Genomic and clinical characterization of early T-cell precursor lymphoblastic lymphoma.

Early T-cell precursor phenotype acute lymphoblastic leukemia (ETP-ALL) is a subtype of T-ALL with a unique immunophenotype and genetic abnormalities distinct from conventional T-ALL. A subset of T lymphoblastic lymphoma (T-LLy) also demonstrates the early T-cell precursor immunophenotype and may be a counterpart of ETP-ALL. Unlike ETP-ALL, the incidence, clinical features, and genomic features of ETP-LLy are unknown. We reviewed the immunophenotyping data of 218 T-LLy patients who enrolled in the Children's Oncology Group AALL0434 clinical trial and identified 9 cases (4%) exhibiting a definitive ETP immunophenotype. We performed single-nucleotide polymorphism array profiling on 9 ETP-LLy and 15 non-ETP T-LLy cases. Compared with non-ETP T-LLy, ETP-LLy showed less frequent deletion of 9p (CKDN2A/B), more frequent deletion of 12p (ETV6) and 1p (RPL22), and more frequent absence of biallelic T-cell receptor γ deletions. Recurrent abnormalities previously described in ETP-ALL such as deletions of 5q and 13q and gain of 6q were not observed in ETP-LLy cases. There were no failures of therapy among the ETP-LLy subtype with a 4-year event-free survival of 100%. Overall, ETP-LLy does not exhibit unifying genetic alterations but shows some distinct genomic features from non-ETP T-LLy suggesting that ETP-LLy may be a distinct entity from non-ETP T-LLy.

Genomic landscape of cutaneous follicular lymphomas reveals 2 subgroups with clinically predictive molecular features.

Primary cutaneous follicle center lymphomas (PCFCLs) are indolent B-cell lymphomas that predominantly remain skin restricted and manageable with skin-directed therapy. Conversely, secondary cutaneous involvement by usual systemic follicular lymphoma (secondary cutaneous follicular lymphoma [SCFL]) has a worse prognosis and often necessitates systemic therapy. Unfortunately, no histopathologic or genetic features reliably differentiate PCFCL from SCFL at diagnosis. Imaging may miss low-burden internal disease in some cases of SCFLs, leading to misclassification as PCFCL. Whereas usual systemic FL is well characterized genetically, the genomic landscapes of PCFCL and SCFL are unknown. Herein, we analyzed clinicopathologic and immunophenotypic data from 30 cases of PCFCL and 10 of SCFL and performed whole-exome sequencing on 18 specimens of PCFCL and 6 of SCFL. During a median follow-up of 7 years, 26 (87%) of the PCFCLs remained skin restricted. In the remaining 4 cases, systemic disease developed within 3 years of diagnosis. Although the SCFLs universally expressed BCL2 and had BCL2 rearrangements, 73% of the PCFCLs lacked BCL2 expression, and only 8% of skin-restricted PCFCLs had BCL2 rearrangements. SCFLs showed low proliferation fractions, whereas 75% of PCFCLs had proliferation fractions >30%. Of the SCFLs, 67% had characteristic loss-of-function CREBBP or KMT2D mutations vs none in skin-restricted PCFCL. Both SCFL and skin-restricted PCFCL showed frequent TNFRSF14 loss-of-function mutations and copy number loss at chromosome 1p36. These data together establish PCFCL as a unique entity with biological features distinct from usual systemic FL and SCFL. We propose 3 criteria based on BCL2 rearrangement, chromatin-modifying gene mutations (CREBBP, KMT2D, EZH2, and EP300), and proliferation index to classify cutaneous FL specimens based on the likelihood of concurrent or future systemic spread.

Genomic Analyses Identify Recurrent Alterations in Immune Evasion Genes in Diffuse Large B-Cell Lymphoma, Leg Type.

Cutaneous diffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas with a poor prognosis. To elucidate their genetic bases, we analyzed exome sequencing of 37 cutaneous DLBCLs, including 31 DLBCLs, leg type (DLBCL-LT) and 6 cutaneous DLBCLs-not otherwise specified (DLBCL-NOS). As reported previously, 77% of DLBCL-LT harbor NF-κB-activating MYD88 mutations. In nearly all MYD88-wild-type DLBCL-LT, we found cancer-promoting mutations that either activate the NF-κB pathway through alternative genes (NFKBIE or REL) or activate other canonical cancer pathways (BRAF, MED12, PIK3R1, and STAT3). After NF-κB, the second most commonly mutated pathway putatively enables immune evasion via mutations predicted to downregulate antigen processing (B2M, CIITA, HLA) or T-cell co-stimulation (CD58). DLBCL-LT have little genetic overlap with the genetically heterogeneous DLBCL-NOS. Instead, they resemble primary central nervous system and testicular large B-cell lymphomas (primary central nervous system lymphomas and primary testicular lymphomas). Like primary central nervous system lymphomas/primary testicular lymphomas, 40% of DLBCL-LT (vs. 0% of DLBCLs-not otherwise specified) harbored PDL1/PDL2 translocations, which lead to overexpression of PD-L1 or PD-L2 in 50% of the cases. Collectively, these data broaden our understanding of cutaneous DLBCLs and suggest novel therapeutic approaches (e.g., BRAF or PI3K inhibitors). Additionally, they suggest novel treatment paradigms, wherein DLBCL-LT can be targeted with strategies (e.g., immune checkpoint blockers) currently being developed for genomically similar primary central nervous system lymphomas/primary testicular lymphomas.

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

AIMS: Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). METHODS: For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. RESULTS: We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. CONCLUSIONS: We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay.

Genetic evaluation of juvenile xanthogranuloma: genomic abnormalities are uncommon in solitary lesions, advanced cases may show more complexity.

Juvenile xanthogranuloma is a rare histiocytic proliferation primarily affecting infants and young children, characterized by aberrant infiltration of histiocyte-derived cells in the skin, soft tissues and more rarely, visceral organs. Juvenile xanthogranuloma is generally considered to be a benign disorder; most lesions are solitary cutaneous nodules that resolve spontaneously without treatment. However, cases with extracutaneous involvement, multiple lesions, and/or systemic disease often require aggressive therapy. Though molecular studies have provided evidence of clonality in juvenile xanthogranuloma, in support of a neoplastic process, little is known about the genetic profile of juvenile xanthogranuloma. We used molecular inversion probe array technology to evaluate the genomic characteristics (copy number alterations or copy neutral-loss of heterozygosity) of 21 archived cases of juvenile xanthogranuloma (19 solitary, 1 diffuse cutaneous, 1 systemic). Four cases (19%) showed acquired, clonal alterations. Two lesions from a case of diffuse cutaneous juvenile xanthogranuloma showed distinct profiles: JXG-1a contained trisomy 5 and 17 and JXG-1b contained loss of heterozygosity in 5q. The systemic juvenile xanthogranuloma (JXG-2) showed multiple genomic alterations. Only two of 19 solitary juvenile xanthogranulomas showed abnormal genomic profiles: JXG-3 showed gains on 1q and 11q and JXG-4 showed a 7.2 Mb loss in 3p. No recurrent abnormalities were observed among these cases. The presence of non-recurrent copy number alterations in a subset of samples implies that copy number changes are unlikely driving pathogenesis in juvenile xanthogranuloma, but may be acquired during disease progression. The presence of genomic abnormalities in more advanced cases (ie, systemic and diffuse cutaneous juvenile xanthogranuloma) supports this notion, particularly as the advanced cases of juvenile xanthogranuloma presented more genomic complexity.

Genomic analysis of follicular dendritic cell sarcoma by molecular inversion probe array reveals tumor suppressor-driven biology.

Follicular dendritic cell sarcoma is a rare malignant neoplasm of dendritic cell origin that is currently poorly characterized by genetic studies. To investigate whether recurrent genomic alterations may underlie the biology of follicular dendritic cell sarcoma and to identify potential contributory regions and genes, molecular inversion probe array analysis was performed on 14 independent formalin-fixed, paraffin-embedded samples. Abnormal genomic profiles were observed in 11 out of 14 (79%) cases. The majority showed extensive genomic complexity that was predominantly represented by hemizygous losses affecting multiple chromosomes. Alterations of chromosomal regions 1p (55%), 2p (55%), 3p (82%), 3q (45%), 6q (55%), 7q (73%), 8p (45%), 9p (64%), 11q (64%), 13q (91%), 14q (82%), 15q (64%), 17p (55%), 18q (64%), and 22q (55%) were recurrent across the 11 samples showing abnormal genomic profiles. Many recurrent genomic alterations in follicular dendritic cell sarcoma overlap deletions that are frequently observed across human cancers, suggesting selection, or an active role for these alterations in follicular dendritic cell sarcoma pathogenesis. In support of a tumor suppressor-driven biology, homozygous deletions involving tumor suppressor genes CDKN2A, RB1, BIRC3, and CYLD were also observed. Neither recurrent gains nor amplifications were observed. This genomic characterization provides new information regarding follicular dendritic cell sarcoma biology that may improve understanding about the underlying pathophysiology, provide better prognostication, and identify potential therapeutic markers for this rare disease.

Genomic analysis of adult B-ALL identifies potential markers of shorter survival.

B lymphoblastic leukemia (B-ALL) in adults has a higher risk of relapse and lower long-term survival than pediatric B-ALL, but data regarding genetic prognostic biomarkers are much more limited for adult patients. We identified 70 adult B-ALL patients from three institutions and performed genome-wide analysis via single nucleotide polymorphism (SNP) arrays on DNA isolated from their initial diagnostic sample and, when available, relapse bone marrow specimens to identify recurring copy number alterations (CNA). As B-cell developmental genes play a crucial role in this leukemia, we assessed such for recurrent deletions in diagnostic and relapse samples. We confirmed previous findings that the most prevalent deletions of these genes occur in CDKN2A, IKZF1, and PAX5, with several others at lower frequencies. Of the 16 samples having paired diagnostic and relapse samples, 5 showed new deletions in these recurrent B-cell related genes and 8 showed abolishment. Deletion of EBF1 heralded a significant negative prognostic impact on relapse free survival in univariate and multivariate analyses. The combination of both a CDKN2A/B deletion and an IKZF1 alteration (26% of cases) also showed a trend toward predicting worse overall survival compared to having only one or neither of these deletions. These findings add to the understanding of genomic influences on this comparably understudied disease cohort that upon further validation may help identify patients who would benefit from upfront treatment intensification.

C/EBPß-1 promotes transformation and chemoresistance in Ewing sarcoma cells.

CEBPB copy number gain in Ewing sarcoma was previously shown to be associated with worse clinical outcome compared to tumors with normal CEBPB copy number, although the mechanism was not characterized. We employed gene knockdown and rescue assays to explore the consequences of altered CEBPB gene expression in Ewing sarcoma cell lines. Knockdown of EWS-FLI1 expression led to a decrease in expression of all three C/EBPß isoforms while re-expression of EWS-FLI1 rescued C/EBPß expression. Overexpression of C/EBPß-1, the largest of the three C/EBPß isoforms, led to a significant increase in colony formation when cells were grown in soft agar compared to empty vector transduced cells. In addition, depletion of C/EBPß decreased colony formation, and re-expression of either C/EBPß-1 or C/EBPß-2 rescued the phenotype. We identified the cancer stem cell marker ALDH1A1 as a target of C/EBPß in Ewing sarcoma. Furthermore, increased expression of C/EBPß led to resistance to chemotherapeutic agents. In summary, we have identified CEBPB as an oncogene in Ewing sarcoma. Overexpression of C/EBPß-1 increases transformation, upregulates expression of the cancer stem cell marker ALDH1A1, and leads to chemoresistance.

Acute Leukemia and Concurrent Mediastinal Germ Cell Tumor: Case Report and Literature Review.

There is a known association of primary nonseminomatous mediastinal germ cell tumors (NSMGCT) and hematologic malignancy in younger males not linked to treatment. When combined these two rare entities convey a very poor prognosis. Here we report a 16-year-old male with an anterior mediastinal mass diagnosed as a malignant germ cell tumor based on elevation of serologic markers. He was found to have acute leukemia with megakaryocytic differentiation several days later. We focus our report on the pathologic findings, including a review of the literature, and a novel molecular analysis of the germ cell tumor.

Genomic Copy Number Analysis of HER2-Equivocal Breast Cancers.

OBJECTIVES: Guidelines for HER2 testing define an equivocal range for HER2 using two approved testing methods, immunohistochemistry (IHC) and in situ hybridization (ISH). We investigated genome-wide copy number alterations in this subgroup. METHODS: Ten breast cancers with equivocal HER2 status by both IHC and ISH were analyzed by single-nucleotide polymorphism cytogenomic microarray (SNP array). DNA ploidy analysis by flow cytometry was performed on nine cases with sufficient material remaining. RESULTS: SNP array analysis showed uniform gain of chromosome 17 (polysomy) in one case and segmental copy number gains encompassing HER2 and the centromere in five other cases. Flow cytometry revealed hyperdiploidy in six cases, all but one of which also had HER2 gains on SNP array. Although there was no evidence of HER2 amplification by SNP array, six cases showed amplification of other genomic regions, including known oncogenes in four cases. CONCLUSIONS: A combination of hyperdiploidy and segmental copy number gains contributes to HER2 ISH-equivocal results in most breast cancers. Cases in which HER2 copy number gain is not corroborated by genomic analysis suggest the presence of other contributing variables influencing ISH results. Genomic copy number analysis also implicates non-HER2 oncogenic drivers in many cases that are HER2 equivocal.

Low miR-187 expression promotes resistance to chemoradiation therapy in vitro and correlates with treatment failure in patients with esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) has a poor prognosis and is increasing in incidence in many western populations. Neoadjuvant chemoradiation therapy (CRT) followed by surgery is increasingly the standard of care for locally advanced EAC; however, resistance to treatment is a significant clinical problem. The identification of both novel biomarkers predicting response to treatment and novel therapeutic targets to enhance the efficacy of CRT are key to improving survival rates in EAC. In this study we performed global microRNA (miRNA) profiling of pre-treatment EAC biopsies and identified 67 miRNA significantly altered in patients who are resistant to CRT. One of these miRNA, miR-187, was significantly decreased in pre-treatment EAC tumors from patients having a poor response to neoadjuvant CRT, highlighting downregulation of miR-187 as a potential mechanism of treatment resistance in EAC. In vitro, miR-187 was demonstrated to play a functional role in modulating sensitivity to X-ray radiation and cisplatin in EAC and its dysregulation was demonstrated to be due to chromosomal alterations. In vitro, miR-187 altered expression of a diverse array of pathways, including the immune regulator complement component 3 (C3), serum levels of which we have previously demonstrated to predict patient response to CRT. In vivo, expression of C3 was significantly increased in tumors from patients having a poor response to CRT. This study highlights for the first time a role for miR-187 as a novel biomarker of response to CRT and a potential therapeutic target for enhancing the efficacy of CRT in EAC.

Two Unrelated Burkitt Lymphomas Seven Years Apart in a Patient With X-Linked Lymphoproliferative Disease Type 1 (XLP1).

OBJECTIVES: We describe a rare case of a male child with X-linked lymphoproliferative disease type 1 (XLP1) who presented with Burkitt lymphoma (BL) when he was 6 years old, achieved a complete response to therapy, and developed a second BL after seven years. METHODS: Diagnostic H&E stained slides and ancillary studies were reviewed for both lymphomas. B-cell clonality by PCR and SNP array studies were performed on both specimens. RESULTS: Both lymphomas were Epstein-Barr virus (EBV) negative. Flow cytometry showed λ light chain restriction in the initial BL and κ light chain restriction in the subsequent BL. B-cell clonality testing indicated that the two lymphomas are not clonally related. SNP array analysis of the second BL showed genomic changes that were not present in the first BL. CONCLUSIONS: These results confirm that these two tumors represent unrelated BLs. Pathologists and clinicians should be aware that second lymphomas in XLP1 patients may represent new neoplasms rather than late relapses.

Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations.

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers.

Pediatric B-Cell Lymphoma With Lymphoblastic Morphology, TdT Expression, MYC Rearrangement, and Features Overlapping With Burkitt Lymphoma.

Burkitt lymphoma (BL) and B-lymphoblastic lymphoma are subtypes of pediatric non-Hodgkin lymphoma with different presenting features, treatment, and outcomes. This case report documents a 5-year-old female who presented with B-cell lymphoma with lymphoblastic morphology, terminal deoxynucleotidyl transferase expression, MYC rearrangement, and features overlapping with BL. Genomic microarray analysis identified a gain on the long arm of chromosome 1 without other definitive changes. She was treated according to a BL protocol and remains in remission 16-months after initial diagnosis.

Observations of the genomic landscape beyond 1p19q deletions and EGFR amplification in glioma.

BACKGROUND: With recent advancements in molecular techniques, the opportunities to gather whole genome information have increased, even in degraded samples such as FFPE tissues. As a result, a broader view of the genomic landscape of solid tumors may be explored. Whole genome copy number and loss of heterozygosity patterns can advance our understanding of mechanisms and complexity of various tumors. RESULTS: Genome-wide alterations involving copy number changes and loss of heterozygosity were identified in 17 glioma samples with positive FISH results for 1p19q co-deletions (n = 9) or EGFR amplification (n = 8). Gliomas positive for 1p19q co-deletions did not have other frequently recurrent genomic alterations. Additional copy-number alterations were observed in individual cases, and consisted primarily of large-scale changes, including gains or losses of entire chromosomes. The genomic architecture of EGFR amplified gliomas was much more complex, with a high number of gains and losses across the genome. Recurrent alterations in EGFR amplified gliomas were both focal, such as CDKN2A homozygous deletions, and large, such as chromosome 10 loss. CONCLUSIONS: Microarray enabled a broader picture of the genomic alterations occurring in glioma cases. Gliomas with 1p19q co-deletion had a relatively quiet genome, apart from the selected co-deletion. Additional alterations in isolated cases, involved primarily larger aberrations. On the other hand, EGFR amplified cases tended to be more complex and have specific abnormalities associated with the EGFR amplification. Furthermore, 1p19q co-deletions and EGFR amplification associated copy number changes appeared to often be mutually exclusive.

Streamlining the OncoScan® Array Procedure for Use in a Clinical Laboratory.

Microarray analysis has found tremendous utility in the clinical laboratory testing for detection of copy number changes (CNCs) and loss of heterozygosity (LOH). Recently the OncoScan® array was introduced as a tool for identification of CNCs and LOH in formalin-fixed paraffin-embedded oncology samples. The objective of this study was to identify steps in the OncoScan procedure that could be modified to make the process more efficient and technician-friendly in the clinical laboratory setting. Eighteen samples previously processed according to the manufacturer-recommended protocol were reprocessed using a modified protocol. The two primary modifications to the protocol included the elimination of a brief "chill and spin" step and an adjustment to the overnight hybridization temperature to allow for simultaneous hybridization of OncoScan and CytoScan® arrays. A comparison of paired samples processed using both protocols showed that our modified protocol performs similarly to the manufacturer-recommended protocol, yielding equivalent quality control metrics and calls.

Copy number assessment by competitive PCR with limiting deoxynucleotide triphosphates and high-resolution melting.

BACKGROUND: DNA copy number variation is associated with genetic disorders and cancer. Available methods to discern variation in copy number are typically costly, slow, require specialized equipment, and/or lack precision. METHODS: Multiplex PCR with different primer pairs and limiting deoxynucleotide triphosphates (dNTPs) (3-12 µmol/L) were used for relative quantification and copy number assessment. Small PCR products (50-121 bp) were designed with 1 melting domain, well-separated Tms, minimal internal sequence variation, and no common homologs. PCR products were displayed as melting curves on derivative plots and normalized to the reference peak. Different copy numbers of each target clustered together and were grouped by unbiased hierarchical clustering. RESULTS: Duplex PCR of a reference gene and a target gene was used to detect copy number variation in chromosomes X, Y, 13, 18, 21, epidermal growth factor receptor (EGFR), survival of motor neuron 1, telomeric (SMN1), and survival of motor neuron 2, centromeric (SMN2). Triplex PCR was used for X and Y and CFTR exons 2 and 3. Blinded studies of 50 potential trisomic samples (13, 18, 21, or normal) and 50 samples with potential sex chromosome abnormalities were concordant to karyotyping, except for 2 samples that were originally mosaics that displayed a single karyotype after growth. Large cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) (CFTR) deletions, EGFR amplifications, and SMN1 and SMN2 copy number assessments were also demonstrated. Under ideal conditions, copy number changes of 1.11-fold or lower could be discerned with CVs of about 1%. CONCLUSIONS: Relative quantification by restricting the dNTP concentration with melting curve display is a simple and precise way to assess targeted copy number variation.

Neural tube defects and atypical deletion on 22q11.2.

The 22q11.2 deletion syndrome (22q11.2DS) is a common microdeletion disorder. Most of the patients show the common 3 Mb deletion but proximal 1.5 Mb deletion and unusual deletions located outside the common deleted region, have been detected particularly with the advance of comparative cytogenomic microarray technologies. The individuals reported in the literature with unusual deletions involving the 22q11 region, showed milder facial phenotypes, decreased incidence of cardiac anomalies, and intellectual disability. We describe two sibs with an atypical 0.8 Mb microdeletion of chromosome 22q11 who both showed myelomeningocele and mild facial dysmorphisms. The association between neural tube defect and the clinical diagnosis of Di George anomaly/velocardiofacial syndrome is well documented in the literature, but not all cases had molecular studies to determine breakpoint regions. This report helps to narrow a potential critical region for neural tube defects associated with 22q11 deletions.

Two cases of Scimitar syndrome associated with multiple congenital skeletal anomalies and lacking abnormalities by genomic microarray analysis.

Scimitar syndrome is a congenital anomaly occurring in approximately 1/50,000 births, consisting of partial anomalous pulmonary venous return, right lung hypoplasia, and several associated defects. The condition generally has significant morbidity and mortality, but the underlying cause is poorly understood. In this report, we describe 2 autopsy cases of Scimitar syndrome associated with multiple skeletal anomalies and attempt to characterize possible genetic abnormalities in this condition. In light of these findings, we discuss the embryology and direct timing during development of the anomalies associated with this syndrome.