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Background:Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases.Methods:We examined the genomic landscape of a patient cohort of LMs (n = 30 cases) that underwent comprehensive genomic profiling using a large-panel next-generation sequencing assay. Immunohistochemical analyses were completed in parallel.Results:These LMs had low mutational burden with hotspot PIK3CA mutations (n = 20) and NRAS (n = 5) mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal LM harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable radiologic complete response, as defined in RECIST1.1, to the PI3Kα inhibitor alpelisib within the context of a personalized N-of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive to alpelisib treatment in vitro, which was demonstrated by a concentration-dependent drop in measurable impedance, an assessment of cell status.Conclusions:Our findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations.Funding:R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953.Clinical trial number:NCT03941782

BackgroundLymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases.MethodsWe examined the genomic landscape of a patient cohort of LMs (n=30 cases) that underwent comprehensive genomic profiling (CGP) using a large-panel next generation sequencing (NGS) assay. Immunohistochemical analyses were completed in parallel.ResultsThese LMs had low mutational burden with hotspot PIK3CA mutations and NRAS mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal lymphatic malformation harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable complete response to the PI3Kα inhibitor alpelisib within the context of a personalized N-of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive in vitro to alpelisib in a concentration-dependent manner.ConclusionsOur findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations.FundingR.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953.Clinical trial numberNCT03941782

Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases.We examined the genomic landscape of a patient cohort of LMs (These LMs had low mutational burden with hotspotOur findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations.R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953.NCT03941782.

Background This study evaluated the feasibility of a technology-enhanced group-based fitness intervention for adolescent and young adult (AYA) survivors of childhood cancer. Procedure AYA survivors ages 13-25 years were randomized to the intervention (eight in-person group sessions with mobile app and FitBit followed by 4 weeks of app and FitBit only) or waitlist control. Assessments were at 0, 2, 3, 6, and 9 months. Feasibility was evaluated by enrollment, retention, attendance, app engagement, and satisfaction. Secondary outcomes included physical activity, muscular strength/endurance, cardiorespiratory fitness, health-related quality of life, and fatigue. Results A total of 354 survivors were mailed participation letters; 68 (19%) were screened, of which 56 were eligible and 49 enrolled (88% of those screened eligible, 14% of total potentially eligible). Forty-nine survivors (Mage = 18.5 years, 49% female) completed baseline assessments and were randomized (25 intervention, 24 waitlist). Thirty-seven (76%) completed the postintervention assessment and 32 (65%) completed the final assessment. On average, participants attended 5.7 of eight sessions (range 1-8). Overall intervention satisfaction was high (M = 4.3, SD = 0.58 on 1-5 scale). Satisfaction with the companion app was moderately high (M = 3.4, SD = 0.97). The intervention group demonstrated significantly greater improvement in lower body muscle strength compared to the waitlist postintervention, and small but not statistically significant changes in other secondary measures. Conclusions A group-based intervention with a mobile app and fitness tracker was acceptable but has limited reach due to geographical barriers and competing demands experienced by AYA survivors.

Background Nausea is one of the most commonly reported side effects in patients receiving chemotherapy. Patients who experience nausea during chemotherapy may also experience depression, metabolic imbalances, dehydration, decreased ability to function, and treatment delays, which can ultimately affect outcomes. Objectives This study aimed to determine the efficacy of a cool damp washcloth with peppermint essential oil versus a cool damp washcloth alone on the self-reported intensity of nausea in patients receiving chemotherapy in the outpatient ambulatory setting. Methods 79 adult patients receiving chemotherapy were recruited from an outpatient ambulatory infusion center in the southeastern United States. Patients were separated into two groups (no scent and peppermint) and asked to rate the intensity of their chemotherapy-induced nausea at pre- and postintervention using the Baxter Retching Faces pictorial scale. Findings The results demonstrated that the use of peppermint oil was effective in decreasing the intensity of nausea experienced by patients compared to a cool washcloth alone.

The risk of developing skin cancer is elevated among childhood cancer survivors (CCS), particularly among those treated with radiation. This survey study examined the skin cancer surveillance behaviors of 94 CCS. Approximately 48% of CCS had ever conducted skin self-examination (SSE) and 31% had ever received a physician skin examination. Rates of physician skin examination were 2.5 times higher among CCS treated with radiation compared to those without radiation. However, rates of SSEs did not differ based on treatment history. These findings highlight the need to promote skin cancer surveillance as an important aspect of CCS survivorship care.

Severe bleeding in acute immune thrombocytopenic purpura (ITP) is rare but can cause significant complications to the patient. Here we report the case of a pediatric patient with acute ITP and hematuria refractory to anti-D immune globulin, high dose intravenous immunoglobulin G, and high dose steroids. Her hematuria was successfully treated with recombinant factor VIIa (rFVIIa). While further investigation on the use of rFVIIa in ITP is warranted, this case report contributes to the pediatric literature for its use during the course of an initial presentation of ITP with hemorrhagic complications.

The Cancer Institute of New Jersey Acute Lymphoblastic Leukemia trial (CINJALL) employed a post-induction regimen centered on intensive oral antimetabolite therapy, with no intravenous methotrexate (MTX). Fifty-eight patients enrolled between 2001 and 2005. A high rate of induction death (n = 3) or induction failure (n = 1) was observed. Among those who entered remission, five-year DFS is 80 ± 8.9% for those at standard risk of relapse and 76 ± 7.8% for high-risk patients, with median follow up over six years. The estimated cumulative incidence of testicular relapse among boys was elevated (13 ± 7.2%) compared to the rate observed on contemporary protocols. We conclude that post-induction therapy using intensive oral antimetabolites for children with acute lymphoblastic leukemia (ALL) can result in overall long-term DFS comparable to that observed among children treated with regimens including intravenous MTX. However, an increased risk of late extramedullary relapse among boys was observed, supporting the prevailing opinion that high-dose MTX improves outcome for children with ALL.

Stroke is a well-known complication of sickle cell disease (SCD). It is estimated to occur in approximately 11% of patients with SCD by the age of 20. The most frequent cause of cerebrovascular accident (CVA) is blockage of the intracranial internal carotid and middle cerebral arteries. Hemorrhagic stroke is less common, occurring in approximately 3% of children by age 20. Transcranial Doppler (TCD) is the standard test for prediction of stroke risk in children with sickle cell anemia. The authors present a case of a 12-year-old female with SCD transferred to their institution after suffering a catastrophic intracranial hemorrhage. Her most recent TCD was normal 6 months prior to her admission.

The Children's Cancer Group (CCG) 1952 clinical trial for children with standard-risk acute lymphoblastic leukemia (SR-ALL) compared intrathecal (IT) methotrexate (MTX) with IT triples (ITT) (MTX, cytarabine, and hydrocortisone sodium succinate [HSS]) as presymptomatic central nervous system (CNS) treatment. Following remission induction, 1018 patients were randomized to receive IT MTX and 1009 ITT. Multivariate analysis identified male sex, hepatomegaly, CNS-2 status, and age younger than 2 or older than 6 years as significant predictors of isolated CNS (iCNS) relapse. The 6-year cumulative incidence estimates of iCNS relapse are 3.4% +/- 1.0% for ITT and 5.9% +/- 1.2% for IT MTX; P = .004. Significantly more relapses occurred in bone marrow (BM) and testicles with ITT than IT MTX, particularly among patients with T-cell phenotype or day 14 BM aspirate containing 5% to 25% blasts. Thus, the estimated 6-year event-free survivals (EFS) with ITT or IT MTX are equivalent at 80.7% +/- 1.9% and 82.5% +/- 1.8%, respectively (P = .3). Because the salvage rate after BM relapse is inferior to that after CNS relapse, the 6-year overall survival (OS) for ITT is 90.3% +/- 1.5% versus 94.4% +/- 1.1% for IT MTX (P = .01). It appears that ITT improves presymptomatic CNS treatment but does not improve overall outcome.

Myelodysplastic syndromes (MDS) and myeloproliferative syndromes (MPS) of childhood are a heterogeneous group of clonal disorders of hematopoiesis with overlapping clinical features and inconsistent nomenclature. Although a number of genetic conditions have been associated with MDS and MPS, the overall contribution of inherited predispositions is uncertain. We report a retrospective study examining clinical features, genetic associations, and outcomes in 167 children with MDS and MPS. Of these patients, 48 had an associated constitutional disorder. One hundred one patients had adult-type myelodysplastic syndrome (A-MDS), 60 had juvenile myelomonocytic leukemia (JMML), and 6 infants with Down syndrome had a transient myeloproliferative syndrome (TMS). JMML was characterized by young age at onset and prominent hepatosplenomegaly, whereas patients with A-MDS were older and had little or no organomegaly. The most common cytogenetic abnormalities were monosomy 7 or del(7q) (53 cases); this was common both in patients with JMML and those with A-MDS. Leukemic transformation was observed in 32% of patients, usually within 2 years of diagnosis. Survival was 25% at 16 years. Favorable prognostic features at diagnosis included age less than 2 years and a hemoglobin F level of less than 10%. Older patients tended to present with an adult-type MDS that is accommodated within the French-American-British system. In contrast, infants and young children typically developed unique disorders with overlapping features of MDS and MPS. Although the type and intensity of therapy varied markedly in this study, the overall outcome was poor except in patients with TMS.

Purpose: To assess translocation breakpoint distribution within the MLL genomic breakpoint cluster region (bcr), 40 cases of de novo leukemia in children were examined by karyotype and Southern blot analysis. Patients and Methods: Criteria for inclusion were karyotypic or molecular rearrangement of chromosome band 1 1q23. Of the 40 cases, 31 occurred in infants. Twenty cases were acute lymphoblastic leukemia (ALL), 17 were acute myeloid leukemia (AML), and 3 were biphenotypic. Results: Karyotype identified 27 cases with translocation of chromosome band 11q23 and 2 with abnormalities of band 1 1q1 3 but not 11q23. Southern blot analysis showed rearrangement within the MLL genomic bcr in 38 of the 40 cases. In these 38, additional probe-restriction digest combinations localized MLL genomic breakpoints to the 5' portion of the bcr in 14 cases and to the 3' portion in 18; material was insufficient for further localization to 5' or 3' within the bcr in 6 cases. In the two remaining cases, both with t(4;11)(q21 ;q23), one breakpoint mapped 5' of the bcr between intron 3 and exon 5, whereas the other breakpoint was neither within nor 5' of the MLL genomic bcr. Conclusions: Suggested trends warranting investigation in more patients were breakpoint sites in the 3' bcr in AML and in patients older than 12 months. The distribution of MLL genomic breakpoints within the bcr in de novo leukemia in children is distinct from that in adults, where the breakpoints cluster in the 5' portion of the bcr.

Translocations of the MLL gene at chromosome band 11q23 are the most common cytogenetic alterations in de novo leukemia in infants and in leukemia related to chemotherapy with DNA topoisomerase II inhibitors. Experiments on knock-in mice suggest that additional mutational events may by required for full leukemogenesis. Therefore, we used single-strand conformation polymorphism analysis and an allele-specific restriction enzyme assay to investigate the frequency of KRAS and NRAS mutations in 32 pediatric leukemias with translocation of the MLL gene. Of 25 de novo cases, 13 were acute lymphoblastic leukemia (ALL), 10 were acute myeloid leukemia (AML), and 2 were biphenotypic. Three secondary leukemias were AML, 1 was biphenotypic, 1 was ALL, and 2 were diagnosed as myelodysplasia. The frequency of RAS mutations was 2 of 10 in de novo AML. Both mutations occurred in infant monoblastic variants. RAS mutations were otherwise absent in this series. This is the first report of congenital leukemias where translocation of the MLL gene and RAS mutation coexist. The frequency of RAS mutations in de novo AMLs with MLL gene translocations is similar to that in other forms of AML, but RAS mutations play a limited role in lymphoid and treatment-related leukemias with similar translocations.

We examined clinical, morphologic, and cytogenetic features and ALL-1 (MLL, Htrxl, HRX) gene rearrangements in 17 cases of secondary leukemia that occurred 11 months to 9 years from diagnoses of primary cancers in children who received topoisomerase II inhibitors or developed secondary leukemias typical of those associated with this therapy. Primary diagnoses included nine solid tumors and eight leukemias. Ten secondary leukemias were acute myeloid leukemia (AML), one was of mixed lineage, two were acute lymphoblastic leukemia (ALL), and four presented as myelodysplasia. Of 15 cases with 11q23 involvement, 11 (73%) were cytogenetically identifiable; four cases had molecular rearrangement only. By Southern blot, rearrangements within the ALL-1 gene were similar to sporadic cases. The results of this analysis suggest the following: (1) In most pediatric cases of topoisomerase II inhibitor-associated leukemia, there is disruption of the breakpoint cluster region of the ALL-1 gene at chromosomal band 11q23. (2) Exposure histories vary in secondary 11q23 leukemia, as the only topoisomerase II inhibitor was dactinomycin in one case, and, in another case, no topoisomerase II inhibitor was administered. (3) There is clinical, morphologic, cytogenetic, and molecular heterogeneity in pediatric secondary 11q23 leukemia. (4) There are some survivors of pediatric secondary 11q23 leukemia, but the outcome is most often fatal.

Eighteen patients with Fanconi anemia (FA) with evidence of bone marrow (BM) aplasia underwent allogenic BM transplants (BMT) from matched sibling donors (MSD). Median age at BMT was 7.6 years. Conditioning consisted of low-dose cyclophosphamide (CY; 5 mg/kg x 4 days) and thoracoabdominal irradiation (TAI; 400 cGy). Graft-versus-host disease (GVHD) prophylaxis included cyclosporin A and prednisone. In addition antithymocyte globulin (ATG) was administered in the pretransplant period to promote engraftment and in the posttransplant period for additional GVHD prophylaxis. Engraftment occurred rapidly (median, 12 days for an absolute neutrophil count > or = 0.5 x 10(9)/L; median, 22 days for platelet count > or = 50 x 10(9)/L). Seventeen patients have sustained engraftment and are transfusion-independent, with Lansky scores of 100% at median follow-up of 27 months. One patient developed graft failure 4 months after initial engraftment and required a second BM infusion. None of the patients developed acute GVHD; 3 patients (16%) developed chronic GVHD. BMT is a feasible option for FA patients having an MSD and should be performed at a young age and early in the course of the disease, before the development of complications. We believe the addition of ATG to the transplant regimen of low-dose CY, TAI, and cyclosporin was responsible for improvement in the survival of FA patients undergoing BMT. The regimen was well tolerated and was associated with a low incidence of complications including GVHD.

Summary There have only been a few reports documenting the use of umbilical cord blood as a source of stem cells for haemopoietic reconstitution. We report our experience with a child with Fanconi anaemia (FA) who underwent a stem cell transplant using umbilical cord blood cells from his HLA matched sibling. Although the engraftment was somewhat slow, it was complete and comparable to other transplants performed in FA patients using HLA matched sibling marrow. There was no graft-versus-host disease. The post-transplant period was uncomplicated and, at a follow-up of 36 months, this child is well with normal blood counts and immune function.

Numerical abnormalities of chromosome 7 were detected by using fluorescence labeled in situ hybridization (FISH) procedure with a centromere-specific probe in four cases of acute myeloid leukemia (AML) and three cases of myelodysplastic syndromes (MDS). Comparison of these results with classical cytogenetic (CC) data demonstrated a good correlation between the two methods. FISH confirmed the finding of monosomy 7 in all patients who demonstrated this abnormality by CC. Two AML patients who did not show monosomy 7 by CC were unexpectedly found to contain this abnormality in 39.8% and 17% cells when examined by FISH. Given that our modified FISH method consistently yielded >96% hybridization efficiency, these findings constitute an unexpected but real presence of monosomy 7 in a substantial number of interphase cells that had remained undetected by classical karyotyping. Finally, a number of maturing myeloid cells including granulocytes also demonstrated monosomy 7 by FISH, thereby confirming the ability of malignant cells to undergo differentiation. We conclude that FISH constitutes a highly sophisticated molecular technique which can be extremely useful in select cases for detecting ‘masked monosomy 7’ as well as helping to determine the lineage of terminally mature cells in AML, thereby providing a handle on the effects of cytokines or chemotherapy on normal vs leukemic clones.

The Children's Cancer Group 1952 (CCG-1952) clinical trial studied the substitution of oral 6-thioguanine (TG) for 6-mercaptopurine (MP) and triple intrathecal therapy (ITT) for intrathecal methotrexate (IT-MTX) in the treatment of standard-risk acute lymphoblastic leukemia. After remission induction, 2027 patients were randomized to receive MP (n = 1010) or TG (n = 1017) and IT-MTX (n = 1018) or ITT (n = 1009). The results of the thiopurine comparison are as follows. The estimated 7-year event-free survival (EFS) for subjects randomized to TG was 84.1% (± 1.8%) and to MP was 79.0% (± 2.1%; P = .004 log rank), although overall survival was 91.9% (± 1.4%) and 91.2% (± 1.5%), respectively (P = .6 log rank). The TG starting dose was reduced from 60 to 50 mg/m2 per day after recognition of hepatic veno-occlusive disease (VOD). A total of 257 patients on TG (25%) developed VOD or disproportionate thrombocytopenia and switched to MP. Once portal hypertension occurred, all subjects on TG were changed to MP. The benefit of randomization to TG over MP, as measured by EFS, was evident primarily in boys who began TG at 60 mg/m2 (relative hazard rate [RHR] 0.65, P = .002). The toxicities of TG preclude its protracted use as given in this study. This study is registered at http://clinicaltrials.gov as NCT00002744.

Proliferation kinetics of both leukemia and a variety of solid tumors have been assessed after in vivo infusions of the thymidine analogues, iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU). In acute myeloid leukemia (AML), these data indicate that the pretherapy cell cycle time (Tc) of myeloblasts is a prognostic indicator for remission duration since patients with slowly cycling myeloblasts had more durable remissions. The presence of in vivo differentiation detected from the day 7 biopsy after chemotherapy was also of favorable prognosis as these individuals had statistically significant improvement in their remission duration

Expression of transforming growth factor-beta (TGF-beta), which inhibits the proliferation of hematopoietic progenitors, was investigated simultaneously with cell cycle characteristics in 63 bone marrow biopsies from 23 cases with acute promyelocytic leukemia (APL). Bromodeoxyuridine (BrdU) was administered to every patient (17 newly diagnosed) for determination of the labeling index (LI) and the durations of S-phase (Ts) and the cell cycle (Tc) of leukemic promyelocytes. APL cases had lower LI both in the bone marrow aspirate (6.1% v 11.4%, P = .008) and biopsy (21.1% v 28.0%, P = .001) and longer Tc (93.6 hours v 56.0 hours, P = .002) when compared with other French-American-British subtypes. TGF-beta expression (detected by a monoclonal anti-TGF-beta 2/beta 3 antibody) was dramatically high, especially in interstitial areas of the biopsies. S-phase cells were found as geographically restricted islands of proliferation (GRIPs) in 20 of 22 cases. Weekly biopsies showed an increment in TGF-beta on day 7 of therapy in 13 of 17 cases, while in vivo differentiation was noted in 9 of 15. We conclude that the presence of high TGF-beta expression may explain the biologic basis for the slowly cycling nature of leukemic promyelocytes in APL as well as the unique clustering of S- phase cells observed in GRIPs.

Presence of megakaryocytic cells in patients with myeloid disorders were investigated by staining plastic embedded biopsy sections with an anti-Factor VIII antibody (AFA). Two hundred and fifty cases were studied, 207 of whom had acute myeloid leukemia (AML) while 43 had myelodysplastic syndromes (MDS). Abnormal clusters of AFA positive cells indicating multilineage disease were identified in 17% with primary AML ( 30 175 ), 38% with secondary AML ( 12 32 ) and 42% cases of MDS ( 18 43 ). Biological characteristics of these 60 AFA positive cases were investigated. No unique differences in cell cycle characteristics following bromodeoxyuridine (BrdU) were identified. We confirm several recent reports that the incidence of multilineage involvement in AML is substantial.

In the Western World, potentially fatal megaloblastic anaemia in children is usually caused by lack of Cbl known as Juvenile Cobalamin Deficiency (JCD), which leads to hematological and neurological disturbances. The classic cause of JCD is malabsorption, the inability of the small intestine to absorb the essential molecule from dietary sources. Consensus is lacking on which laboratory tests are the best for explaining the etiology of JCD due to the myriad of potential causes (Carmel et al, 2003). Underlying mechanisms vary in different parts of the world and in different socioeconomic groups (Grasbeck 2006). However, patients may go improperly diagnosed for long periods because the serial exclusion testing for various causes can take months. Consequently, developmental delay is potentially common but easily overlooked in young children. Two main forms of inherited JCD exist: Imerslund-Grasbeck syndrome (IGS; OMIM261100; Imerslund 1960; Grasbeck et al, 1960) and Intrinsic factor Deficiency (IFD; OMIM261000; Katz et al, 1972). In IGS, deleterious mutations in either CUBN (Aminoff et al, 1999) or AMN (Tanner et al, 2003) cause malabsorption of Cbl. In IFD, mutations in the GIF gene itself are causative (Yassin et al, 2004; Tanner et al, 2005). All three genes can be sequenced in search of mutations in genomic DNA (Tanner et al, 2004, 2005). However, the screening task is daunting given that CUBN has 67 exons and AMN is very GC-rich, complicating the PCR-based analysis. To our knowledge commercial screening is not offered and research based testing is not widely available. Especially for under-served socioeconomic groups, affordable genetic testing may be years away. During our ongoing genetic studies of JCD, we have encountered four patients of West-African ancestry. Three were from the USA and one was referred to us from the UK. Sequencing of GIF revealed that all alleles with African roots carried the same c.183_186delGAAT frameshift mutation (M61fs), suggesting either a founder event or mutational recurrence. We undertook the present study to clarify this question. Genomic DNA samples were isolated from peripheral blood after informed consent by standard phenol-chloroform-ethanol-precipitation. Families 8 (Tanner et al, 2005) and 55 (Yassin et al, 2004) were described previously and are of West African decent. Family 33 is Jamaican with African ancestry living in the UK and family 39 is from the US East Coast, with the father reporting African-Native American and the mother reporting European ancestry. All four patients were given Cbl replacement therapy by parenteral Cbl injection and no longer show any clinical symptoms. Control DNA samples from 93 African-American and 93 Caucasian with no evidence of JCD were collected from the metropolitan Columbus area under a separate IRB approved protocol. For mutation analysis, we amplified GIF exon 2 from genomic DNA by PCR and analyzed it as previously described (Tanner et al, 2005). Genotyping was performed with the three microsatellite markers GIF M1 (~28 kb proximal of GIF), GIF M3b (intron 7), and GIF M4 (~2kb distal), and analyzed using fluorescent-labeled primers as described (Tanner et al, 2004). PCR conditions and primer sequences are available upon request. To elucidate if the haplotype carrying the c.183_186delGAAT mutation is an ancestral founder event, we genotyped the three flanking markers GIF M1, GIF M3b, and GIF M4 in the four families. Based on a parsimonious analysis requiring the least number of recombinations and implying linkage disequilibrium across this 40 kb region, we inferred the disease haplotype as 180-M61fs-235-168. This ancestral haplotype was found unaltered in three out of seven parents and can be explained in the remaining four by one recombination each (Fig 1). Naturally, this assumption is not taking into account the true haplotype frequencies in Africans. Thus, we studied DNA samples from 93 African-American (AA) and 93 European-American (EA) controls. After excluding the presence of the mutation in all controls, we constructed definitive haplotypes based on homozygosity and then possible haplotypes based on the observed definitive haplotypes, again using the parsimony principle. Through the resulting haplotype frequencies (Supplemental Table I), we found that none of the 93 EA control samples had the ancestral haplotype 180-235-168 on which the mutation probably arose, while only two of the 93 AA control samples carried 180-235-168 (~1%). Allele 180 of marker GIF M1 was found among 14/186 (7.5%) alleles in the AA controls, and was not detected in the EA controls (Supplemental Table I). Likewise, allele 168 of marker GIF M4 was seen in 10/186 AA alleles (5.4%) with no occurrence in EA. This allele was found in strong linkage disequilibrium with M61fs in the JCD parents with six out of seven carrying it (Fig 1). Because the ancestral haplotype 180–235-168 existed only in AA and is quite rare amongst them (~1%), we concluded that the c.183_186delGAAT mutation very likely represents a founder event for JCD patients of the Sub-Saharan West-African lineage. This mutation was never detected in ~120 cases of JCD other than these 4 cases of African ancestry. Moreover, we have not encountered any AA patient so far that had any other JCD mutation, although the numbers are still small (n=4). Fig 1 Pedigrees of four patients affected by intrinsic factor deficiency (IFD, filled symbols) due to the c.183_186delGAAT frameshift mutation (M61fs) in the intrinsic factor gene, GIF. Haplotyping with markers GIF M1, GIF M3b, and GIF M4 flanking mutation ... In summary, we identified a specific GIF mutation to be responsible for all JCD cases of West-African origin so far. The strong association of a particular mutation with a medically under-served minority could be used to their immediate benefit. It allows for quick and easy genetic testing in a disease that is notoriously difficult to diagnose but easy to treat, once the correct diagnosis is made. We believe that one simple PCR-based test in suspected cases of JCD in patients of West-African origin should be considered early during diagnosis.

Aberrant or anomalous anatomy is an under appreciated risk for venous thromboembolic events (VTE). Five adolescents with VTE and predisposing anatomic abnormalities are presented. In three cases, knowledge of the underlying anatomic abnormalities resulted in changes in treatment and management. In two other cases, failure to consider or correct the underlying defect resulted in recurrent thrombosis or post-thrombotic complications. Few case reports are found in the pediatric literature, but a MEDLINE search across all age groups suggests these anomalies are frequently found when appropriate radiological imaging is obtained.

Aminopterin offers advantages over the related antifolate, methotrexate, including greater potency, complete bioavailability, and more consistent accumulation and metabolism by patients' blasts. This current trial was done to document the toxicity of the aminopterin within a multiagent therapeutic regimen for children with newly diagnosed ALL.Patients at high risk of relapse were non-randomly assigned to therapy including oral aminopterin 4 mg/m(2), in two doses 12 h apart, in place of methotrexate 100 mg/m(2) in four divided doses.Thirty-two patients, 22 with pre-B ALL and ten with T-lineage ALL, have been treated with aminopterin, with median follow up of 40 months. Hematologic, mucosal and hepatic toxicity has been tolerable and reversible. There have been no toxic deaths among patients in remission. During weekly AMT therapy, higher mean neutrophil counts were observed among patients who were wild type for polymorphisms in methylene tetrahydrofolate reductase and methionine synthase reductase.Aminopterin can be safely incorporated in multiagent therapy for patients with ALL, in place of systemic methotrexate, without causing excessive toxicity. These results support a larger trial comparing the efficacy and toxicity of aminopterin and methotrexate in therapy for patients with ALL.

Purpose: To evaluate the response rate, survival, and toxicity of mitoxantrone and cytarabine induction, high-dose cytarabine and etoposide intensification, and further consolidation/maintenance therapies, including bone marrow transplantation, in children with relapsed, refractory, or secondary acute myeloid leukemia (AML). To evaluate response to 2-chlorodeoxyadenosine (2-CDA) and etoposide (VP-16) in patients who did not respond to mitoxantrone and cytarabine. Patients and Methods: Patients with relapsed/refractory AML (n = 101) and secondary AML (n = 13) were entered. Results: Mitoxantrone and cytarabine induction achieved a remission rate of 76% for relapsed/refractory patients and 77% for patients with secondary AML, with a 3% induction mortality rate. Cytarabine and etoposide intensification exceeded the acceptable toxic death rate of 10%. The response rate of 2-CDA/VP-16 was 8%. Two-year overall survival was estimated at 24% and was better than historical control data. Patients with secondary AML had similar outcomes to relapsed or refractory patients. Initial remission longer than 1 year was the most important prognostic factor for patients with primary AML (2-year survival rate, 75%), whereas for patients with primary AML, with less than 12 months of initial remission, survival was 13% and was similar to that of refractory patients (6%). Conclusion: Mitoxantrone and cytarabine induction is effective with reasonable toxicity in patients with relapsed/refractory or secondary AML. The cytarabine and etoposide intensification regimen should be abandoned because of toxicity. Patients with relapsed AML with initial remissions longer than 1 year have a relatively good prognosis.

Neonatal alloimmune thrombocytopenia with intracranial hemorrhage is a reported phenomenon. While most of the hemorrhages are noted to be either intraventricular or intraparenchymal, the authors describe the case of a fourth-ventricle hemorrhage with extension into the spinal column down the cervical spinal cord secondary to maternal anti-human platelet antigen (HPA-1a) antibody.

Protein C replacement therapy with a monoclonal antibody purified, virus inactivated protein C concentrate was carried out in nine infants (three male, six female) with severe congenital protein C deficiency and life-threatening purpura fulminans and/or thrombosis associated with disseminated intravascular coagulation (DIC). Eight infants were homozygous for protein C deficiency; one was a compound heterozygote. The treatment period varied from 22 days to three years. The half-life of protein C was found to be as short as two to three hours during activation of the coagulation system, increasing to approximately ten hours after stabilization. During the acute phase, protein C levels of 0.10 to 0.25 IU/mL were associated with elevated markers of coagulation activation indicating DIC, while protein C levels greater than 0.25 were associated with normalization of coagulation markers. No product-related side effects were reported. Episodes of bleeding or purpura recurred in all patients who were switched to oral anticoagulant therapy, necessitating reinstatement of protein C replacement therapy, either as needed to control symptoms, or on a long-term prophylactic schedule, alone or in addition to oral anticoagulation. Home treatment with protein C concentrate allowed a near-normal life-style for patients who otherwise would be hospitalized for long periods of time.

Examination of the proliferative characteristics of myeloblasts was undertaken in situ in bone marrow (BM) biopsies of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) following sequential infusions of iodo- (IUdR) and bromodeoxyuridine (BrdU). The ability to identify S-phase cells which have incorporated both or either one of the labels in vivo by using two monoclonal antibodies in vitro permitted the measurement of labeling index (LI) and durations of S-phase (Ts) and the total cell cycle (Tc) both from the BM aspirates and biopsies. While the LI is 2–3 times higher in biopsies, Ts and Tc are fairly comparable in the two samples in 8/10 cases ( p = 0.02 and 0.003 respectively). Advantages associated with the determination of cell cycle parameters in BM biopsies have been discussed at length.

The antifolate aminopterin (AMT) may offer clinical advantages over the related compound methotrexate (MTX), due to its pharmacodynamic and pharmacokinetic properties (Cancer Chemother Pharmacol2006;57:826). These properties may lead to greater ease of administration and less pharmacodynamic variability than is seen with oral MTX. In our phase II trial, we demonstrated that AMT at its MTD (4 mg/m2/week in two doses, 12 h apart) had significant activity among children with relapsed/refractory ALL (Clin Cancer Res2005; 11: 8089). The phase IIB trial was conducted to test whether AMT can be safely incorporated at its MTD in multiagent therapy in place of oral MTX for children with newly diagnosed ALL at high risk (HR) of relapse by NCI criteria. The backbone of intensive antimetabolite-based therapy has been previously published (JCO1996;14:2803, Cancer Chemother Pharmacol2006;57:826). Thirty-three HR patients have been treated with AMT. With median follow up of 2.5 years, 2-yr EFS is 85% ± 7%. Seventeen have completed all planned therapy, and all have completed the Consolidation phase of weekly AMT at its MTD (along with mercaptopurine twice daily and intrathecal therapy every other week), allowing a description of the toxicity possibly attributable to AMT in the setting of multiagent ALL therapy. Oral AMT had complete bioavailability (compared to IV AMT kinetics on the Phase I and II trials). Bone marrow penetration by AMT, as indicated by steady state RBC AMT concentrations, is identical to that of MTX when MTX is given at 25-fold higher doses (100 mg/m2/week). During maintenance therapy, RBC AMT is proportional to the average weekly AMT dose, but was not predictive of hematologic toxicity. Hematologic toxicity among HR patients is similar to that observed among concurrent standard risk patients who receive identical therapy except for MTX in place of AMT and no delayed intensification. HR patients who are homozygous wild-type for both polymorphisms in the gene for methyl-tetrahydrofolate reductase (MTHFR C677T and A1298C) had higher mean neutrophil counts during weekly AMT therapy than those with one or more polymorphic allele (2687 ± 333, N=7 vs. 1613 ± 83, N=22; P

Aminopterin (AMT) was replaced by methotrexate (MTX) in the therapy for patients with acute lymphoblastic leukemia (ALL) because of fears that the former antifolate produced excessive and unpredictable toxicity. Regardless of the presumed risk, AMT has greater accumulation and metabolism by leukemic blasts than MTX in vitro and had 100% bioavailability in early clinical trials. Therefore we are using AMT as a replacement for MTX to treat newly diagnosed patients with ALL at high risk (HR) of relapse by NCI criteria. The Cancer Institute of NJ ALL trial (CINJALL) is based an earlier regimen using intensive antimetabolite therapy (100 mg/m2 MTX in 4 divided doses weekly or every other week for 76 weeks) delivered orally on an outpatient basis. With 7 years median follow up, the 5-year EFS for the 106 patients treated on this earlier protocol is 84% ±3% (personal communication, N. Winick, UT Southwestern Medical Center). HR patients on CINJALL are non-randomly assigned to receive a single delayed intensification and oral AMT at its MTD, 4 mg/m2 in two divided doses 12 hours apart, in place of oral MTX (given to standard risk (SR) patients). There is no intravenous MTX or AMT on either arm. To date, 44 children have enrolled: 15 on the SR (MTX) arm (median age 3 years; range 1–9) and 29 on the HR (AMT) arm (median age 14 years; range 2–21). As of this analysis, median follow up is 85 weeks (range 0–177 weeks). Six HR and one SR patient have completed all scheduled therapy. Twenty HR and 14 SR patients have completed the 12-week consolidation, which includes weekly divided-dose antifolate therapy. The mean AMT AUC, 0.64 ± 0.24 mmol·hr/L, was not statistically different from the AUC after IV AMT at the same dose on the Phase I trial, confirming complete bioavailability. RBC antifolate was measured after a minimum of 8 weeks of therapy as an indication of marrow exposure. The lack of significant difference between mean RBC MTX and RBC AMT (240 ± 63 vs. 211 ± 22 pmol/ml RBC), despite a 25-fold difference in weekly dose, supports the pharmacodynamic equivalence of the two drugs at this dose and schedule. Mean CSF MTX 2-6h after oral MTX was significantly greater than CSF AMT (15.8 ± 2.3 vs 3.8 ± 1.3 nM; P < 0.001). Focusing on toxicity in consolidation, where the the two arms differ only in the antifolate given, there was no significant difference between the MTX and AMT arms in mean WBC count, hemoglobin or platelets. HR patients were no more likely than SR patients to experience >10% body weight loss, admission to the hospital or treatment interruption for treatment-related toxicity, neurotoxicity, SGOT >5 x normal, bilirubin > 2x normal, ANC