Antonio R. Perez-Atayde

Late cardiac effects of doxorubicin therapy for acute lymphoblastic leukemia in childhood.

BACKGROUND Cardiotoxicity is a recognized complication of doxorubicin therapy, but the long-term effects of doxorubicin are not well documented. We therefore assessed the cardiac status of 115 children who had been treated for acute lymphoblastic leukemia with doxorubicin 1 to 15 years earlier in whom the disease was in continuous remission. METHODS Eighteen patients received one dose of doxorubicin (45 mg per square meter of body-surface area), and 97 received multiple doses totaling 228 to 550 mg per square meter (median, 360). The median interval between the end of treatment and the cardiac evaluation was 6.4 years. Our evaluation consisted of a history, 24-hour ambulatory electrocardiographic recording, exercise testing, and echocardiography. RESULTS Fifty-seven percent of the patients had abnormalities of left ventricular afterload (measured as end-systolic wall stress) or contractility (measured as the stress-velocity index). The cumulative dose of doxorubicin was the most significant predictor of abnormal cardiac function (P less than 0.002). Seventeen percent of patients who received one dose of doxorubicin had slightly elevated age-adjusted afterload, and none had decreased contractility. In contrast, 65 percent of patients who received at least 228 mg of doxorubicin per square meter had increased afterload (59 percent of patients), decreased contractility (23 percent), or both. Increased afterload was due to reduced ventricular wall thickness, not to hypertension or ventricular dilatation. In multivariate analyses restricted to patients who received at least 228 mg of doxorubicin per square meter, the only significant predictive factors were a higher cumulative dose (P = 0.01), which predicted decreased contractility, and an age of less than four years at treatment (P = 0.003), which predicted increased afterload. Afterload increased progressively in 24 of 34 patients evaluated serially (71 percent). Reported symptoms correlated poorly with indexes of exercise tolerance or ventricular function. Eleven patients had congestive heart failure within one year of treatment with doxorubicin; five of them had recurrent heart failure 3.7 to 10.3 years after completing doxorubicin treatment, and two required heart transplantation. No patient had late heart failure as a new event. CONCLUSIONS Doxorubicin therapy in childhood impairs myocardial growth in a dose-related fashion and results in a progressive increase in left ventricular afterload sometimes accompanied by reduced contractility. We hypothesize that the loss of myocytes during doxorubicin therapy in childhood might result in inadequate left ventricular mass and clinically important heart disease in later years.

TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors

Inflammatory myofibroblastic tumors (IMTs) are neoplastic mesenchymal proliferations featuring an inflammatory infiltrate composed primarily of lymphocytes and plasma cells. The myofibroblastic cells in some IMTs contain chromosomal rearrangements involving the ALK receptor tyrosine-kinase locus region (chromosome band 2p23). ALK-which is normally restricted in its expression to neural tissues-is expressed strikingly in the IMT cells with 2p23 rearrangements. We now report a recurrent oncogenic mechanism, in IMTs, in which tropomyosin (TPM) N-terminal coiled-coil domains are fused to the ALK C-terminal kinase domain. We have cloned two ALK fusion genes, TPM4-ALK and TPM3-ALK, which encode approximately 95-kd fusion oncoproteins characterized by constitutive kinase activity and tyrosylphosphorylation. Immunohistochemical and molecular correlations, in other IMTs, implicate non-TPM ALK oncoproteins that are predominantly cytoplasmic or pre- dominantly nuclear, presumably depending on the subcellular localization of the ALK fusion partner. Notably, a TPM3-ALK oncogene was reported recently in anaplastic lymphoma, and TPM3-ALK is thereby the first known fusion oncogene that transforms, in vivo, both mesenchymal and lymphoid human cell lineages.

Primary Renal Neoplasms with the ASPL-TFE3 Gene Fusion of Alveolar Soft Part Sarcoma A Distinctive Tumor Entity Previously Included among Renal Cell Carcinomas of Children and Adolescents

The unbalanced translocation, der(17)t(X;17)(p11.2;q25), is characteristic of alveolar soft part sarcoma (ASPS). We have recently shown that this translocation fuses the TFE3 transcription factor gene at Xp11.2 to ASPL, a novel gene at 17q25. We describe herein eight morphologically distinctive renal tumors occurring in young people that bear the identical ASPL-TFE3 fusion transcript as ASPS, with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. A relationship between these renal tumors and ASPS was initially suggested by the cytogenetic finding of a balanced t(X;17)(p11.2;q25) in two of the cases, and the ASPL-TFE3 fusion transcripts were then confirmed by reverse transcriptase-polymerase chain reaction. The morphology of these eight ASPL-TFE3 fusion-positive renal tumors, although overlapping in some aspects that of classic ASPS, more closely resembles renal cell carcinoma (RCC), which was the a priori diagnosis in all cases. These tumors demonstrate nested and pseudopapillary patterns of growth, psammomatous calcifications, and epithelioid cells with abundant clear cytoplasm and well-defined cell borders. By immunohistochemistry, four tumors were negative for all epithelial markers tested, whereas four were focally positive for cytokeratin and two were reactive for epithelial membrane antigen (EMA) (one diffusely, one focally). Electron microscopy of six tumors demonstrated a combination of ASPS-like features (dense granules in four cases, rhomboid crystals in two cases) and epithelial features (cell junctions in six cases, microvilli and true glandular lumens in three cases). Overall, although seven of eight tumors demonstrated at least focal epithelial features by electron microscopy or immunohistochemistry, the degree and extent of epithelial differentiation was notably less than expected for typical RCC. We confirmed the balanced nature of the t(X;17) translocation by fluorescence in situ hybridization in all seven renal tumors thus analyzed, which contrasts sharply with the unbalanced nature of the translocation in ASPS. In summary, a subset of tumors previously considered to be RCC in young people are in fact genetically related to ASPS, although their distinctive morphological and genetic features justify their classification as a distinctive neoplastic entity. Finally, the finding of distinctive tumors being associated with balanced and unbalanced forms of the same translocation is to our knowledge, unprecedented.

Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy

Mutations in PRKAG2, the gene for the gamma 2 regulatory subunit of AMP-activated protein kinase, cause cardiac hypertrophy and electrophysiologic abnormalities, particularly preexcitation (Wolff-Parkinson-White syndrome) and atrioventricular conduction block. To understand the mechanisms by which PRKAG2 defects cause disease, we defined novel mutations, characterized the associated cardiac histopathology, and studied the consequences of introducing these mutations into the yeast homologue of PRKAG2, Snf4. Although the cardiac pathology caused by PRKAG2 mutations Arg302Gln, Thr400Asn, and Asn488Ile include myocyte enlargement and minimal interstitial fibrosis, these mutations were not associated with myocyte and myofibrillar disarray, the pathognomonic features of hypertrophic cardiomyopathy caused by sarcomere protein mutations. Instead PRKAG2 mutations caused pronounced vacuole formation within myocytes. Several lines of evidence indicated these vacuoles were filled with glycogen-associated granules. Analyses of the effects of human PRKAG2 mutations on Snf1/Snf4 kinase function demonstrated constitutive activity, which could foster glycogen accumulation. Taken together, our data indicate that PRKAG2 mutations do not cause hypertrophic cardiomyopathy but rather lead to a novel myocardial metabolic storage disease, in which hypertrophy, ventricular pre-excitation and conduction system defects coexist.

Central giant cell lesions of the jaws: A clinicopathologic study

The biologic behavior of central giant cell lesions of the jaws ranges from quiescent to aggressive with destructive expansion. To date, these variations have not been explained by the findings of routine histologic examination. This retrospective clinicopathologic study of giant cell lesions was performed to search for histologic correlates of biologic behavior. Lesions in 17 patients were classified clinically as nonaggressive (group I) or aggressive (group II). In general, group II lesions affected children at an earlier age, were larger at the time of diagnosis, and recurred more frequently. The following histologic parameters were assessed: fractional surface area occupied by giant cells (FSA), relative size index of giant cells (RSI), stromal characteristics, mitotic index, inflammatory cells, and hemosiderin content. Histologic differences between the two groups were not as clear as the differences in biologic behavior. However, aggressive lesions had a higher RSI, and recurrent giant cells lesions had a higher RSI and FSA; these parameters warrant further study. In addition, electron microscopic differences in a small number of aggressive and nonaggressive lesions were documented.

Gamma-globulin treatment of acute myocarditis in the pediatric population.

BACKGROUND Myocardial damage in myocarditis is mediated, in part, by immunological mechanisms. High-dose intravenous gamma-globulin (IVIG) is an immunomodulatory agent that is beneficial in myocarditis secondary to Kawasaki disease, as well as in murine myocarditis. Since 1990, the routine management of presumed acute myocarditis at Childrens Hospital, Boston, and Childrens Hospital, Los Angeles, has included administration of high-dose IVIG. METHODS AND RESULTS We treated 21 consecutive children presenting with presumed acute myocarditis with IVIG, 2 g/kg, over 24 hours, in addition to anticongestive therapies. A comparison group comprised 25 recent historical control patients meeting identical eligibility criteria but not receiving IVIG therapy. Left ventricular function was assessed during five time intervals: 0 to 7 days, 1 to 3 weeks, 3 weeks to 3 months, 3 to 6 months, and 6 to 12 months. At presentation, the IVIG and non-IVIG groups had comparable left ventricular enlargement and poor fractional shortening. Compared with the non-IVIG group, those treated with IVIG had a smaller mean adjusted left ventricular end-diastolic dimension and higher fractional shortening in the periods from 3 to 6 months (P = .008 and P = .033, respectively) and 6 to 12 months (P = .072 and P = .029, respectively). When adjusting for age, biopsy status, intravenous inotropic agents, and angiotensin-converting enzyme inhibitors, patients treated with IVIG were more likely to achieve normal left ventricular function during the first year after presentation (P = .03). By 1 year after presentation, the probability of survival tended to be higher among IVIG-treated patients (.84 versus .60, P = .069). We observed no adverse effects of IVIG administration. CONCLUSIONS These data suggest that use of high-dose IVIG for treatment of acute myocarditis is associated with improved recovery of left ventricular function and with a tendency to better survival during the first year after presentation.

Congenital Mesoblastic Nephroma t(12;15) Is Associated with ETV6-NTRK3 Gene Fusion Cytogenetic and Molecular Relationship to Congenital (Infantile) Fibrosarcoma

Morphological, cytogenetic, and biological evidence supports a relationship between congenital (infantile) fibrosarcoma (CFS) and congenital mesoblastic nephroma (CMN). These tumors have a very similar histological appearance, and they are both associated with polysomies for chromosomes 8, 11, 17, and 20. Recently, CFS was shown to contain a novel t(12; 15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion. The aims of this study were to determine whether congenital mesoblastic nephroma contains the t(12;15)(p13;q25) translocation and ETV6-NTRK3 gene fusion and whether ETV6-NTRK3 fusions, in CMN and CFS, antedate acquisition of nonrandom chromosome polysomies. To address these aims, we evaluated 1) ETV6-NTRK3 fusion transcripts by reverse transcriptase polymerase chain reaction and sequence analysis, 2) genomic ETV6-region chromosomal rearrangement by fluorescence in situ hybridization, and 3) chromosomal polysomies by karyotyping and fluorescence in situ hybridization. We report ETV6-NTRK3 fusion transcripts and/or ETV6-region rearrangement in five of six CMNs and in five of five CFSs. The ETV6-NTRK3 fusion transcripts and/or ETV-region chromosome rearrangements were demonstrated in two CMNs and one CFS that lacked chromosome polysomies. These findings demonstrate that t(12;15) translocation, and the associated ETV6-NTRK3 fusion, can antedate acquisition of chromosome polysomies in CMN and CFS. CMN and CFS are pathogenetically related, and it is likely that they represent a single neoplastic entity, arising in either renal or soft tissue locations.

A role for Lin28 in primordial germ-cell development and germ-cell malignancy

The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwart efforts to investigate molecular mechanisms of germ-cell specification. stella (also called Dppa3) marks the rare founder population of the germ lineage. Here we differentiate mouse embryonic stem cells carrying a stella transgenic reporter into putative PGCs in vitro. The Stella+ cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella+ cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing, is essential for proper PGC development. Furthermore, we show that Blimp1 (also called Prdm1), a let-7 target and a master regulator of PGC specification, can rescue the effect of Lin28 deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Overexpression of Lin28 promotes formation of Stella+ cells in vitro and PGCs in chimaeric embryos, and is associated with human germ-cell tumours. The differentiation of putative PGCs from embryonic stem cells in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ-cell development and malignancy.

Transgenic Mice Overexpressing Mutant PRKAG2 Define the Cause of Wolff-Parkinson-White Syndrome in Glycogen Storage Cardiomyopathy

Background—Mutations in the &ggr;2 subunit (PRKAG2) of AMP-activated protein kinase produce an unusual human cardiomyopathy characterized by ventricular hypertrophy and electrophysiological abnormalities: Wolff-Parkinson-White syndrome (WPW) and progressive degenerative conduction system disease. Pathological examinations of affected human hearts reveal vacuoles containing amylopectin, a glycogen-related substance. Methods and Results—To elucidate the mechanism by which PRKAG2 mutations produce hypertrophy with electrophysiological abnormalities, we constructed transgenic mice overexpressing the PRKAG2 cDNA with or without a missense N488I human mutation. Transgenic mutant mice showed elevated AMP-activated protein kinase activity, accumulated large amounts of cardiac glycogen (30-fold above normal), developed dramatic left ventricular hypertrophy, and exhibited ventricular preexcitation and sinus node dysfunction. Electrophysiological testing demonstrated alternative atrioventricular conduction pathways consistent with WPW. Cardiac histopathology revealed that the annulus fibrosis, which normally insulates the ventricles from inappropriate excitation by the atria, was disrupted by glycogen-filled myocytes. These anomalous microscopic atrioventricular connections, rather than morphologically distinct bypass tracts, appeared to provide the anatomic substrate for ventricular preexcitation. Conclusions—Our data establish PRKAG2 mutations as a glycogen storage cardiomyopathy, provide an anatomic explanation for electrophysiological findings, and implicate disruption of the annulus fibrosis by glycogen-engorged myocytes as the cause of preexcitation in Pompe, Danon, and other glycogen storage diseases.

Idiopathic steatohepatitis in childhood: a multicenter retrospective study.

OBJECTIVE To describe the clinical, laboratory, and histopathologic features of idiopathic steatohepatitis in children. STUDY DESIGN Retrospective review of all liver biopsies performed at Boston Childrens Hospital, Massachusetts General Hospital, and the University of Massachusetts Medical Center from 1991 to 1994. Chart review was performed when biopsies demonstrated steatosis. RESULTS Eighty-two patients had biopsy-proven hepatic steatosis. Fourteen patients had fatty liver without evidence of inherited, infectious, autoimmune, endocrinologic, toxicologic, or iatrogenic causes. All 14 patients were obese, averaging 159% of ideal body weight (range, 121% to 222%). Nine patients initially had transient abdominal pain, two had hepatomegaly, and one was identified by incidental laboratory evaluation. These 12 patients had biopsies because of persistent elevations of aminotransferase levels. Two other patients without risk factors for steatosis were identified at staging laparotomy for Hodgkin lymphoma. The 10 boys and 4 girls had an average age of 13.5 years (range, 10 to 18 years). Aminotransferase elevations were modest, with aspartate aminotransferase and alanine aminotransferase values averaging 77 +/- 38 IU and 129 +/- 73 IU, respectively. All had imaging studies demonstrating diffuse fatty change. Histologic examination of biopsy specimens revealed varying degrees of steatosis with inflammation and fibrosis. CONCLUSION Idiopathic steatohepatitis occurs predominantly or exclusively in obese peripubertal children. This entity represents a frequent reason for liver biopsy in this age group. The degree of steatosis, fibrosis, and inflammation does not correlate with symptoms or signs, and significant liver injury with bridging fibrosis may be present.