year after completion of treatment no ovarian re-covery was seen (Fig. 3A). The two patients with SCT withoutradiotherapy had a conditioning regime including cyclophospha-mide/fludarabine or busulfan/melphalan and they have not recov-ered hormonally 36 and 8 months respectively after completion of treatment.The patients with ALL also showed a significant decline inAMH after 3 months but the hormonal status improved duringmaintenance therapy (low-dose chemotherapy), as shown inFigure 3B. There was a tendency for higher levels of AMH andinhibin B after treatment than at diagnosis in those who hadcompleted their 2½ years of treatment (Fig. 3B). The levels of FSH, LH, and E2 remained stable throughout the treatment in thepatients with ALL (Fig. 3B right panel). DISCUSSION The present study of young female cancer patients before andshortly after onset of puberty demonstrated the occurrence of anacute reduction of AMH and inhibin B as early as 3 months afterstart of chemotherapy, and the results from this interim analysisconfirm a recent published study similar to ours from Broughamet al. [26]. All patients received a multimodality treatment com-prising two to six different drugs during the first 3 months of therapy. Interestingly, the degree of hormonal decline was notpredicted by the dose and type of chemotherapy during the initialmonths of treatment, or by the patients’ age or occurrence of menarche. Regarding the largest group of patients, those withALL, there was a trend towards higher AMH levels at diagnosisand detectable levels after 3 months of treatment in females8 years or older compared to the lower levels in the youngerpatients.The uniform decrease of AMH and inhibin B after 3 months of treatment, independent of gonadotoxic treatment or not, does notpredict whether ovarian function will be affected in the future.Whether an early decrease reflects a permanent reduction of the 040801201600255075100  A B C D E F G H I J    E   2  p  m  o   l   /   L   F   S   H  a  n   d   L   H   I   U   /   L 01530456001234  A B C D E F G H I J    i  n   h   i   b   i  n   B  p  g   /  m   L   A   M   H  n  g   /  m   L AB  AMH and inhibin B over time (A-J) FSH, LH, and E2 over time (A-J) 01530456001234  A B C D E F G H I    i  n   h   i   b   i  n   B  p  g   /  m   L   A   M   H  n  g   /  m   L 9 9 9 9 9 8 6 6 4 7 7 7 7 5 5 4 4 4 4 040801201600255075100  A B C D E F G H I    E   2  p  m  o   l   /   L   F   S   H  a  n   d   L   H   I   U   /   L Fig. 3.  Mean levels of AMH (black), inhibin B (blue), LH (orange), FSH (red), and E2 (green) before start of cancer treatment (time point A)and during and after treatment in 3–4-month intervals (time points B–J). Error bars indicate SD. The number of samples for each time point isshown in italic under the X-axis on the left panels. The lines in the upper corner on the left panels illustrate the individual duration of treatment(dotted lines depicts maintenance treatment where small dots depicts oral low dose chemotherapy).  A : Levels of the indicated hormones in ninefemales before start and during and after cancer treatment including irradiation below the diaphragm and/or stem cell transplantation.  B : Levelsof the hormones over time in seven females who received relatively homogeneous treatment for acute lymphatic leukemia (ALL). Maintenancetreatment with low-dose chemotherapy was initiated in all patients between time points C and D. Ovarian Response to Pediatric Cancer Therapy 5 Pediatr Blood Cancer   DOI 10.1002/pbc
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