Comparative Genomic Hybridization Studies Research Articles

EP07.01-001 Molecular Profiling Predicts Outcomes in Patients With Resected Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer that rarely develop distant metastasis but often invade surrounding areas. Due to the limited therapeutic options available, the prognosis of these patients is poor, particularly in those with sarcomatoid histology. Moreover, trimodality treatment of resectable MPM is still controversial with high heterogeneity of clinical outcomes among resected patients and the lack of predictive biomarkers. Genomic studies have showed that MPM tumors are characterized by poor mutational landscapes characterized by minute mutations, rather than driver mutations, that are often missed in comparative genomic hybridization and next-generation sequencing studies.

RUNX1 associated Familial Platelet Disorder with Myeloid Malignancy (FPD-MM) in Children: A Novel New Phenotype with Juvenile and Chronic Myelomonocytic Leukemia (JMML/CMML) Characteristics

Introduction:RUNX1 (aka AML1; 21q22.12) is indispensable in the establishment of definitive hematopoiesis in humans. Activating RUNX1 mutations are associated with both Acute Myeloid and Lymphoblastic Leukemias (AML, ALL). On the other hand, hypofunctioning RUNX1 mutations cause dominantly inherited Familial Platelet Disorder (FPD). RUNX1 FPD has a high risk for progression to pancytopenia, myeloproliferative disorders (MPD) or AML, hence the new WHO category FPD with myeloid malignancy (FPD-MM). Those with MM carry mutations in other genes seen in AML, MDS. It is a relatively rare disorder with ~75 affected kindreds reported worldwide (Sood, et al. Blood 2017). Detailed reviews of pediatric cases are few.Case Histories: We encountered two children with RUNX1 associated thrombocytopenia; the mutations are novel. The first family is that of 14 yr old AAF, presenting with fainting- blood counts are shown in Table 1; fetal hemoglobin (HbF) was elevated; bone marrow was hypercellular with 6% type 1 blasts, extreme paucity of megakaryocytes, erythroid hyperplasia and large numbers of sea blue histiocytes. The high HbF suggested JMML while the monocyte CD16;14 profile (95.6% CD14+ cells) was similar to that seen in the adult type Chronic Myelomonocytic Leukemia (CMML). Her mother has pancytopenia without excess blasts in the marrow. The second case presented with neonatal thrombocytopenia; father has history of excessive bruising.Results: Blood counts and values for HbF are listed in Table 1.Molecular testing:Case1: A Myeloid gene panel showed RUNX1 - NM_001754.4:c.501delT, p.Ser167Argfs*9; PHF6 - NM_032458.2:c.902dupA, p.Tyr301*; CUX1- NM_001202543.1:c.2378delC, p.Pro793Argfs*26. No mutations were noted in PTPN11, CBL or RAS genes, the latter confirmed by JMML panel done at University of California, San Francisco. UCSF panel identified a mutation in SH2B3, a gene linked to erythrocytosis not caused by JAK2 mutations. Her mother has the same RUNX1 mutation, thus identifying a germline mutation of RUNX1 in her and her child but not the PHF6, CUX1 or the SH2B3 mutations seen in her daughter. A half sibling is unaffected and is a potential transplant donor for the mother.Case2: No coding sequence mutations were detected in genes associated with familial thrombocytopenia including ETV6, GATA1 and RUNX1. Array Comparative Genomic Hybridization studies (Prevention Genetics) identified a heterozygous deletion of the entire exon 5 of RUNX1.To understand the complex findings in family 1 additional studies were done- DRAQ5, CD71, Fetal Hb staining showed that NRBC in Case 1 contained predominantly high HBF cells. LIN28B was markedly elevated in the proband but not the mother (HbF- normal); LIN28B expression was normal in Case 2.Treatment/Outcome: In Case 1, low dose decitabine therapy resulted in the control of MPD features with good Hb recovery and normalization of the monocyte CD16;14 profiles. There was no platelet response to decitabine nor to a course of valproic acid. The child died of fulminant acute graft vs host disease affecting the liver following a 4/6 cord mismatch transplantation. Mother continues to show moderately severe pancytopenia requiring frequent transfusion support. The second child is symptom free with mild thrombocytopenia.Discussion: The hybrid JMML/CMML features in the index child are likely caused by the concurrent CUX1/PHF6/SH2B3 mutations. We are unable to establish if these are true de novo mutations as the father was not available for study; she had no full siblings. Neither high HbF nor high LIN28B are known feature of FPD by itself nor CMML or Polycythemia Vera (p Vera). Recently, the high HbF in JMML has been linked to high expression of LIN28B. SH2B3 mutation may have contributed to the high erythroid proliferation observed in our case. Induced CUX1 haploinsufficiency in mice causes MPD akin to CMML and megakaryocytic (Meg) proliferation (An N, et al. Blood 2018). The virtual absence of Megs in our case indicates that the CUX1 mutation was unable to overcome the Meg ploidization defect caused by the RUNX1 mutation. PHF6 mutations occur in T-ALL and AML but have not been linked to high HbF.Conclusions: Normal HbF and normal LIN28B expression in the mother of Case1 and in Case2 indicate that increased LIN28B is linked to the high HbF in Case 1 and that high LIN28B itself is a consequence of the malignant transformation caused by the concurrent CUX1/PHF6/SH2B3 mutations. DisclosuresChitlur:Baxter, Bayer, Biogen Idec, and Pfizer: Honoraria; Novo Nordisk Inc: Consultancy. Ravindranath:AGIOS: Other: Site Investigator for Pyruvate Kinase Deficiency.

Familial Chiari Type 1: A Molecular Karyotyping Study in a Turkish Family and Review of the Literature

The etiology of Chiari I malformation (CMI) has not been fully elucidated. Therefore, we performed a genetic study of a Turkish family in which 3 sisters had a diagnosis of CMI with or without syringomyelia. In a family with 7 children, 4 daughters complained of occipital headaches. In 2 of these daughters, CMI had been diagnosed during their 30s, and CMI plus syrinx had been diagnosed in the other daughter in her 40s. Cranial magnetic resonance imaging of the fourth daughter who had developed headaches during her 30s showed normal findings. Because the other siblings in the family were asymptomatic, radiological examinations were not performed. The family had a history of distant consanguineous marriage between parents. Additionally, the father had died, and the mother was asymptomatic, with radiologically normal findings. Array comparative genome hybridization studies were performed for 12 persons from 3 generations of this family. None of the 12 cases examined harbored copy number variations. This family with 3 sisters having CMI suggested a possible autosomal recessive single-gene etiology. Cases of familial CMI are unusual but important to study because they could reveal the specific genes involved in posterior fossa/foramen magnum structure and function and provide insights into the cause of sporadic cases.

Microcephaly, Deafness, and Renal Dysplasia: A Case of Barakat Syndrome

Background: Barakat syndrome is a rare autosomal dominant disorder characterized by hypoparathyroidism, sensorineural deafness, and renal disease, collectively known as HDR syndrome. This disease is caused by the mutation of GATA3 gene located on chromosome 10p15. GATA3 is involved in the embryonic development of kidneys, inner ears, parathyroid glands, and central nervous systems.Case report: Herein, we presented a 20-month-old female with seizure and microcephaly, congenital left kidney dysplasia, hypoparathyroidism, and bilateral sensorineural deafness. Her laboratory tests were consistent with hypoparathyroidism, and the chromosomal study revealed a deletion in chromosome 10. The patient was diagnosed as a case of Barakat syndrome based on her clinical and laboratory tests. The microarray-based comparative genomic hybridization study of the patient was compatible with the monosomy of 10p15.3p13 and trisomy of 12p13.33p13.33.Conclusion: It is important to be aware of rare inherited conditions like Barakat syndrome (HDR syndrome) in a patient with abnormal presentations, such as seizure, neurodevelopmental delay, kidney defects associated with hearing loss, and clinical abnormalities associated with hypoparathyroidism.

ANGELMAN SYNDROME: THE CLINICAL CASE OF COMPLEX CHROMOSOMAL ABERRATION IN A CHILD WITH A RETARDATION OF PSYCHOPHYSICAL DEVELOPMENT

In the structure of the genetic causes of Angelman syndrome, homogeneous disomy of paternal origin on chromosome 15 with translocation is about 1%. It is accompanied by unexpressed phenotypic manifestations, with a low incidence of convulsive paroxysms and satisfactory physical development of patients. The article presents a clinical case of complex chromosomal aberration of chromosome 15 in a child with Angelman syndrome and congenital malformations (nonunion of the thoracic lymphatic duct). During the examination, a whole range of different research methods were used: somatogenetic, clinical and genealogical, cytogenetic, molecular genetic, instrumental, clinical laboratory, biochemical, and others. A psycho-neurological examination of this child was also conducted. Based on a cytogenetic examination and a comparative genomic hybridization study, an abnormal male karyotype was detected in the patient: de novo: 45, XY, der (15) t (15; 15) (q10; q10) dup (15) (q13q11). Using the FISH method, the isodicentric chromosome 15 of parental origin was found, which was formed by two cen15 and contained two copies of the critical region (15) (q11q13) of Prader-Willi / Angelman syndrome, which is commonly called the “Proximal inverted duplication - invdup (15)”. Phenotypic manifestations in partial trisomy along the long arm of paternal origin with isodicentric chromosome 15 with inverted duplication of the proximal region are described. As a result of the work, it was determined that children with congenital malformations and delayed psychophysical development, as well as their parents necessarily need medical genetic counseling and the use of modern molecular genetic diagnostic methods for verification of the diagnosis. Sick children, the karyotype of which has an isodicentric chromosome of 15 paternal origin, which contains two copies of the critical region (15) (q11q13) of Prader-Willi / Angelman syndrome, may have marked main clinical symptoms and craniofacial dysmorphisms characteristic of Angelman syndrome.

Whole-exome sequencing demonstrates recurrent somatic copy number alterations and sporadic mutations in specialized stromal tumors of the prostate

In a previous array comparative genomic hybridization study, we detected common deletions of chromosomes 13 and 14 in prostatic stromal sarcoma and stromal tumor of uncertain malignant potential (STUMP). In this study, we performed whole-exome sequencing (WES) and fluorescence in situ hybridization to explore somatic mutations in 1 low-grade stromal sarcoma, 1 high-grade stromal sarcoma, and 12 STUMPs including 5 cases of degenerative atypia type, 1 myxoid type, 1 phyllodes type, and 5 cases of recently described round cell type. WES was successful on 13 cases that revealed frequent somatic copy number alterations including losses of chromosomes 13 (11 cases), 14 (11 cases), and 1p (9 cases), and partial or complete loss of chromosome 10 (7 cases). Fluorescence in situ hybridization was done on 9 cases and showed compatible chromosome 13 copy numbers with the WES results. STUMPs and the low-grade stromal sarcoma carried moderate tumor mutation burdens that ranged from 1.23 to 7.24 mutations per megabase, while the high-grade stromal sarcoma harbored a significantly higher mutation burden (11.55 mutations per megabase). Sporadic somatic mutations were observed, but no recurrent driver mutations could be discerned. In conjunction with prior array comparative genomic hybridization, we have demonstrated the consistent gene dosage profiles that support the clonal nature and the concept of specialized stromal tumors of the prostate as a distinctive tumor entity.

Unexpected frequency of genomic alterations in histologically normal colonic tissue from colon cancer patients.

As shown by genomic studies, colorectal cancer (CRC) is a highly heterogeneous disease, where copy number alterations (CNAs) may greatly vary among different patients. To explore whether CNAs may be present also in histologically normal tissues from patients affected by CRC, we performed CGH + SNP Microarray on 15 paired tumoral and normal samples. Here, we report for the first time the occurrence of CNAs as a common feature of the histologically normal tissue from CRC patients, particularly CNAs affecting different oncogenes and tumor-suppressor genes, including some not previously reported in CRC and others known as being involved in tumor progression. Moreover, from the comparison of normal vs paired tumoral tissue, we were able to identify three groups: samples with an increased number of CNAs in tumoral vs normal tissue, samples with a similar number of CNAs in both tissues, and samples with a decrease of CNAs in tumoral vs normal tissue, which may be likely due to a selection of the cell population within the tumor. In conclusion, our approach allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal tissue, suggesting that tumorigenic genetic lesions are already present in histologically normal colonic tissue and that the use in array comparative genomic hybridization (CGH) studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, which may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5181-0) contains supplementary material, which is available to authorized users.

Abstract 1534: Amplification of PITPNC1 affects breast cancer progression

Abstract Introduction Identification of driver mutations at single gene level and determination of their respective contribution to the enhanced invasive potential of cancer cells, via expression profiling and functional assays, are indispensable steps toward elucidating breast cancer pathobiology. In our previous array comparative genomic hybridization studies, gain in a region of chromosome 17, 17q23.3-24.3, was frequently observed in invasive duct carcinoma (IDC) patients with lymph node metastasis. Particularly, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), found in 17q24.2, was associated with higher histological grade, larger tumor size, positive HER2 staining, and poorer prognosis from the analysis of a NKI dataset. PITPNC1 is involved in signal transduction and intracellular lipid transport, and may be an essential part of the epidermal growth factor signaling pathway. Additionally, amplification of PITPNC1 via the loss of microRNA-126 suppression has been implicated in metastatic angiogenesis and colonization. In this study, we aim to assess the role of PITPNC1 in the development of aggressive breast cancer by evaluating its effects on breast cancer progression and invasion. Method A total of 21 samples - 13 pure duct carcinoma in situ (DCIS), 8 IDC with or without lymph node metastasis - were used for the preliminary round of this study. Formalin-fixed and paraffin-embedded tissue blocks were microdissected and whole genome amplified using ligation-mediated PCR. Amplified DNA was subjected to quantitative real-time PCR. Copy number alterations of PITPNC1 were calculated with Livak method, using B2M as the reference gene. Lipofectamine transfection overexpressing PITPNC1 in non-invasive MCF-10A and invasive MCF7, MD-MB-231 cell lines was prepared for subsequent in vitro assays. Proliferation and migration capabilities associated with PITPNC1 overexpression is being evaluated using MTT assay and transwell migration assay. Result From preliminary qPCR data, PITPNC1 was shown to be amplified in DCIS samples (p = 0.0025) and IDC samples (p = 0.0093). The 95% confidence intervals of fold difference against normal breast tissue control are [7.1, 23.4] and [5.7, 24.4] respectively. PITPNC1 overexpressing MCF-10A cells exhibit a higher proliferation rate in culture compared to controls. Conclusion Consistent amplification of PITPNC1 is seen in both DCIS and IDC cases. However, it is still unclear if PITPNC1 amplification is a result of a single mutational event occurring early in cancer progression or a cumulative effect occurring over time. Additional qPCR on paired samples against a pooled control would be necessary to determine its contribution to invasion. The regularity of copy number gain in the PITPNC1 locus and its association with HER2 expression highlight its predictive potential as a novel biomarker for tumorigenesis or invasion. Citation Format: Peiqi Wang, Ranju Nair, Nisha Kanwar, Grace Cheung, Susan J. Done. Amplification of PITPNC1 affects breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1534.

De novo deletions and duplications of 17q25.3 cause susceptibility to cardiovascular malformations.

BackgroundGenomic disorders resulting from deletion or duplication of genomic segments are known to be an important cause of cardiovascular malformations (CVMs). In our previous study, we identified a unique individual with a de novo 17q25.3 deletion from a study of 714 individuals with CVM.MethodsTo understand the contribution of this locus to cardiac malformations, we reviewed the data on 60,000 samples submitted for array comparative genomic hybridization (CGH) studies to Medical Genetics Laboratories at Baylor College of Medicine, and ascertained seven individuals with segmental aneusomy of 17q25. We validated our findings by studying another individual with a de novo submicroscopic deletion of this region from Cytogenetics Laboratory at Cincinnati Children’s Hospital. Using bioinformatic analyses including protein-protein interaction network, human tissue expression patterns, haploinsufficiency scores, and other annotation systems, including a training set of 251 genes known to be linked to human cardiac disease, we constructed a pathogenicity score for cardiac phenotype for each of the 57 genes within the terminal 2.0 Mb of 17q25.3.ResultsWe found relatively high penetrance of cardiovascular defects (~60 %) with five deletions and three duplications, observed in eight unrelated individuals. Distinct cardiac phenotypes were present in four of these subjects with non-recurrent de novo deletions (range 0.08 Mb–1.4 Mb) in the subtelomeric region of 17q25.3. These included coarctation of the aorta (CoA), total anomalous pulmonary venous return (TAPVR), ventricular septal defect (VSD) and atrial septal defect (ASD). Amongst the three individuals with variable size duplications of this region, one had patent ductus arteriosus (PDA) at 8 months of age.ConclusionThe distinct cardiac lesions observed in the affected patients and the bioinformatics analyses suggest that multiple genes may be plausible drivers of the cardiac phenotype within this gene-rich critical interval of 17q25.3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0291-0) contains supplementary material, which is available to authorized users.

Association of chromosome 19 to lung cancer genotypes and phenotypes.

The Chromosome 19 Consortium, a part of the Chromosome-Centric Human Proteome Project (C-HPP, http://www.C-HPP.org ), is tasked with the understanding chromosome 19 functions at the gene and protein levels, as well as their roles in lung oncogenesis. Comparative genomic hybridization (CGH) studies revealed chromosome aberration in lung cancer subtypes, including ADC, SCC, LCC, and SCLC. The most common abnormality is 19p loss and 19q gain. Sixty-four aberrant genes identified in previous genomic studies and their encoded protein functions were further validated in the neXtProt database ( http://www.nextprot.org/ ). Among those, the loss of tumor suppressor genes STK11, MUM1, KISS1R (19p13.3), and BRG1 (19p13.13) is associated with lung oncogenesis or remote metastasis. Gene aberrations include translocation t(15, 19) (q13, p13.1) fusion oncogene BRD4-NUT, DNA repair genes (ERCC1, ERCC2, XRCC1), TGFβ1 pathway activation genes (TGFB1, LTBP4), Dyrk1B, and potential oncogenesis protector genes such as NFkB pathway inhibition genes (NFKBIB, PPP1R13L) and EGLN2. In conclusion, neXtProt is an effective resource for the validation of gene aberrations identified in genomic studies. It promises to enhance our understanding of lung cancer oncogenesis.

Abstract P3-06-28: Alterations in HER2 status and outcome following neoadjuvant chemotherapy in HER2-positive breast cancer (BC)

Abstract Background: Emerging data suggest that chemotherapy and anti-HER2 agents can change HER2 status in HER2+ BC and that this may have prognostic implications. The findings suggest a role for post-treatment (tx) retesting of HER2 status in surgical specimens. However, few studies have specifically examined this phenomenon, and direct comparisons of post-tx HER2 testing modalities are lacking. Furthermore, the biologic basis for HER2 alterations remains poorly understood. The aims of this study were to systematically evaluate the effects of neoadjuvant chemotherapy (NAT) including anti-HER2 agents on HER2 protein expression (P-EXP) and gene amplification (G-AMP), and to examine the potential prognostic impact of HER2 alterations on outcomes in HER2+ BC. Design: We retrospectively identified 129 patients with HER2+ BC who received NAT at our institution. HR (ER and PR) and HER2 P-EXP were evaluated by immunohistochemistry (IHC) and HER2 G-AMP by fluorescence in situ hybridization (FISH) in pre- and post-tx samples. Pathologic tumor responses were categorized as no residual disease (RD; no invasive or in situ cancer), residual ductal carcinoma in situ (DCIS) only, and residual invasive carcinoma. Pathologic complete response (pCR) was defined as DCIS only or no RD. Association between groups was determined by Fisher exact test. For survival analysis, Kaplan Meier curves were constructed and significance in curve separation was assessed by log rank test. Results: Mean follow-up was 52.6 months (range 6-162). Eighty-four (65%) cases had residual invasive cancer, 21 (16%) residual DCIS only and 24 (19%) no RD. Post-tx HER2 status was available in 70 cases with residual invasive cancer, and 27 (39%) of these showed reduced HER2 P-EXP (staining intensity 0, 1+, 2+) by IHC. Twenty-one (78%) of 27 cases with reduced HER2 P-EXP retained HER2 G-AMP (IHC-/FISH+), and 6 (22%) were negative for both HER2 P-EXP and G-AMP (IHC-/FISH-). The subset of IHC-/FISH- patients showed 100% 5-year recurrence free survival (RFS), whereas IHC-/FISH+ and IHC+ (intensity 3+) cases demonstrated similarly decreased 5-year RFS of 67% and 71%, respectively. There was a trend towards reduced HER2 expression in HR+ versus HR- cases (46% vs 26%, p=0.19). HR-HER2+ tumors were more likely to achieve pCR than HR+HER2+ cases (48% vs 26%; p=0.014, OR=0.375). In the HR- subset, RFS was significantly better in patients with no RD compared to those with residual invasive cancer (p=0.047); this relationship was not observed in the HR+ group (p=0.693). Conclusion: A significant fraction of pre-tx HER2+ BC demonstrate reduced HER2 P-EXP following NAT. In a subset of these, post-tx decreased HER2 P-EXP is associated with retained HER2 G-AMP, suggestive of HER2 protein downregulation. In another subset, decreased HER2 P-EXP with associated lack of HER2 G-AMP indicates selection for HER2 non-amplified clones in HER2 heterogeneous tumors. Array comparative genomic hybridization studies are in progress to further elucidate these mechanisms. Lastly, our results suggest that post-tx evaluation of HER2 status by FISH in addition to IHC may have prognostic and/or predictive value in HER2+ BC. Further studies in larger prospective study populations are needed to confirm our findings. Citation Format: Dianna Ng, Gregor Krings, Christina Yau, Kristie White, Jie Hou, Anthony Chua, James P Grenert, Yunn-Yi Chen. Alterations in HER2 status and outcome following neoadjuvant chemotherapy in HER2-positive breast cancer (BC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-28.

Angiogenesis in primary hyperparathyroidism.

Angiogenesis can be described as a formation of new vessels from the existing microvasculature and is a process of great importance to the tumor development. Parathyroid tissue can trigger spontaneous induction of angiogenesis in vitro and in vivo models in a vascular endothelial growth factor (VEGF)-dependent manner. Autotransplantated parathyroid tissue after thyroidectomy is able to form new vasculature and produce parathormone, maintaining calcium homeostasis. A great amount of factors contributes to the process of new vessel formation in primary hyperparathyroidism, such as VEGF, transforming growth factor β, and angiopoietins. Studies demonstrated that markers for angiogenesis can be useful in distinguishing between parathyroid hyperplasia and neoplasia, due to the increased angiogenesis in parathyroid proliferative lesions compared with parathyroid adenomas. These factors include, inter alia, VEGF, VEGFR2, CD105, and fibroblast growth factor-2. Although these differences appear promising in the differential diagnosis, there is an overlap between benign and malignant parathyroid lesions and there is no definite cutoff value. Loss of heterozygosity and comparative genomic hybridization studies revealed chromosomal regions frequently altered in parathyroid tumorigenesis at 9p21, 1p21-22, 1p35-36, and 11q13. Therefore, immunohistochemistry and genetic testing should be an additional diagnostic marker in combination with the traditional criteria. A better understanding of angiogenesis in primary hyperparathyroidism could result in more precise assessment of diagnosis and more effective treatment, especially in those cases, in which the commonly used parameters are insufficient.

A case of Sotos syndrome presented with end-stage renal disease due to the posterior urethral valve

Sotos syndrome (SS, OMIM 117550) is characterized by prenatal and postnatal overgrowth with multiple congenital anomalies. However, there have been few cases of growth retardation caused by renal failure from infancy. We report a case of dysplasia of the bilateral kidneys with renal failure and poor postnatal growth. A 2-month-old boy visited the emergency room owing to poor oral intake and abdominal distension. He was born at the gestational age of 38 weeks with a birth weight of 4,180 g. After birth, he had feeding difficulty and abdominal distension. Upon physical examination, his height and weight were in less than the 3rd percentile, while his head circumference was in the 50th percentile on the growth curve. He also showed a broad and protruding forehead and high hairline. Blood laboratory tests showed severe azotemia; emergent hemodialysis was needed. Abdominal ultrasonography revealed bilateral renal dysplasia with multiple cysts and diffuse bladder wall thickening. A posterior urethral valve was suggested based on vesicoureterography and abdominal magnetic resonance findings. Results of a colon study to rule out congenital megacolon did not reveal any specific findings. The conventional karyotype of the patient was 46, XY. Array comparative genomic hybridization study revealed a chromosome 5q35 microdeletion including the NSD1 gene, based on which SS was diagnosed. We describe a case of SS presenting with end stage renal disease due to posterior urethral valve. The typical somatic overgrowth of SS in the postnatal period was not observed due to chronic renal failure that started in the neonatal period.

Sorafenib Is Contributing to Salvage Chemotherapy and Is Effective Given Alone for Maintenance in AML FLT3 ITD Patients Relapsing Post Allo Hematopoietic Stem Cell Transplantation

Normal karyotype AML patients with FLT3 ITD have a higher risk of relapse than those lacking this mutation (Estey EH, Am J Hematol. 2013). Our comparative genomic hybridization study (77 patients) showed that AML cases with as compared to those without FLT3 ITD had significantly lower number of amplifications or deletions (number of total CNV aberrations: 18±4 vs 51±8, p=0.003). The latter observation in concert with that of others (Thiede, Blood 2002) suggest that FLT3 ITD mutation if present in normal karyotype patients is a key player in the pathogenesis of leukaemia and targeting this mutation may be used successfully in FLT3 ITD positive patients relapsing post HSCT.This was also the case in the two patients presented in this study.Patient UPN 952, male, 53 years old, AML M4 (FAB), normal karyotype, received alloHSCT in 2 CR after completion of two lines of remission induction therapies (first: DA3+7 (Daunorubicin, Ara-C), HAM, high dose Ara-C; second: ICE (IDA, Ara-C, VP-16)) and then promptly transplanted. He relapsed 56 days after transplantation.Patient UPN 938, female, 50 years old, AML, normal karyotype, received as an induction DA 3+7, and for consolidation HAM and high dose Ara-C and completed only one course of the maintenance therapy and transplanted in CR.Both cases were transplanted from unrelated donors (10/10 HLA A, B, C, DR and DQ alleles matched), they received myeloablative conditioning (i.v. Busulfan and Cyclophosphamide) and Cyclosporin A as GvHD prophylaxis. At relapse they received salvage chemotherapy tailored to their biological performance (UPN 938 having Age Adjusted Charlson Comorbidity Index 3 received FLAG and UPN 952 with the index 9, ECOG 3, DA2+5). Both cases received in addition Sorafenib (two times 400 mg per day). The response was prompt and the marrow was free from blasts beginning from 11 day post chemotherapy. UPN 952 received as a maintenance only one course of 6-TG with low dose of Ara-C. Due to the substantial comorbidity and liver toxicity WHO3 further chemotherapy treatment was terminated. The patient was left only on Sorafenib (2 times 200 mg per day). UPN 938 was receiving the maintenance therapy (AML protocol) in 6 – 8 weeks intervals based on low dose Ara-C with 6TG or DNR and also Sorafenib (2 times 200 mg per day).In both cases FLT3 signalling pathway (FLT3 Pathway Mutation PCR Array, SABiosciences, Qiagen) revealed a lack of any additional mutation at the check points in FLT3, KRAS, HRAS, NRAS, MEK1, PIK3CA, BRAF and PTEN genes. UPN 938 had in addition to FLT3 ITD mutation c.1807_1808insATGAATATGATCTCAAAT (p.K602_W603insYEYDLK) insertion which relevance was not so far describe. Sorafenib resistance mutations were not found.The patients were on Sorafenib for 8 (UPN 938) and 9 months (UPN 952).The course of UPN938 was uncomplicated. The second case showed mild aGvHD symptoms evolving into cGvHD (skin lesions and dry eye) slowed down with rapamicine.Up to now the patients are in CR and free from FLT3 ITD. The main observation points:- Sorafenib with chemotherapy tailored to the biologic performance of patients contributes to the efficient salvage in patients with relapsing FLT3 ITD positive AML post HSCT and is also effective given alone as a maintenance.- Side effects were seen in one out of two patients likely associated with the blocking of VEGFR signalling transmission (hand foot skin rash, von Willebrandt factor activity – 388%).Conclusion: 1.The use of multikinase inhibitor (Sorafenib) contributes effectively to salvage therapy in FLT3+ AML patients relapsing post alloHSCT2.Sorafenib given alone is able to maintain long-lasting remission3.Side effects are individually dependentSupported by NBiR CellsTherpy grant and Byer Health Care DisclosuresNo relevant conflicts of interest to declare.

Distinct Patterns of Genomic Alterations in Adult T-Cell Leukemia-Lymphoma Endemic in the Western World

Introduction: Acute T-cell leukemia/lymphoma (ATLL) is a highly aggressive malignancy caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL carries a dismal prognosis and is generally incurable with conventional chemotherapy. ATLL is challenging to study at the molecular level, in part due to its complex genetic alterations likely resulting from years of HTLV-I driven T-cell proliferation and accumulation of genetic damage prior to malignant transformation. While no specific chromosome or genetic abnormalities have been proven to contribute to the pathogenesis of ATLL, older comparative genomic hybridization (CGH) studies performed in Japanese patients have demonstrated frequent genetic lesions (gains and losses) involving specific chromosomal regions, thus limited information exists about the chromosomal abnormalities occurring in the African ATLL variant commonly seen in the Western World.Methods: In this study, we used a high-density oligo array 244K platform CGH platform (Agilent Technologies) with an average resolution of 8.9 Kb, to perform a comprehensive genomic analysis of 47 ATLL patient tumor specimens obtained from African-descendants in the United States, Caribbean and Brazil. Patients were sub-classified as acute-type (A) ATLL (n=31), lymphomatous (L) (n=8), chronic (n=7, six whom had unfavorable features), and one with smouldering type according to Shimoyama criteria. DNA samples were extracted from peripheral blood mononuclear cells or tumor samples from these patients and checked for quality.Results: ATLL tumors exhibited complex genomic abnormalities and high copy number changes (CNCs). The average of copy number (CN) aberrations per sample was 238 in the L-group vs. 114 in acute/unfavorable chronic (A/UC) group. However, many chromosomal alterations were observed in this cohort, which had not been previously reported by other studies. The common CNCs were gains at 1q21-q44, 3, 3p, 7q22-q36, 8, 18, 19p13.1-p13.3, 21q21.1-q22.3, 22q12-q13 and losses at 5q13.2-q32, 6q11-q15, 9q13-q21. Gains in the 14 q32 (IGH) regions and losses in the 7p14.1 (TCRG), 7q34 (TCRB) and 14q11.2 (TCRA) regions involving small DNA segments were frequently observed. Genomic losses involving at least one or more known or candidate tumor suppressor genes were found in nearly all tumors, including some genes not previously implicated in ATLL. Some of the most significant gene or locus specific losses occurring in at least 20 % of the tumors in aggressive ATLL subtypes (A/UC and L) are summarized in Table 1. Losses of CDKN2A and CDKN2B tumor suppressor genes have been previously implicated in ATLL and other cancers. Several other genes found by this analysis have been implicated in apoptosis or cancer (i.e. CBLB, ANKRD11, IKZF1, and EPC1). IMMP2L deletion was associated with shorter survival time (2.3 weeks) compared with those cases without this gene deletion (29 weeks) in the A/UC group (p=0.005). ANKRD11 homo- or heterozygous deletions were seen in 37% of L-type and 19% of acute-type cases, and were associated with a shorter survival (13 vs. 43 weeks, p=<0.05) in the A group. CPN2/LRRC15 locus gains in 32% of A-type were linked to poorer survival (16 vs. 42 weeks, p=0.05).Table 1GENEFUNCTIONLOSSES (n) GAINS (n) NRXN3Membrane receptor, cell adhesion23 A/UC 3 LNSIMMP2LMitochondrial inner membrane peptidase16 A/UC* 5 L4 A/UCCDKN2A/ CDKN2B (P16INK4/p15INK4b)Cyclin-dependent kinase inhibitors, tumor suppressors16 A/UC 2 L1 A/UC 1 LCBLBE3 ubiquitin protein ligase12 A/UC 1 LNSANKRD11Transcriptional inhibitor, co-activator of p536 A/UC* 3 LNSCPN2CarboxypeptidaseNS10 A/UC* 2 LIKZF1Zinc-finger DNA binding proteins , lymphocyte differentiationNS6 A/UC 3 LINSIG1Endoplasmic reticulum membrane protein, intracellular lipid metabolismNS10 A/UC 1 LEPC1Member of the polycomb group family, transcriptional activator and repressor10 A/UC 2 LNS* are genomic imbalances associated with a statistically significant reduction in survival. NS: non-significant.Conclusion: In sum, using a high resolution CGH array we observed distinct patterns ofgenetic aberrations in ATLL endemic in the Western World. We have successfully narrowed the genomic regions containing potential candidate genes that could be relevant to the pathogenesis of this fatal disease. Functional studies are required to determine the role of some of these genes in the pathogenesis of ATLL. DisclosuresO’Hara:BioDiscovery, Inc.: Employment.

Expression of domains for protein-protein interaction of nucleotide excision repair proteins modifies cancer cell sensitivity to platinum derivatives and genomic stability.

Nucleotide excision repair (NER) is involved in the repair of DNA damage caused by platinum derivatives and has been shown to decrease the cytotoxic activity of these drugs. Because protein-protein interactions are essential for NER activity, we transfected human cancer cell lines (A549 and HCT116) with plasmids coding the amino acid sequences corresponding to the interacting domains between excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum, complementation group A (XPA), as well as ERCC1 and xeroderma pigmentosum, complementation group F (XPF), all NER proteins. Using the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and annexin V staining, we showed that transfected A549 cells were sensitized 1.2-2.2-fold to carboplatin and that transfected HCT116 cells were sensitized 1.4-5.4-fold to oxaliplatin in vitro. In addition, transfected cells exhibited modified in vivo sensitivity to the same drugs. Finally, in particular cell models of the interaction between ERCC1 and XPF, DNA repair was decreased, as evidenced by increased phosphorylation of the histone 2AX after exposure to mitomycin C, and genomic instability was increased, as determined by comparative genomic hybridization studies. The results indicate that the interacting peptides act as dominant negatives and decrease NER activity through inhibition of protein-protein interactions.

Chromophobe renal cell carcinoma with neuroendocrine differentiation/morphology: A clinicopathological and genetic study of three cases

Chromophobe renal cell carcinoma (ChRCC) with neuroendocrine differentiation/morphology (NED/NEM) is exceedingly rare. We present three cases of ChRCC with NED/NEM, two of which showed positivity for neuroendocrine markers on immunohistochemical analysis. Patients ranged in age from 49 to 79 years (mean: 64.3 years). One of the three patients died of metastatic disease to multiple organs. Of the remaining two patients, one is currently alive without disease and the other is alive with disease. Histologically, all three tumors were composed of conventional ChRCC and NEM showed glandular and rosette formation. Immunohistochemically, tumor cells were positive for CK7, KAI1, E-cadherin, and c-kit in both ChRCC and neuroendocrine areas in three cases. CD56 and synaptophysin immunoreactivity were detected in two cases; in only the neuroendocrine area in one case and in both components in the other. Neuroendocrine granules were ultrastructurally observed at both neuroendocrine and conventional areas of ChRCC. Array comparative genomic hybridization (CGH) study indicated losses of chromosomes 1, 2, 6, 10, 17, 21, and Y in both conventional ChRCC and NED in one case. In addition, losses of chromosomes 1, 2, 4, 6, 9, 10, 13, 16p, 17, and 21 were observed in both components of the remaining one tumor. Furthermore, loss of chromosome 5 was identified only in the neuroendocrine area in this case. We concluded that the neuroendocrine area may reflect dedifferentiation within ChRCC. It is possible that losses of chromosomes 4, 5, and 16p may be involved in the neuroendocrine differentiation or progression of ChRCC.

Recurrent chromosomal aberrations in intravenous leiomyomatosis of the uterus: high-resolution array comparative genomic hybridization study.

Uterine intravenous leiomyomatosis (IVL) is a distinct smooth muscle neoplasm with a potential of clinical aggressiveness due to its ability to extend into intrauterine and extrauterine vasculature. In this study, chromosomal alterations analyzed by oligonucleotide array comparative genomic hybridization were performed in 9 cases of IVL. The analysis was informative in all cases with multiple copy number losses and/or gains observed in each tumor. The most frequent recurrent loss of 22q12.3-q13.1 was observed in 6 tumors (66.7%), followed by losses of 22q11.23-q13.31, 1p36.13-p33, 2p25.3-p23.3, and 2q24.2-q32.2 and gains of 6p22.2, 2q37.3 and 10q22.2-q22.3, in decreasing order of frequency. Copy number variants were identified at 14q11.2, 15q11.1-q11.2, and 15q26.2. Genes mapping to the regions of loss include CHEK2, EWS, NF2, PDGFB, and MAP3K7IP1 on chromosome 22q, HEI10 on chromosome 14q, and succinate dehydrogenase subunit B, E2F2, ARID1A KPNA6, EIF3S2 , PTCH2, and PIK3R3 on chromosome 1p. Regional losses on chromosomes 22q and 1p and gains on chromosomes 12q showed overlaps with those previously observed in uterine leiomyosarcomas. In addition, presence of multiple chromosomal aberrations implies a higher level of genetic instability. Follow-up polymerase chain reaction (PCR) sequencing analysis of MED12 gene revealed absence of G> A transition at nucleotides c.130 or c.131 in all 9 cases, a frequent mutation found in uterine leiomyoma and its variants. In conclusion, this is the first report of high-resolution, genome-wide investigation of IVL by oligonucleotide array comparative genomic hybridization. The presence of high frequencies of recurrent regional loss involving several chromosomes is an important finding and likely related to the pathogenesis of the disease.

Elucidation of Distinctive Genomic DNA Structures in Patients with 46,XX Testicular Disorders of Sex Development Using Genome Wide Analyses

Although several genes, including the SRY gene, are involved in testicular differentiation, the entire mechanism of this differentiation remains unclear. We performed genome wide analysis in patients with 46,XX testicular disorders of sex development to comprehensively elucidate the mechanisms oftesticular differentiation. Whole genomic DNA was extracted from the peripheral blood of 4 patients with 46,XX testicular disorders of sex development who were SRY negative. Genomic DNA was hybridized to a GeneChip® human mapping 250K array set. Compared to normal female data, we detected common loss of heterozygosity and copy number variation regions in 4 patients using Genotyping Console™ software. Loss of heterozygosity was detected in 19 regions of 11 chromosomes. Atotal of 27 genes or nearby genomic areas were included in the applicable regions. Copy number loss was recognized in 13 regions of 10 chromosomes, and these regions included 55 genes. Copy number gain was detected in 6 regions of 4chromosomes, which included the upstream region of the SOX3 gene. The regions with loss of heterozygosity did not contain genes associated with testicular differentiation. However, the upstream area of the SOX3 gene, which is located in Xq27.1, was included in the region of copy number gain. These results suggest that high expression of the SOX3 gene led to testicular differentiation despite SRY gene loss. As this applicable area is not within a coding region, genome wide analyses were valuable for detecting thenovel regions associated with testicular differentiation.

Intracystic papillary carcinoma of breast: interrelationship with in situ and invasive carcinoma and a proposal of pathogenesis: array comparative genomic hybridization study of 14 cases

Classifying intracystic papillary carcinoma under invasive or in situ ductal carcinoma is still a matter of debate. The purpose of this study was to explore the genomic relationship of this tumor to its concurrent invasive ductal carcinoma and ductal carcinoma in situ using array comparative genomic hybridization. Intracystic papillary carcinoma cases were classified into three categories: pure, with concurrent ductal carcinoma in situ or with concurrent invasive ductal carcinoma. Each component was dissected using laser capture microdissection. DNA was extracted and array comparative genomic hybridization was performed. The test of difference in copy number changes among the three tumors was carried out using CGHMultiArray. Intracystic papillary carcinoma clustered with four of five concurrent ductal carcinoma in situ cases and with two of two invasive ductal carcinoma cases. Intracystic papillary carcinoma showed the highest proportions of genome copy number aberration, followed by ductal carcinoma in situ, and then by invasive ductal carcinoma (P=0.06). Comparing intracystic papillary carcinoma with invasive ductal carcinoma vs without invasive ductal carcinoma, the former had 11q22.1–23.3 loss (P=0.031) and chr5 gain (P=0.085), and was enriched with matrix metalloproteinase genes. Comparing intracystic papillary carcinoma with ductal carcinoma in situ vs without ductal carcinoma in situ, the former had gain in 5q35.3 (P=0.041), 8q24.3 (P=0.041) and 21q13.2 to 21q13.31 (P=0.011). Comparing intracystic papillary carcinoma with ductal carcinoma in situ, the latter acquired a group of genes involved in cell adhesion and motility, whereas intracystic papillary carcinoma differentially expressed genes that are involved in papillary carcinomas of other organs (thyroid and kidney). We conclude that the overall molecular change in intracystic papillary carcinoma is closer to ductal carcinoma in situ than to invasive ductal carcinoma, which may explain the indolent behavior of this tumor. We offer herein a proposal of intracystic papillary carcinoma pathogenesis through its relation to invasive ductal carcinoma and ductal carcinoma in situ.