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Evidence suggests that the practice of sharing clinicians’ notes with patients via online patient portals may increase patient engagement and improve patient–clinician relationships while requiring little change in providers’ workflow. Authors examined clinical social workers’ experiences and attitudes related to open psychotherapy notes using focus groups and telephone interviews. Twenty-four of 29 eligible therapists agreed to open their notes to patients, and nine participated in this study. Participants were generally positive about their experiences and reported few disruptions to their workload or practice. However, they were hesitant to bring up notes to patients during sessions, and they discussed the benefits of open therapy notes mostly hypothetically. The five therapists who did not share notes worried that open notes would be detrimental to therapeutic relationships, patient well-being, and workflow. However, the concern they discussed most often related to the electronic health record rather than to open notes, because therapy notes are visible to all authorized clinicians as part of the general medical record. Future research is needed to deepen our understanding of the risks and benefits of open psychotherapy notes and to inform development of training programs to support therapists in opening notes.

Taking advantage of the deep targeted sequencing capabilities of next generation sequencers, we have developed a novel two step insertion deletion (indel) detection algorithm (IDA) that can determine indels from single read sequences with high computational efficiency and sensitivity when indels are fractionally less compared to wild type reference sequence. First, it identifies candidate indel positions utilizing specific sequence alignment artifacts produced by rapid alignment programs. Second, it confirms the location of the candidate indel by using the Smith-Waterman (SW) algorithm on a restricted subset of Sequence reads. We demonstrate that IDA is applicable to indels of varying sizes from deep targeted sequencing data at low fractions where the indel is diluted by wild type sequence. Our algorithm is useful in detecting indel variants present at variable allelic frequencies such as may occur in heterozygotes and mixed normal-tumor tissue.

The DNA mismatch repair (MMR) pathway corrects specific types of DNA replication errors that affect microsatellites and thus is critical for maintaining genomic integrity. The genes of the MMR pathway are highly conserved across different organisms. Likewise, defective MMR function universally results in microsatellite instability (MSI) which is a hallmark of certain types of cancer associated with the Mendelian disorder hereditary nonpolyposis colorectal cancer. (Lynch syndrome). To identify previously unrecognized deleted genes or loci that can lead to MSI, we developed a functional genomics screen utilizing a plasmid containing a microsatellite sequence that is a host spot for MSI mutations and the comprehensive homozygous diploid deletion mutant resource for Saccharomyces cerevisiae. This pool represents a collection of non-essential homozygous yeast diploid (2N) mutants in which there are deletions for over four thousand yeast open reading frames (ORFs). From this screen, we identified a deletion mutant strain of the PAU24 gene that leads to MSI. In a series of validation experiments, we determined that this PAU24 mutant strain had an increased MSI-specific mutation rate in comparison to the original background wildtype strain, other deletion mutants and comparable to a MMR mutant involving the MLH1 gene. Likewise, in yeast strains with a deletion of PAU24, we identified specific de novo indel mutations that occurred within the targeted microsatellite used for this screen.

Childhood leukemia, which accounts for > 30% of newly diagnosed childhood malignancies, is one of the leading causes of death for children with cancer. Genome-wide studies using microarray chips to identify copy number changes in human cancer are becoming more common. In this pilot study, 45 pediatric leukemia samples were analyzed for gene copy aberrations using novel molecular inversion probe (MIP) technology. Acute leukemia subtypes included precursor B-cell acute lymphoblastic leukemia (ALL) (n = 23), precursor T-cell ALL (n = 6), and acute myeloid leukemia (n = 14). The MIP analysis identified 69 regions of recurring copy number changes, of which 41 have not been identified with other DNA microarray platforms. Copy number gains and losses were validated in 98% of clinical karyotypes and 100% of fluorescence in situ hybridization studies available. We report unique patterns of copy number loss in samples with 9p21.3 (CDKN2A) deletion in the precursor B-cell ALL patients, compared with the precursor T-cell ALL patients. MIPs represent an attractive technology for identifying novel copy number aberrations, validating previously reported copy number changes, and translating molecular findings into clinically relevant targets for further investigation.

Molecular inversion probe (MIP) technology has been demonstrated to be a robust platform for large-scale dual genotyping and copy number analysis. Applications in human genomic and genetic studies include the possibility of running dual germline genotyping and combined copy number variation ascertainment. MIPs analyze large numbers of specific genetic target sequences in parallel, relying on interrogation of a barcode tag, rather than direct hybridization of genomic DNA to an array. The MIP approach does not replace, but is complementary to many of the copy number technologies being performed today. Some specific advantages of MIP technology include: less DNA required (37 ng vs. 250 ng), DNA quality less important, more dynamic range (amplifications detected up to copy number 60), allele-specific information "cleaner" (less SNP cross-talk/contamination), and quality of markers better (fewer individual MIPs versus SNPs needed to identify copy number changes). MIPs can be considered a candidate gene (targeted whole genome) approach and can find specific areas of interest that otherwise may be missed with other methods.

We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing.

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.

Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the leading cause of death for children with cancer. Genomic instability events contribute to tumorigenesis and have been used to classify and risk stratify adult and pediatric cancers. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution, and can detect both gene copy number and loss of heterozygosity (LOH) events in clinical samples. Studying pediatric leukemia samples with MIP technology may identify new molecular alterations that could prove useful in risk stratification and discovery of new therapeutic targets for childhood leukemia. Objective: To use MIP technology to identify novel areas of allelic imbalance in childhood leukemia. Methods: DNA was extracted from leukemia blasts at diagnosis (n=45, 23 pre-B ALL, 14 AML, 7 pre-T ALL, 1 Burkitt’s). DNA was also extracted from normal peripheral blood collected at remission to use as paired germline controls. The MIP assay was run with a customized Affymetrix 24K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). DNA required for this assay was limited to 75 ng per sample. Copy number changes and LOH were identified by comparing probe signal intensity between leukemia and normal germline samples. Clinical cytogenetic data (karyotype and FISH analysis) was used as a control to confirm findings from known areas of allelic imbalance. Results: Each leukemia sample had unique patterns of allelic imbalance distributed across all chromosomes. MIPs identified all clinically reported cytogenetic copy number changes for each sample, in addition to areas of allelic imbalance not clinically reported. Samples had recurring areas of copy number changes and LOH events shared by all leukemia types. MIPs detected areas of allelic imbalance in both previously described and novel genes. Areas of recurring genomic deletions included: ATR (3q23), TLX3 (5q35.1), ADRB3 (8p12), CDKN2A (9p21.3), DOCK8 (9p24.3), PAX5 (9p13.2), PTPN11 (12q24.13), C3AR1 (12p13.31), TCRA (14q11.2), AKT1 (q14q32.33). Areas of recurring genomic amplification included: SLC2A9 (4p16.1), RAI14 (5p13.2), CDH12 (5p14.3), PMCHL1(5p14.3), AURKB (17p13.1). These findings are being validated with Quantitative Real-Time PCR. Conclusions: MIPs represent a novel genomic technology that can identify previously unreported gene copy number and LOH events in childhood leukemia. Unique and overlapping areas of allelic imbalance were found in both childhood ALL and AML clinical samples. The shared genomic regions of allelic imbalance between different leukemia types may represent a common molecular mechanism of leukemogenesis that warrants further investigation. Analysis of more childhood leukemia samples through Pediatric Cooperative Groups may help to determine how common these areas of allelic imbalance are in children and whether they are of prognostic significance. Further exploration of these copy number and LOH events may help us to better understand the biology of childhood leukemia and its clinical behavior.

Background: Follicular lymphoma, derived from germinal center B cells, is one of the most common types of lymphoma. Follicular lymphoma like many other malignancies demonstrates genomic instability, a phenomenon where tumor cells accumulate various genetic changes including point mutations, genomic deletions and gene amplifications. Such genomic aberrations may contribute to cancer phenotype such as transformation. Previous studies of follicular lymphoma have shown a combination of cytogenetic - aneuploidy type events from gross chromosomal alterations to smaller deletions and amplifications. For example, deletions in chromosome 6q may be associated with follicular lymphoma aggressiveness. Efforts to identify these changes have used traditional karyotyping, comparative genomic hybridization (CGH) or traditional genetic markers (microsatellites) for loss of heterozygosity (LOH) analysis. Characterization of of genomic instability in follicular lymphoma has not been thoroughly investigated on a genomic scale. Objectives: We have employed a novel genomic technology relying on molecular inversion probes (MIPs) to conduct a large scale analysis of gene copy aberrations in follicular lymphoma. MIPs technology enables querying any gene sequence in the genome at a high resolution for allele-specific gene copy alterations and polymorphisms simultaneously, a significant improvement over other genomic methods such as array CGH. We are using this genomic technology to delineate concordant gene copy aberrations in follicular lymphoma that may influence a variety of clinical outcomes using a probe set with over 7,000 cancer-related genes among 25,000 loci. Methods: DNA was extracted from a cohort of follicular lymphoma clinical samples obtained at the time of diagnosis and prior to treatment. B-cell lymphoma cells were purified using a Rosette purification scheme, genomic DNA was isolated from the purified tumor cell populations from 37 individual lymphoma samples and the cohort was analyzed with a MIP cancer probe set described above. Each probe contains two unique recognition sequences to a genomic DNA target and a unique barcode sequence. The molecular inversion probe assay was conducted. The final products were hybridized to a generic barcode microarray overnight and interrogated by an Affymetrix scanner. Barcode array data was preprocessed and gene copy number was extrapolated for each molecular inversion probe. Concordant gene copy aberrations were determined across the sample set. Results: Follicular lymphoma samples from pretreated patients have common patterns of chromosomal losses, gains and multiple gene deletions with large overlap in some areas of deletion. Common large scale chromosomal deletions and amplifications included regions of chromosome 6 and 18. Multiple individual genes were found to be commonly amplified or deleted as well including BTK which is suspected to play a role in follicular lymphoma. Conclusions: MIP technology has been used to analyze follicular lymphoma for highly sensitive and specific gene copy analysis. We identified concordant gene copy alterations found across the majority of samples in the cohort, suggesting that these aberrations may play a role in follicular lymphoma development. We are pursuing additional studies to confirm these findings.

9530 Background: Leukemia accounts for ∼40% of newly diagnosed pediatric malignancies, and relapsed leukemia is the leading cause of death in childhood cancer. Genomic instability events contribute to neoplastic development and have been used to classify and risk stratify non-leukemic adult and pediatric tumors. Analyzing leukemic blasts for gene copy changes with advanced molecular techniques could prove useful in further risk stratifying and developing new treatment strategies for pediatric leukemia. Methods: Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel with high specificity and sensitivity at the highest genomic resolution, and were originally designed for single nucleotide genotyping. The MIP assay was adapted to analyze both gene copy number and loss of heterozygosity (LOH) events in pediatric leukemia samples (pre-B ALL, T-ALL, AML). DNA was extracted (100 ng) from paired bone marrow (diagnosis) and peripheral blood (remission) samples (n = 40). The MIP assay was run with a customized Affymetrix 20K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). Gene copy number changes were identified by comparing probe signal intensity between leukemia samples and normal cell-lines. LOH events were determined by identifying genotype changes between matched leukemic and remission samples. Results: Each sample had unique patterns of multiple gene copy changes and LOH events distributed across all chromosomes. Additionally, samples were found to have overlapping copy number changes and LOH regardless of leukemia type. AML samples had fewer LOH events and could be separated by unsupervised clustering from the other leukemia samples. Conclusions: MIPs represent novel genotyping technology that can be adapted for gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of LOH and deleted genes may represent a common molecular mechanism that requires further investigation. No significant financial relationships to disclose.

Background: Leukemia is the most common pediatric malignancy, accounting for nearly 40% of all new childhood cancer. The cure rates for pediatric ALL have increased to more than 80% and the cure rates for pediatric AML now approach 50%. Much of this progress can be attributed to cytogenetic and molecular risk stratification with subsequent randomized control trials. Genomic instability events may also serve as relevant prognostic biomarkers. A novel high-throughput genomic technology called Molecular Inversion Probes (MIPs) quantifies genomic instability, gene copy number and allelic imbalances at the highest genomic resolution. MIPs can analyze genetic target sequences in parallel with high specificity and sensitivity. Further classifying leukemic blasts using more precise molecular techniques could prove useful in further risk stratifying and developing new treatment strategies. Objective: To adapt MIPs to characterize and define unique molecular subtypes of childhood leukemia. Methods: DNA was extracted from AML and ALL clinical samples obtained at diagnosis. 400 ng of leukemic DNA was mixed with a previously synthesized MIP cancer probe set (667 probes representing all human chromosomes from exon sequences of 298 cancer genes); each probe contains two unique recognition sequences to targeted genomic DNA and a unique barcode (tag) sequence. A thermostable polymerase and ligase were added, and the mixture was denatured. Probe homology sequences were then annealed to complementary genome sites. Standard PCR reagants were added including one fluorescent-labeled primer and unlabeled primer. The reaction was thermocycled and probes circularized in the nucleotide specific “gap fill” reaction were amplified. The PCR mixture was hybridized to a barcode microarray overnight. The chip was stained and washed, and then interrogated by an Affymetrix scanner via argon laser excitation at 488-nm. ‘Cluster along Chromosomes’ analysis was performed to detect significant amplifications or deletions. Results: Childhood ALL and AML samples had unique patterns of multiple gene deletions. Additionally, overlap was found in several of these deleted genes. Significantly deleted genes in common between ALL and AML samples included: RAP1GA1, HYAL1, HSPA9B, CSF1R, TNF, NOTCH4, FANCE, CCND3, BAG2, PLAGL1, MAD1L1, POLM, POLD2, IGFBP3, PSD, SUFU, CYP17A1, MXI1, HRAS, IGF2, RRM1, BRCA2, TP53, ERBB2, ERCC2, and POLD1. Conclusions: MIPs represent a novel technology for highly sensitive and specific gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of deleted genes in both ALL and AML may represent a common molecular mechanism that requires further investigation. We currently are adapting MIPs for an expanded cancer probe set and are exploring new techniques to determine loss of heterozygosity (LOH) in pediatric leukemia.