Florette K. Hazard

Genomic analysis of fibrolamellar hepatocellular carcinoma

Pediatric tumors are relatively infrequent, but are often associated with significant lethality and lifelong morbidity. A major goal of pediatric cancer research has been to identify key drivers of tumorigenesis to eventually develop targeted therapies to enhance cure rate and minimize acute and long-term toxic effects. Here, we used genomic approaches to identify biomarkers and candidate drivers for fibrolamellar hepatocellular carcinoma (FL-HCC), a very rare subtype of pediatric liver cancer for which limited therapeutic options exist. In-depth genomic analyses of one tumor followed by immunohistochemistry validation on seven other tumors showed expression of neuroendocrine markers in FL-HCC. DNA and RNA sequencing data further showed that common cancer pathways are not visibly altered in FL-HCC but identified two novel structural variants, both resulting in fusion transcripts. The first, a 400 kb deletion, results in a DNAJB1-PRKCA fusion transcript, which leads to increased cAMP-dependent protein kinase (PKA) activity in the index tumor case and other FL-HCC cases compared with normal liver. This PKA fusion protein is oncogenic in HCC cells. The second gene fusion event, a translocation between the CLPTM1L and GLIS3 genes, generates a transcript whose product also promotes cancer phenotypes in HCC cell lines. These experiments further highlight the tumorigenic role of gene fusions in the etiology of pediatric solid tumors and identify both candidate biomarkers and possible therapeutic targets for this lethal pediatric disease.

Pregnancy outcomes in women with chronic endometritis and recurrent pregnancy loss

OBJECTIVE To evaluate the prevalence of chronic endometritis (CE) in women with recurrent pregnancy loss (RPL) and compare pregnancy outcomes in women with and without CE. DESIGN Case-control observational study. SETTING Academic fertility practice. PATIENT(S) Women with two or more pregnancy losses. INTERVENTION(S) Hematoxylin and eosin (H & E) staining was performed on all endometrial biopsies and plasma cells were identified by morphology. Immunohistochemical (IHC) staining for CD138 was later applied to all tissue samples. Charts were reviewed to evaluate the outcome of the next clinical intrauterine pregnancy. MAIN OUTCOME MEASURE(S) Miscarriage rate and live birth rate. RESULT(S) A total of 107 women met inclusion criteria. The use of CD138 IHC staining resulted in a significantly higher prevalence of CE compared with the use of H & E staining and morphological assessment alone (56% [60/107] vs. 13% [14/107]). The 51 women with untreated CE were compared with the 45 women without CE by CD138 staining. Among those women with a subsequent pregnancy, the live birth rate in the next clinical intrauterine pregnancy after endometrial evaluation was 67.6% (23/34) in women with untreated CE and 87.1% (27/31) in women without CE. Age, body mass index (BMI), results of RPL evaluation, and number of prior losses were not significantly different between the two groups. CONCLUSION(S) CD138 IHC staining of endometrial biopsies in women with RPL provides increased sensitivity when screening for CE compared with H & E staining and morphological assessment alone. Untreated CE may contribute to poor pregnancy outcomes and deserves further investigation in a larger cohort.

First Trimester Miscarriage Evaluation

Miscarriage is a relatively common occurrence for otherwise healthy women. Despite its frequency, evaluation for cause is rare. The most common cause of miscarriage is sporadic chromosome errors. Chromosomal analysis of the miscarriage offers an explanation in at least 50% of cases. Conventional cytogenetic evaluation can only be done on fresh tissue, so it is critical that the treating physician consider genetic testing at the time of the miscarriage. Ultrasound can estimate the gestational age at the time of miscarriage and identify major abnormalities in some embryos. A careful pathological examination can add to the evaluation by ruling out rare disorders with the highest recurrence risk. A multidisciplinary approach to miscarriage evaluation is essential to understanding the cause and risk of recurrence. A thorough evaluation of a miscarriage, in combination with emotional support, can often provide the necessary reassurance and confidence as the patient prepares for her next pregnancy.

Ovarian surface epithelial neoplasms in the pediatric population: incidence, histologic subtype, and natural history.

Surface epithelial neoplasms account for a small but significant proportion of pediatric ovarian tumors. The overall incidence, prevalence of histologic subtypes, and natural history of these neoplasms has not been thoroughly evaluated. A retrospective review of the pathology archives of Stanford University School of Medicine yielded 69 surface epithelial ovarian tumors in 64 pediatric patients 18 years of age or younger from 1974 to 2010. Tumors comprised benign (57.8%), borderline/low malignant potential (LMP) (37.5%), and malignant (4.7%) subgroups and exhibited serous, mucinous, and mixed histology; there were no clear cell, pure endometrioid, or transitional (Brenner) tumors. In addition, no high-grade carcinomas were identified. Clinical follow-up data were available in a subset of patients (maximum follow-up, 22 y). Similar numbers of recurrences were found in each of the 3 subgroups. However, overall survival was 100% for benign and borderline/LMP tumors and 50% for carcinomas. The type of surgical management and the use of chemotherapy varied; 2 patients with borderline/LMP tumors were treated by sterilizing procedures and/or chemotherapy. These data suggest that surface epithelial neoplasms comprise a small but significant proportion of ovarian tumors in the pediatric population, and they exhibit a marked preponderance for benign, borderline, and low-grade malignant subgroups. In contrast to their adult counterpart, high-grade serous carcinoma in children is extraordinarily rare and not seen in this series. Given this difference, uniform treatment modalities with consideration for ovarian conservation and fertility preservation should be rigorously adopted in any pediatric patient with a suspected ovarian surface epithelial neoplasm.

Evaluation of intestinal biopsies for pediatric enteropathy: a proposed immunohistochemical panel approach.

Congenital enteropathies are rare disorders with significant clinical consequences; however, definitive diagnosis based on morphologic assessment of duodenal biopsies with routine stains alone is often impossible. To determine the role of immunohistochemistry (IHC) in the evaluation for microvillous inclusion disease, congenital tufting enteropathy (intestinal epithelial dysplasia), and enteroendocrine cell dysgenesis, a series of duodenal biopsies from 26 pediatric patients with chronic/intractable diarrhea was retrospectively reviewed. IHC stains for CD10, EpCAM, chromogranin, and villin were performed on all biopsies, and the results were correlated with hematoxylin and eosin and ultrastructural findings using electron microscopy, when available. Biopsies from 2 patients diagnosed with microvillous inclusion disease at the time of original biopsy demonstrated diffuse CD10-positive cytoplasmic inclusions within enterocytes and normal expression of EpCAM and chromogranin. Biopsies from 3 patients, including 2 siblings with confirmed EPCAM mutations, demonstrated complete loss of EpCAM expression and normal expression of CD10 and chromogranin; electron microscopic evaluation revealed characteristic ultrastructural findings of tufting enteropathy. Biopsies from 1 patient with a confirmed NEUROG3 mutation demonstrated an absence of intestinal enteroendocrine cells by chromogranin staining, consistent with enteroendocrine cell dysgenesis. Four patients’ biopsies displayed nonspecific staining patterns for CD10 and/or EpCAM with normal expression of chromogranin, and 16 patients’ biopsies exhibited normal expression for all 3 markers. Villin stains demonstrated heterogenous brush border labeling with nonspecific cytoplasmic reactivity, a pattern variably present throughout the biopsy series. In conclusion, the routine use of an IHC panel of CD10, EpCAM, and chromogranin is warranted in patients meeting specific age and/or clinical criteria, as the morphologic findings of congenital enteropathies may be subtle, focal, or inapparent on routine stains.

Liver Pathology in Infantile Mitochondrial DNA Depletion Syndrome

Mitochondrial DNA (mtDNA) depletion syndrome is a relatively novel cause of hepatic dysfunction in the pediatric population. It is caused by mutations in either mtDNA or nuclear DNA (nDNA) that result in a quantitative reduction in mtDNA and, in turn, dysfunctional oxidative phosphorylation. In infants, it results in the hepatocerebral phenotype, characterized by hyperbilirubinemia, coagulopathy, lactic acidosis, hypoglycemia, lethargy, encephalopathy, developmental delay, and hypotonia. Three infants diagnosed with mtDNA depletion syndrome at The Childrens Hospital of Philadelphia were identified, and their clinical presentation, disease course, and histologic and ultrastructural features of liver samples (pre- and postmortem) were characterized. While a different mutant gene was identified in each child, they all showed clinical evidence of metabolic dysfunction soon after birth and expired by 1 year of age. Steatosis, cholestasis, and cytoplasmic crowding by atypical mitochondria were consistent pathologic liver findings. Other findings included hepatocyte hypereosinophilia, fibrosis, and hemosiderosis. This analysis provides insight into the important clinical signs/symptoms and histopathologic and ultrastructural features of mtDNA depletion syndrome in infants and young children. Knowledge of these characteristics will facilitate early recognition and appropriate treatment of this rare disorder. Additionally, ultrastructural evaluation of liver samples by electron microscopy is an important diagnostic component of hepatic dysfunction caused by metabolic abnormalities. This type of analysis should be routinely employed in the setting of unexplained cholestasis, especially when accompanied by steatosis and hepatocyte hypereosinophilia.

Predictors of acute chemotherapy‐associated toxicity in patients with Ewing sarcoma

Ewing sarcoma (ES) is a malignant tumor of bone and soft tissue of children and young adults. Patients with ES are treated with intensive chemotherapy regimens. We describe predictors of acute chemotherapy‐associated toxicity in this population.

Congenital Pancreatoblastoma: Report of an Atypical Case and Review of the Literature

Pancreatoblastoma is a rare malignant tumor of the pancreas mostly diagnosed in childhood. The clinical presentation and outcome of infantile and congenital pancreatoblastoma have not been clearly elucidated. This report describes our recent institutional experience with an unusual case of congenital pancreatoblastoma. Review of the scientific literature identifies approximately 200 cases of pancreatoblastoma. We describe the 9 infantile (aged 3 mo and younger) and 4 congenital cases previously reported and summarize their clinical presentation and outcome. We also define the close association of infantile/congenital pancreatoblastoma and Beckwith-Wiedemann syndrome (50%) versus all affected age groups (4.5%).

Comparison of Latino and Non-Latino Patients with Ewing Sarcoma

Ewing sarcoma (ES) is a malignancy of bone and soft tissue in children and adults. Previous registry‐based studies indicate that Latino patients with ES have inferior outcomes compared to non‐Latino patients, though an etiology for this difference could not be identified. To explore possible differences that might underlie this disparity, we conducted a retrospective study to compare clinical characteristics, tumor features, healthcare access, and treatment outcomes between Latino and non‐Latino patients with ES.

Tissue microarrays from bone marrow aspirates for high-throughput assessment of immunohistologic markers in pediatric acute leukemia.

Gene expression profiling studies have been employed to investigate prognostic subgroups in pediatric acute leukemia. Tissue microarrays (TMAs) are useful for high-throughput analysis of protein expression of target genes in acute leukemia samples and for validation of gene microarray analysis. Using cryopreserved samples of pediatric acute leukemia bone marrow aspirates, we constructed TMA from as few as 1 million cells. Bone marrow core biopsies from the same patients were included on the same TMA for comparison. A panel of 15 immunohistochemical markers typically used for diagnosis as well as those targeting recently characterized, prognostically relevant molecules of interest in pediatric acute leukemia was used to evaluate protein expression. Staining results confirm that suspension cells from bone marrow aspirates can be effectively used to derive protein expression data from multiple cases simultaneously with comparable efficacy to that of biopsy tissue. This method allows for new markers of diagnostic, prognostic, or therapeutic importance to be screened on large numbers of study patients. Furthermore, this technique may facilitate the inclusion of small samples, aspirates, and body fluids in large-scale studies of protein expression in clinical trials and protocols in which tissue biopsies are often unavailable.