10502 Background: Patients with INSS stage III neuroblastoma represent a heterogeneous population with respect to disease presentation and prognosis and controversy exists regarding the most effective treatment algorithms. Patients ≥18 months of age with INSS Stage 3 tumors that are unfavorable histology (UH) and MYCN-non-amplified ( MYCN-NA) represent a small cohort of patients with an outcome intermediate of those with favorable histology tumors and MYCN amplified tumors. The presence of Segmental Chromosome Aberrations (SCA) may predict outcome; however, their impact specifically in this cohort of patients has not been reported. Methods: Eligible patients enrolled on therapeutic protocols A3973 (n=34), ANBL0532 (n=27), and biology protocol ANBL00B1 (n=101 with 29 treated on A3973/ANBL0532) with stage III disease, MYCN–NA, UH and age ≥18 months at diagnosis were analyzed. Copy number alterations and loss of heterozygosity (LOH) for relevant loci were scored for gains/losses by two independent reviewers. Results: The 5-year EFS/OS for children ≥18 months with stage III, MYCN–NA, UH disease treated on A3973 and ANBL0532 was 73.0±8.1%/87.9±5.9% and 61.4±10.2%/ 73.0±9.2%, respectively, with no statistical differences in EFS or OS between the two cohorts (p=0.1286 and p=0.2180, respectively). In the combined cohort of patients enrolled on A3973 and ANBL0532, statistically significant differences were found (p(s) <0.0001) in patients with CR/VGPR (n=39) and PR (n=13) having better outcomes than <PR (n=5) (5-year EFS: 74.0±7.6% vs. 75.0±12.5% vs. 0%; 5-year OS: 84.4±6.2% vs. 100% vs. 20.0±17.9%). Subjects with chromosome 11q loss/LOH had an inferior outcome in comparison to those without 11q loss/LOH (10-year EFS: 44.4+/-24.1% vs. 78.1+/-9.4%, p=0.01; 10-year OS: 62.4+/-15.9% vs. 85.9+/-7.8%, p=0.02)). Patients with 1p loss/LOH and 2p gain also showed trend towards worse event-free survival (p=0.086 and p=0.088, not statistically significant) but not in overall survival. Conclusions: High-risk therapy that included single myeloablative therapy led to an 81.6±5.3%5-year OS in patients ≥18 months with UH and MYCN–NA stage III neuroblastoma. Response to therapy is a powerful predictor of survival and the presence of chromosome 11q loss/LOH is also associated with inferior outcomes. These patients should continue to be treated on high-risk clinical trials.
Synpolydactyly 1, also called syndactyly type II (SDTY2), is a genetic limb malformation characterized by polydactyly with syndactyly involving the webbing of the third and fourth fingers, and the fourth and fifth toes. It is caused by heterozygous alterations in HOXD13 with incomplete penetrance and phenotypic variability. In our study, a five-generation family with an SPD phenotype was enrolled in our Rare Disease Genomics Protocol. A comprehensive examination of three generations using Illumina short-read whole-genome sequencing (WGS) did not identify any causative variants. Subsequent WGS using Pacific Biosciences (PacBio) long-read HiFi Circular Consensus Sequencing (CCS) revealed a heterozygous 27-bp duplication in the polyalanine tract of HOXD13. Sanger sequencing of all available family members confirmed that the variant segregates with affected individuals. Reanalysis of an unrelated family with a similar SPD phenotype uncovered a 21-bp (7-alanine) duplication in the same region of HOXD13. Although ExpansionHunter identified these events in most individuals in a retrospective analysis, low sequence coverage due to high GC content in the HOXD13 polyalanine tract makes detection of these events challenging. Our findings highlight the value of long-read WGS in elucidating the molecular etiology of congenital limb malformation disorders.
It is well known that fetal cells are present in maternal circulation during normal pregnancy. However, the techniques employed to isolate these cells are generally complex and cumbersome. Furthermore, the number of fetal cells isolated from the maternal circulation using these approaches tends to be quite small1-4. Here, we present a case of a pregnancy that was complicated by digynic triploidy in which circulating trophoblasts, both mononucleate and multinucleate, were isolated using a novel high-throughput microfluidic platform. Fluorescence in-situ hybridization (FISH) was then employed to evaluate the number of chromosomes in the cells of the enriched sample. A 32-year-old Caucasian female, gravida 2 para 1, presented at 13 weeks' gestation (by last menstrual period) for first-trimester combined screening. The fetal crown–rump length was consistent with 11 weeks' gestation. The fetal body was noted to be small in comparison to the head and there was suspicion of ventriculomegaly. The placenta was noted to be thin. The patient was offered chorionic villus sampling (CVS) due to the high suspicion of digynic triploidy. She declined CVS at the time but agreed to have non-invasive prenatal screening (NIPS). NIPS indicated a high risk of chromosomal abnormality due to low fetal DNA fraction, but the type of aneuploidy could not be specified. The patient underwent transcervical CVS 2 weeks later without complications. Following CVS, a 14-mL blood sample was collected from the patient, after obtaining informed consent, as part of an ongoing study (IRB #06488, Wright State University, Dayton, OH, USA) evaluating the ability of a novel high-throughput microfluidic platform (LiquidScan™ BioFluidica Inc., San Diego, CA, USA) to positively enrich circulating fetal cells (trophoblasts). The sample was transported overnight from Dayton, Ohio, to San Diego, California, for processing. Routine FISH was performed on the enriched cell sample using chromosome enumerator probes (CEP) (Oxford Gene Technology, Inc., Tarrytown, NY, USA) for chromosomes X, Y and 18 and locus-specific identifier (LSI) probes (Oxford Gene Technology, Inc.) for chromosomes 13 and 21 (Figures 1 and 2). A total of 28 cells with triplicate chromosome complement were identified (Figure 1). Additionally, in 13 cells the number of chromosomes was above that expected for triploidy (Figure 2). Even though it is possible that these entities represent groups of cells, the close apposition of the chromosomes suggests these are more likely to be multinucleate cells originating from the syncytiotrophoblast layer. FISH performed on the CVS sample demonstrated the same complement of chromosomes as that detected in the circulating trophoblasts. The final result on the cultured fetal cells from the CVS sample confirmed a 69,XXX karyotype. The fetus died in the mid second trimester. To our knowledge, this is only the second report of prenatal evaluation of the fetal karyotype in circulating trophoblasts using FISH, which revealed the presence of fetal triploidy4. In our case, FISH analysis of circulating trophoblasts clarified the specific type of aneuploidy in a setting in which the NIPS result was non-specific. Additionally, our results suggest that it may be possible to enrich multinucleate cells of fetal origin. Isolation of fetal cells from maternal circulation has been attempted for many decades with variable success. These efforts have been hampered by the relative low number of circulating fetal cells. There are primarily three types of fetal cells which have been investigated: nucleated red blood cells, lymphocytes and trophoblasts. The trophoblasts have been of the greatest interest in recent years. The numerous methods that have been employed to enrich fetal trophoblasts are beyond the scope of this communication. However, they are mostly comprised of maternal white blood cell depletion methods and positive enrichment strategies1-4. The enrichment method employed in this case was developed by BioFluidica, Inc. and is based on the use of a microfluidic platform. This technique was originally developed to enrich circulating tumor cells5. Briefly, the core technology of this platform consists of a programable microfluidic chip that captures target cells directly from whole blood or other body fluids without the need for preliminary gross separation. The chip can be manufactured so its surface is covered with any antibody or combination of antibodies, which target specific antigens on the cell surface. Whole blood is used without any additional preparation, except for mixing the blood sample with a proprietary non-fixative preservative before it is sent to the laboratory in order to prolong viability of the target cells. When blood is infused into the chip, target cells are selectively captured by the antibodies and remain on the chip while other blood components are extruded from the chip. In this case, mouse anti-huEpCAM antibody was used. The captured cells are eluted from the microfluidic chip following enzymatic release from the microfluidic surface and prepared for future evaluation such as immunocytochemical fluorescence staining, FISH and quantitative polymerase chain reaction. We consider captured cells with positive fluorescent stain for CK7 and negative for CD45 as likely to be of fetal origin. We acknowledge that the number of fetal cells, both mononucleate and multinucleate, could have been enhanced by the fact that CVS was performed prior to the blood draw. We also recognize that much remains to be done to fully evaluate the potential of the methodology used in the presented case. However, we believe that our findings show that microfluidic technology may become clinically useful in the future. The authors thank the entire San Diego-based BioFluidica Scientific Team (Kulvinder Kaur, Janet Cravens Dickerson, Jennifer Barber-Singh, Brenda Garcia Lopez, Dylan Dufek and Judy Muller-Cohn) for their tireless effort without which these results would not be possible. R.M. is an employee of BioFluidica, Inc. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Wolf-Hirschhorn syndrome (WHS) is a rare but recurrent microdeletion syndrome associated with hemizygosity of an interstitial segment of Chromosome 4 (4p16.3). Consistent with historical reports in which overlapping deletions defined a minimal critical region in WHS patients, recent reports from exome sequence analysis have provided further evidence that haploinsufficiency of a specific gene within this critical region, NSD2 (WHSC1), is causal for many features of the syndrome. In this report, we describe three unrelated patients with loss-of-function alterations in NSD2 who presented clinically with WHS features including intrauterine growth retardation and global developmental delay. Two of the three patients also had overlapping features of failure to thrive, short stature, constipation, and hypotonia. This series adds additional cases to expand the phenotypic spectrum of WHS and reports novel NSD2 variants.
In January 2007 the American Medical Association added a new Current Procedural Terminology(R) (CPT) code, 96040, for "Medical Genetics and Genetic Counseling Services." In order to identify the impact of having this new code and to identify issues with implementation of the code, the National Society of Genetic Counselors (NSGC) CPT(R) Working Group surveyed NSGC members using an internet-based survey tool. The majority of respondents (94%) reported being aware of the new code and over half of the respondents (69%) said they were billing for genetic counseling. Approximately 24% of those billing reported using 96040. Many facilities are not using this code and the reported success of billing using 96040 is highly varied. Continued education may be beneficial to encourage reimbursement for 96040 and follow up is needed to assess the ongoing implementation and impact of the new CPT(R) code.
