Gain Of 12p Research Articles

Age, Survival Predictors, and Metastatic Death in Patients With Choroidal Melanoma

To determine whether treatment of choroidal melanoma influences survival by correlating age at death, cause of death, age at treatment, and survival predictors. Prospective cohort study performed at the Liverpool Ocular Oncology Centre, a supraregional, tertiary referral service in England. We included 3072 patients treated for choroidal melanoma from January 15, 1993, through November 23, 2012, and who reside in the mainland United Kingdom. A diagnosis of choroidal melanoma (ie, any uveal melanoma involving the choroid). Largest basal tumor diameter, tumor thickness, TNM stage, ciliary body involvement, extraocular spread, melanoma cytomorphological findings, closed connective tissue loops, mitotic count, chromosome 3 loss, chromosome 6p gain, chromosome 8q gain, age at treatment, age at death, and cause of death. The largest basal tumor diameter correlated with all survival predictors except for chromosome 6p gain. Older age at treatment correlated with ciliary body involvement, extraocular spread, largest basal tumor diameter, tumor thickness, TNM stage, epithelioid cells, chromosome 3 loss, and chromosome 8q gain. A total of 1005 patients had died by the close of the study. The cause of death was metastatic disease due to uveal melanoma in 561 patients. Among the 561 patients, survival time after treatment correlated with sex, basal tumor diameter, ciliary body involvement, extraocular spread, TNM stage, closed loops, and mitotic count. In this group of patients, none of the survival predictors correlated with age at death except for mitotic count, which showed a weak correlation. All survival predictors correlated with an increased likelihood of metastatic melanoma as the cause of death. Patients who are younger at the time of treatment tend to have a smaller, less extensive tumor with a lower degree of malignancy. A tentative explanation for these findings is that ocular treatment prevents tumor growth, dedifferentiation, and metastatic disease in some patients, especially those with a smaller tumor.

MYCN amplification predicts poor outcome for patients with supratentorial primitive neuroectodermal tumors of the central nervous system

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are a rare group of neoplasms occurring in the CNS that includes supratentorial CNS-PNETs, medulloepitheliomas, and ependymoblastomas. While ependymoblastomas frequently carry chromosome 19q13.41 amplification and show aggressive clinical behavior, the biological mechanisms and molecular alterations contributing to the pathogenesis of supratentorial CNS-PNETs remain poorly understood. Moreover, genetic alterations suitable for molecular risk stratification are undefined to date. In order to identify possible molecular markers, we performed multiplex ligation-dependent probe amplification (MLPA) and molecular inversion probe (MIP) analysis on DNA samples of 25 supratentorial CNS-PNETs (median age, 5.35 years; range, 2.41–17.28 years). Tumors with ependymoblastic rosettes (ependymoblastoma/ETANTR) and LIN28A positivity were excluded. MLPA and MIP analysis revealed large losses of genomic material of chromosomes 3, 4, 5, and 13, while frequent gains affected chromosomes 1, 17, 19, 20, and 22. High copy number gains (amplifications) were found in particular at chromosomes 2p24.3 (MYCN, n = 6 cases) and 4q12 (n = 2 cases). Patients with tumors harboring 2p gain or MYCN amplification showed unfavorable overall survival (P = .003 and P = .001, respectively).These markers were independent of the presence of metastases, which was indeed a clinical factor associated with poor overall survival (P = .01) in this series. In the era of the personalized neuro-oncology, the identification of these molecular prognostic markers associated with patient outcome may represent a significant step towards improved patient stratification and risk-adapted therapeutic strategies for patients suffering from supratentorial CNS-PNETs.

A rare case of combined placental site trophoblastic tumour with mature cystic teratoma and mixed germ cell tumour in the testis

<h3>Case report</h3> A 20-year-old male presented with persistent right testicular pain. Following ultrasound detection of testicular nodules and biopsy for intraoperative consultation which yielded germ cell tumour, he underwent radical orchidectomy. A predominantly whitish cyst and a lobulated, variegated nodule were identified. Histology showed a mature cystic teratoma with a focus of infiltrative epithelioid cells containing eosinophilic cytoplasm and pleomorphic nuclei, invading ectatic vessel wall, associated with fibrinoid change. These cells were positive for cytokeratin, human placental lactogen and inhibin, while negative for Melan-A, p63 and alpha-fetoprotein, consistent with placental site trophoblastic tumour (PSTT). The variegated nodule was a mixed germ cell tumour composed of embryonal carcinoma and immature teratoma. <h3>Discussion</h3> Aside from choriocarcinoma, primary trophoblastic tumours such as PSTT, which are derived from intermediate trophoblasts, are extremely rare in the testis. Aside from a case of pure testicular PSTT, two other cases have been described in association with germ cell tumour, of which one is a mature teratoma with PSTT that demonstrated gain of chromosome 12p. The other presented with PSTT in retroperitoneal recurrence of a testicular mixed germ cell tumour. We discuss the features of this tumour in the testis and important differentials in its diagnosis.

The telomere profile distinguishes two classes of genetically distinct cutaneous squamous cell carcinomas

The incidence of skin cancer is increasing worldwide and cutaneous squamous cell carcinomas (SCCs) are associated with considerable morbidity and mortality, particularly in immunosuppressed individuals ('carcinomatous catastrophy'). Yet, molecular mechanisms are still insufficiently understood. Besides ultraviolet (UV)-indicative mutations, chromosomal aberrations are prominent. As telomeres are essential in preserving chromosome integrity, and telomere erosion as well as aberrant spatial telomere distribution contribute to genomic instability, we first established telomere length profiles across the whole tissue and identified normal skin (10/30) harboring discrete epidermal sites (stem cell territories) of evenly short telomeres. Precancerous actinic keratoses (AKs) (17) and SCCs (27) expressed two telomere phenotypes: (i) tissue-wide evenly short to intermediate and (ii) longer and tissue-wide heterogeneous telomere lengths, suggesting two modes of initiation, with one likely to originate in the epidermal stem cells. Although tumor histotype, location, patient gender or age failed to distinguish the two SCC telomere phenotypes, as did telomerase activity, we found a trend for a higher degree of aberrant p53 and cyclin D1 expression with long/heterogeneous telomeres. In addition, we established an association for the short/homogeneous telomeres with a simpler and the heterogeneous telomeres with a more complex karyotype correlating also with distinct chromosomal changes. SCCs (13) from renal transplant recipients displayed the same telomere dichotomy, suggesting that both telomere subtypes contribute to 'carcinomatous catastrophy' under immunosuppression by selecting for a common set (3, 9p and 17q) and subtype-specific aberrations (e.g., 6p gain, 13q loss). As a second mechanism of telomere-dependent genomic instability, we investigated changes in telomere distribution with its most severe form of telomeric aggregates (TAs). We identified a telomere length-independent but progression-dependent increase in cells with small telomere associations in AKs (17/17) and additional TAs in SCCs (24/32), basal cell carcinomas (30/31) and malignant melanomas (15/15), and provide evidence for a reactive oxygen species-dependent mechanism in this UV-induced telomere organization-dependent genomic instability.

Gain of chromosome 6 status does not influence HLA‐expression in uveal melanoma

AbstractPurpose Chromosomal aberrations and the inflammatory phenotype have been identified as predictive factors for survival of uveal melanoma (UM). The mechanism by which these factors are linked remains obscure. We took the human leukocyte antigen (HLA), located on chromosome 6p as marker for inflammation and studied whether aberrations of chromosome 6p influenced the HLA expression in UM.Methods SNP‐array copy number analysis and gene expression profiling were performed on 28 uveal melanomas of patients who underwent enucleation between 1999 and 2004 at the Leiden University Medical Center, Leiden, in The Netherlands. UM protein expression of HLA‐A, ‐B and –DR was measured with immunohistochemistry. The status of chromosome 3, 6p, and gene expression of HLA and HLA regulators were analyzed, as well as protein expression of HLA.Results Gain of 6p was present in 8 cases (29%) and not related with survival. An increased gene expression was seen in the group who died due to metastatic UM for HLA‐A and –B, and B2M. Gene expression correlated with protein expression for HLA‐A, HLA‐B, but not with HLA‐DR. Tumors did not differ in level of HLA protein expression, HLA gene expression, and HLA regulator gene expression with regard to chromosome 6p status.Conclusion High HLA gene‐ and protein expression in UM are not influenced by gain of 6p. Yet for the first time we demonstrated that in UM an increased expression of HLA class I genes correlated with the elevated protein expression of HLA class I.

Gene expression profiling identifies different sub-types of retinoblastoma

Background:Mutation of the RB1 gene is necessary but not sufficient for the development of retinoblastoma. The nature of events occurring subsequent to RB1 mutation is unclear, as is the retinal cell-of-origin of this tumour.Methods:Gene expression profiling of 21 retinoblastomas was carried out to identify genetic events that contribute to tumorigenesis and to obtain information about tumour histogenesis.Results:Expression analysis showed a clear separation of retinoblastomas into two groups. Group 1 retinoblastomas express genes associated with a range of different retinal cell types, suggesting derivation from a retinal progenitor cell type. Recurrent chromosomal alterations typical of retinoblastoma, for example, chromosome 1q and 6p gain and 16q loss were also a feature of this group, and clinically they were characterised by an invasive pattern of tumour growth. In contrast, group 2 retinoblastomas were found to retain many characteristics of cone photoreceptor cells and appear to exploit the high metabolic capacity of this cell type in order to promote tumour proliferation.Conclusion:Retinoblastoma is a heterogeneous tumour with variable biology and clinical characteristics.

Microarray Analysis of Formalin-Fixed Paraffin-Embedded (FFPET) and Frozen (FZT) Uveal Melanoma Detects Clinically Relevant Abnormalities, Including Chromosome 3 Loss of Heterozygosity

Molecular cytogenetic alterations, specifically monosomy 3, are strongly correlated with metastases and death in uveal melanoma (UVM). Although FISH can be used for the identification of monosomy 3, a subset of UVM exhibit only partial loss of chromosome 3 potentially missed with chromosome enumeration probes. Moreover, often limited material available can compromise detection of additional alterations with potential clinical relevance. Microarray analysis is an alternative method for the analysis of such specimens affording whole genome interrogation. In the current study we performed microarray to detect genomic changes in DNA from FFPET and FZT UVM samples (clinical trial NCT00952939). Of the available cases, 23.5% yielded DNA of sufficient quantity and quality to obtain interpretable microarray results, representing 28 patients. All but one case demonstrated significant abnormalities. Gains of 8q, consistent with an apparent i(8)(q10) karyotype, were the most common finding, seen in 21 patients. Monosomy 3 was detected by microarray in 15 cases. A single case showed partial loss of 3 (3q11.2q25.31), the only clinically significant abnormality detected in that case. All cases lacking chromosome 3 abnormalities showed a copy gain of 6p sharing a small 28.18 Mb region of overlap at 6p25.2p21.33. Additional findings included TERT and NEDD9 amplifications, and CDKN2A/B and LUM gene deletions. SNP analysis revealed three cases with unique regions of copy neutral LOH involving 5p15.33q35.3, 15q11.2q21.1 and one case with whole chromosome LOH for chromosomes 3, 4 and 6. The latter was detected in a FZT specimen, while the paired FFPET sample lacked evidence of LOH and showed monosomy 3, trisomy 4 and whole arm gains and losses of 6p and 6q, respectively, suggesting the presence of tumor heterogeneity. Microarray analysis has identified new recurrent abnormalities associated with UVM with potential relevance to UVM biology, diagnosis and prognosis. Conflict of Interest: Roger Schultz is an employee of Perkin Elmer.

Abstract 4702: Chromosome 2p gain and N-Myc amplification predict poor outcome of patients with LIN28 negative supratentorial primitive neuroectodermal tumors of the central nervous system (CNS-PNET)..

Abstract Primitive neuroectodermal tumors of central nervous system (CNS-PNET) which represents 3-7% of all pediatric brain tumors, are a rare group of neoplasms occurring in the CNS and include supratentorial PNETs (s-PNET), medulloepitheliomas and ependymoblastomas. Whereas ependymoblastomas harbour chromosome 19q13.41 amplification, express the neural stem cell marker LIN28 and show aggressive clinical behaviour, the biological mechanisms and molecular alterations contributing to the pathogenesis of s-PNETs remain so far poorly understood. Expression analyses and limited CGH studies indicated that s-PNETs may represent genetically a heterogeneous group with frequent but multiple copy number aberrations. In order to find possible specific molecular and cytogenetic alterations suitable for molecular risk stratification and predictive for the overall survival, we performed a multiplex ligation-dependent probe amplification (MLPA) and molecular inversion probe (MIP) analysis on DNA samples of 25 s-PNET cases of patients (median age 5.35 years; range, 2.41 - 17.28 years). The majority of patients were enrolled in the clinical GPOH HIT2000 trial. MLPA and MIP analysis revealed large losses of genomic material of chromosomes 3, 4, 5, 13, while frequent gains were observed at chromosomes 1, 17, 19, 20 and 22. Recurrent regions of gains were found at chromosome 7q11.2, 7q22.1, 12q13 and losses at 16q23.1, 6p21.1, and 11q11. High-copy number gains (amplifications) were found in particular at chromosome 2p24.3 (N-Myc) (in 6 cases) and 4q21 (in 2 cases). None of the chromosomal alterations identified was found significantly associated with the age of the patients at diagnosis, presence of tumor metastases or presence of postoperative residual tumor. In contrast, patients with tumors harbouring 2p gain or N-Myc amplification showed shorter overall survival (OS) (p=0.0029 and p=0.0012, respectively).These parameters were independent from the presence of metastases, which was indeed the most important clinical factor for unfavourable OS (p=0.0112) in our series. In the era of the personalized neuro-oncology, the identification of molecular marker associated to patients outcome may represent the further significant step towards the patients's risk stratification and the improvement of therapeutic strategies also for patients suffering from s-PNETs. Citation Format: Marco Gessi, Andre' von Bueren, Andras Treszl, Anja zur Muehlen, Stefan Rutkowski, Torsten Pietsch. Chromosome 2p gain and N-Myc amplification predict poor outcome of patients with LIN28 negative supratentorial primitive neuroectodermal tumors of the central nervous system (CNS-PNET). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2013-4702

Changes in MYCN expression in human neuroblastoma cell lines following cisplatin treatment may not be related to MYCN copy numbers

Neuroblastoma is a tumor accounting for approximately 10% of all childhood malignancies and 50% of all childhood cancer-related deaths. MYCN gene copy number variation represents the most important prognostic factor in neuroblastoma. Prognostic significance of MYCN gene expression is more complicated and may depend on other factors such as MYCN gene copy number status. In the present study, we assessed MYCN gene expression using real-time RT-PCR following cisplatin treatment in three human neuroblastoma cell lines (UKF-NB-3, UKF-NB-4 and SK-N-AS) and their cisplatin-resistant counterparts. We also examined MYCN gene status and copy number (gain and amplification) variations using interphase and metaphase fluorescent insitu hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). Only cisplatin-sensitive UKF-NB-4 cells exhibited decreased MYCN expression following treatment with cisplatin. Other sensitive neuroblastoma cells did not exhibit a change in MYCN expression. In contrast, cisplatin-resistant UKF-NB-4 and SK-N-AS cells exhibited increased MYCN expression irrespective of the number of MYCN copies or concentration of cisplatin in the medium. In MYCN-amplified neuroblastoma cells we did not observe any significant change in the number of MYCN copies after cisplatin treatment, whereas MYCN-non-amplified SK-N-AS cells revealed during cisplatin treatment an increased number of MYCN gene copies caused by 2p gain in the majority of cells by FISH. We postulated that cisplatin treatment does not result directly in altered transcription of MYCN. A functional change in MYCN mRNA levels and increased MYCN expression in cisplatin-resistant neuroblastoma cells do not have a clear relationship to MYCN copy numbers. These findings may further contribute to the understanding of cisplatin chemotherapy in connection with MYCN expression, and the possible copy number variations in MYCN neuroblastoma cells may be of importance since targeting of MYCN is being tested as neuroblastoma therapy.

Tumoral EPAS1 (HIF2A) mutations explain sporadic pheochromocytoma and paraganglioma in the absence of erythrocytosis

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are chromaffin-cell tumors that arise from the adrenal medulla and extra-adrenal paraganglia, respectively. The dysfunction of genes involved in the cellular response to hypoxia, such as VHL, EGL nine homolog 1, and the succinate dehydrogenase (SDH) genes, leads to a direct abrogation of hypoxia inducible factor (HIF) degradation, resulting in a pseudo-hypoxic state implicated in PCC/PGL development. Recently, somatic post-zygotic mutations in EPAS1 (HIF2A) have been found in patients with multiple PGLs and congenital erythrocytosis. We assessed 41 PCCs/PGLs for mutations in EPAS1 and herein describe the clinical, molecular and genetic characteristics of the 7 patients found to carry somatic EPAS1 mutations; 4 presented with multiple PGLs (3 of them also had congenital erythrocytosis), whereas 3 were single sporadic PCC/PGL cases. Gene expression analysis of EPAS1-mutated tumors revealed similar mRNA EPAS1 levels to those found in SDH-gene- and VHL-mutated cases and a significant up-regulation of two hypoxia-induced genes (PCSK6 and GNA14). Interestingly, single nucleotide polymorphism array analysis revealed an exclusive gain of chromosome 2p in three EPAS1-mutated tumors. Furthermore, multiplex-PCR screening for small rearrangements detected a specific EPAS1 gain in another EPAS1-mutated tumor and in three non-EPAS1-mutated cases. The finding that EPAS1 is involved in the sporadic presentation of the disease not only increases the percentage of PCCs/PGLs with known driver mutations, but also highlights the relevance of studying other hypoxia-related genes in apparently sporadic tumors. Finally, the detection of a specific copy number alteration affecting chromosome 2p in EPAS1-mutated tumors may guide the genetic diagnosis of patients with this disease.

Molecular pathology of uveal melanoma

Like other cancers, uveal melanomas (UM) are characterised by an uncontrolled, clonal, cellular proliferation, occurring as a result of numerous genetic, and epigenetic aberrations. Signalling pathways known to be disrupted in UM include: (1) the retinoblastoma pathway, probably as a result of cyclin D1 overexpression; p53 signalling, possibly as a consequence of MDM2 overexpression; and the P13K/AKT and mitogen-activated protein kinase/extracellular signal-related kinase pathway pathways that are disturbed as a result of PTEN and GNAQ/11 mutations, respectively. Characteristic chromosomal abnormalities are common and include 6p gain, associated with a good prognosis, as well as 1p loss, 3 loss, and 8q gain, which correlate with high mortality. These are identified by techniques such as fluorescence in situ hybridisation, comparative genomic hybridisation, microsatellite analysis, multiplex ligation-dependent probe amplification, and single-nucleotide polymorphisms. UM can also be categorised by their gene expression profiles as class 1 or class 2, the latter correlating with poor survival, as do BRCA1-associated protein-1 (BAP1) inactivating mutations. Genetic testing of UM has enhanced prognostication, especially when results are integrated with histological and clinical data. The identification of abnormal signalling pathways, genes and proteins in UM opens the way for target-based therapies, improving prospects for conserving vision and prolonging life.

Chromosome 2p gain in monoclonal B‐cell lymphocytosis and in early stage chronic lymphocytic leukemia

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B-cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10(-4) ) and CD38 (P < 1 × 10(-4) ) and ZAP-70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B-cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion.

555 Perinatal Neuroblastoma with a Germline Interstitial 2P Duplication Involving the Mycn Gene: a Case Report

Background/AimsMYCN proto-oncogene is located on chromosome 2p24. MYCN amplification is a poor prognostic factor in neuroblastoma. However, the role of germline MYCN copy number gain is unclear. It is unknown...

Neurofibroma-Like Spindle Cell Melanoma

We present a case that highlights the use of two maneuvers useful in the diagnosis of spindle cell melanoma. A shave biopsy from the cheek of a 58-year-old man demonstrated a thin invasive melanoma of 0.3 mm thickness with a less than 2-mm-wide intra-epidermal component. Below this melanocytic lesion, but not contiguous with it, there was a transected S-100-positive and Melan-A-negative spindle cell proliferation. Upon re-excision, no residuum of conventional melanoma was identified, but a residual spindle cell neoplasm that was 4 mm in diameter, nodular, well-circumscribed, cytologically bland, and S-100 positive was noted. At our consensus conference, our group favored neurofibroma but agreed that spindle cell melanoma could not be excluded based on histopathologic features alone. To further address the differential diagnosis, we performed CD34 staining that demonstrated lack of a CD34 fingerprint. We also completed array-based comparative genomic hybridization, which demonstrated gain of chromosome 6p, loss of 6p and gain of 7. These two methods of analysis support a diagnosis of spindle cell melanoma.

Higher Percentage of FISH-Determined Monosomy 3 and 8q Amplification in Uveal Melanoma Cells relate to Poor Patient Prognosis

To investigate the relation between patient survival and incrementally increasing percentages of fluorescence in situ hybridization-determined complete loss of chromosome 3 (monosomy 3) and gain of chromosome 8q in primary uveal melanoma cells. Clinicopathological factors were related to disease-free survival. Fluorescence in situ hybridization was performed using probes on chromosomes 1, 3, 6, and 8. The percentages of UM cells with monosomy 3 or chromosome 8q gain were classified in groups with incrementally increasing percentages and related to disease-free survival. Correlations between clinical factors and cytogenetic aberrations were also analyzed. Two-hundred twenty choroidal and ciliary body melanomas were analyzed. The following proved to be significant predictors of survival in univariate analysis: older patient age (P = 0.003); large tumor diameter (P < 0.001); mixed cell type (P = 0.001); presence of closed microvascular loops (P < 0.001); loss of chromosome 1p (P = 0.006); monosomy 3 (P < 0.001); gain of 6p (P < 0.001); and gain of chromosome 8q (P < 0.001). Multivariate Cox analysis displayed monosomy 3 (Hazard ratio [HR] 2.83, P = 0.002) and gain of chromosome 8q (HR 3.13, P = 0.002) as the most important independent prognostic factors of poor survival, followed by older patient age (HR 1.02, P = 0.017). Increasing percentages of monosomy 3 and gain of chromosome 8q in tumor cells showed a correlation with worse prognosis (Log-rank test 49.9 and 40.4, both P < 0.001) and increased number of additional copies of 8q correlated with shorter disease-free interval (Log-rank test 45.7, P < 0.001). A high percentage monosomy 3 and chromosome 8q gain in primary UM cells showed a strong relation with poor disease-free survival compared with low percentage aberrations.

Synchronous clear cell renal cell carcinoma and tubulocystic carcinoma: genetic evidence of independent ontogenesis and implications of chromosomal imbalances in tumor progression

Seven percent of renal cell carcinoma (RCC) cases are diagnosed as "unclassified" RCC by morphology. Genetic profiling of RCCs helps define renal tumor subtypes, especially in cases where morphologic diagnosis is inconclusive. This report describes a patient with synchronous clear cell RCC (ccRCC) and a tubulocystic renal carcinoma (TCRC) in the same kidney, and discusses the pathologic features and genetic profile of both tumors. A 67 year-old male underwent CT scans for an unrelated medical event. Two incidental renal lesions were found and ultimately removed by radical nephrectomy. The smaller lesion had multiple small cystic spaces lined by hobnail cells with high nuclear grade separated by fibrous stroma. This morphology and the expression of proximal (CD10, AMACR) and distal tubule cell (CK19) markers by immunohistochemistry supported the diagnosis of TCRC. The larger lesion was a typical ccRCC, with Fuhrman's nuclear grade 3 and confined to the kidney. Molecular characterization of both neoplasms using virtual karyotyping was performed to assess relatedness of these tumors. Low grade areas (Fuhrman grade 2) of the ccRCC showed loss of 3p and gains in chromosomes 5 and 7, whereas oncocytic areas displayed additional gain of 2p and loss of 10q; the high grade areas (Fuhrman grade 3) showed several additional imbalances. In contrast, the TCRC demonstrated a distinct profile with gains of chromosomes 8 and 17 and loss of 9. In conclusion, ccRCC and TCRC show distinct genomic copy number profiles and chromosomal imbalances in TCRC might be implicated in the pathogenesis of this tumor. Second, the presence of a ccRCC with varying degrees of differentiation exemplifies the sequence of chromosomal imbalances acquired during tumor progression.Virtual SlidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1790525735655283

Chromosome 3 Anomalies Investigated by Genome Wide SNP Analysis of Benign, Low Malignant Potential and Low Grade Ovarian Serous Tumours

Ovarian carcinomas exhibit extensive heterogeneity, and their etiology remains unknown. Histological and genetic evidence has led to the proposal that low grade ovarian serous carcinomas (LGOSC) have a different etiology than high grade carcinomas (HGOSC), arising from serous tumours of low malignant potential (LMP). Common regions of chromosome (chr) 3 loss have been observed in all types of serous ovarian tumours, including benign, suggesting that these regions contain genes important in the development of all ovarian serous carcinomas. A high-density genome-wide genotyping bead array technology, which assayed >600,000 markers, was applied to a panel of serous benign and LMP tumours and a small set of LGOSC, to characterize somatic events associated with the most indolent forms of ovarian disease. The genomic patterns inferred were related to TP53, KRAS and BRAF mutations. An increasing frequency of genomic anomalies was observed with pathology of disease: 3/22 (13.6%) benign cases, 40/53 (75.5%) LMP cases and 10/11 (90.9%) LGOSC cases. Low frequencies of chr3 anomalies occurred in all tumour types. Runs of homozygosity were most commonly observed on chr3, with the 3p12-p11 candidate tumour suppressor region the most frequently homozygous region in the genome. An LMP harboured a homozygous deletion on chr6 which created a GOPC-ROS1 fusion gene, previously reported as oncogenic in other cancer types. Somatic TP53, KRAS and BRAF mutations were not observed in benign tumours. KRAS-mutation positive LMP cases displayed significantly more chromosomal aberrations than BRAF-mutation positive or KRAS and BRAF mutation negative cases. Gain of 12p, which harbours the KRAS gene, was particularly evident. A pathology review reclassified all TP53-mutation positive LGOSC cases, some of which acquired a HGOSC status. Taken together, our results support the view that LGOSC could arise from serous benign and LMP tumours, but does not exclude the possibility that HGOSC may derive from LMP tumours.

Papillary Glioneuronal Tumors

This study was designed to evaluate 4 new cases of papillary glioneuronal tumors (PGNTs), 2 of which had atypical histologic features, provides extensive IHC characterization, performed comparative genomic hybridization in 2 of our cases, and reviews the recent literature. The study group comprised 3 women and 1 man, with ages ranging from 12 to 75 years. Patients presented with seizures (n = 3) or muscle spasm (n = 1), and the tumors were located in the supratentorial region of the brain (3 in the frontal and 1 in the parietotemporal lobe). The 2 atypical tumors showed a moderately high mitotic rate (4 and 7/10 HPF, each), vascular endothelial hyperplasia, and necrosis. Tumor cells expressed both neuronal and glial markers, but the degree of neuronal versus glial expression was varied. None of the tumors expressed p53, EGFR wild type/vIII, IDH1, or CD34; however, nestin, galectin-3, and S100 were positive in the tumor cells. No EGFR gene amplification or 1p/19q deletion was found by fluorescence in situ hybridization. Half of the cases revealed PTEN loss by immunohistochemistry, and MGMT methylation was positive in 3 cases by MGMT methylation-specific polymerase chain reaction. Ultrastructurally, either astrocytic or neuronal differentiation was observed, but we could not identify any hybrid cells. An array-based comparative genomic hybridization study revealed loss of 1q, 6p, 8p, 9p, 9q, and 16q and gain of 2q, 3p, 5q, 6p, 7q, 10q, 16q, 19p, and 22q in 2 cases simultaneously. The first patient, who underwent subtotal resection, died because of progression of the tumor within 9 months after surgery; however, 2 patients were symptom free and progression free at 34 and 48 months after gross total resection (the patient 2: plus radiotherapy, the patient 3: no adjuvant chemo- or radiotherapy). The last patient developed seizures after a long symptom-free period (40 mo) with no evidence of tumor recurrence. Our 4 new cases, in conjunction with the literature review, reinforce that PGNTs are tumors that usually occur in young adults (mean age, 24 y); they are most often cystic with a mural nodule or are cystic/solid, supratentorial, closely located with the ventricle, molecularly genetically different from astrocytic or oligodendroglial tumors, and indolent in behavior. Cases (2 of 4 in our study) with atypical histologic features or that occur in advanced age (75 y), and sporadic reports of histologically or biologically aggressive PGNTs, serve to remind pathologists that the full spectrum of PGNTs is as yet unknown.

Novel Germline Genetic Variants Associated with Familial Chronic Lymphocytic Leukemia (CLL)

Abstract 465CLL is among the most heritable of all cancers. To understand the genetic basis of this heritability, we have undertaken a comprehensive genomic analysis of familial CLLs including copy number analysis, gene expression profiling (GEP) and whole exome sequencing (WES). First, we examined whether familial and sporadic cases differ in the spectrum of acquired somatic mutations by WES of tumor and germline DNA of 36 familial CLLs (from 31 affected families). Compared to 55 sporadic CLLs, we observed that the somatic mutation rate in the familial CLLs was similar (mean 0.89 mutations/Mb (range 0.29–3.06) for the sporadics vs mean 0.97/Mb (range 0.11–3.78) for the familials, p=0.40). We also examined the spectrum of somatic mutations by testing for enrichment of 9 recently identified putative tumor drivers in our large CLL sequencing study (reported elsewhere in this meeting). We observed a similar distribution of these recurrent CLL mutations among the 36 familial CLLs as the 55 sporadic CLLs. These results were further confirmed by genotyping of the CLL driver mutations in an additional 32 familial and 67 sporadic CLLs. Collectively, these studies suggest that while the predisposing germline events may differ between familial and sporadic CLL, the spectrum of mutations and pattern of mutagenesis appear similar in the established CLL tumors.We therefore proceeded to examine the genetic characterization of germline DNA to identify predisposing loci, which we hypothesized might be enriched in a familial disease context. We first examined germline copy number variations (CNVs), which have not been previously characterized in this disease. We used high resolution Affymetrix 6.0 SNP arrays to study both tumor and germline DNA of 58 individuals representing 44 different families with CLL and lymphoproliferative disorders (LPDs). We identified two families (A and B) with autosomal dominant inheritance of CLL who carried distinct germline CNVs that affect genes previously implicated in CLL. Members of Family A carried a 525 kb germline deletion targeting DLEU7 at 13q14, but not affecting DLEU2, miR-15a, or miR-16–1. Importantly, by examining the tumor genome from these family members, we observed a uniform loss of the second allele of DLEU7 in 2/2 available CLLs from this family, suggesting an acquired “second hit” of a tumor suppressor gene. These findings underline the complexity of the most common somatically acquired copy number aberration (CNA) in CLL, 13q14 deletion, by demonstrating the role of additional regions other than the heavily investigated miRNA cluster. Members of Family B carried a 720 kb germline gain of 6p25 affecting the IRF4 gene, previously implicated in CLL through the identification of a GWAS risk allele located in the 3’ UTR of IRF4, as well as the recent description of a recurrent somatic mutation affecting 1.5% of CLL cases. In Family B, the coding regions of the four genes located in this 6p gain, namely IRF4, DUSP22, EXOC2 and HUS1B, were sequenced, and no somatic mutations or novel SNPs were identified. However, the 6p gain in Family B represents an allele-specific enrichment of the haplotype carrying the GWAS risk SNP and, as previously described for that allele, results in lower expression of IRF4 in the two CLLs tested in this family. GEP further identified a signature associated with 6p gain that preserved low expression of IRF4 and showed high expression of KLF6. These results demonstrate that germline CNVs may facilitate the “path to cancer” by providing either an allelic deletion of a tumor suppressor or an amplification of a risk allele.As most familial CLL cases have not been accounted for by known SNPs or germline CNVs, we have initiated an in depth analysis of the WES germline results from familial cases compared to both sporadic CLL patients and normal individuals. Candidate variants have been filtered to exclude all SNPs described in the 1000 Genomes project and to focus on highly conserved sites. Thus far we have found that rare germline variants in patients with familial CLL contain a rich source of loci with relevance to B cell biology. Studies in progress are focused on further analysis of informative families and functional analyses of candidate variants. These comprehensive genomic analyses are expected to identify multiple cooperating genetic mechanisms that contribute to CLL pathogenesis, including CNVs and somatic and germline mutations. Disclosures:No relevant conflicts of interest to declare.

Targeted Oligonucleotide Array Assessment of Genomic Copy Number Alterations for Risk Stratification in Chronic Lymphocytic Leukemia

Abstract 1773Risk stratification in chronic lymphocytic leukemia (CLL) is highly desirable and should comprise not only evaluation of clinical features but also molecular prognostic markers. Currently such molecular markers include loss of 17p13, 11q22, 13q14, 6q22, and gain of chromosome 12 as assessed by fluorescence in situ hybridization (FISH) and mutation status of the variable region of the IGH gene (IGHV) by sequencing. In recent years, genome-wide scanning technologies such as array-comparative genomic hybridization (array-CGH) have revealed novel and refined known copy number alterations (CNAs) in the CLL genome. In order to evaluate the potential of array-CGH in prognostication in mature B-cell neoplasms, including CLL, and implement array-CGH in a clinical diagnostic laboratory, a targeted oligonucleotide-based microarray was custom designed to represent genomic regions exhibiting gain/loss in these lymphoid neoplasms. The 4 × 44K formatted array included 2 × 17,348 probes for the 80 selected genomic regions (average resolution of 34kbp), and recommended controls including a 1Mbp genome backbone. DNA extracted from two CLL datasets were submitted to array-CGH using an equimixture of commercially available male/female DNA as a reference. CNAs were detected using Genomics Workbench Lite (Agilent Technologies, Inc.) with the ADM2 algorithm. Analytical sensitivity was assessed by cell line DNA dilution and by FISH (116 specimens) and was 30–40% and 20–25%, respectively. Recurrent CNAs in previously untreated patients, greater than 1.5Mbp in size, were analyzed for association with time to first treatment (TTFT) and overall survival (OS) by the log rank test. Association with IGHV mutation status was tested using the Fisher’s two-sided exact test. In both datasets for untreated specimens, unmutated IGHV negatively correlated with both TTFT and OS significantly (p < 0.05). Gain of chromosome 12 was detected in 11–12% of untreated specimens in both datasets and as expected did not associate with outcome. Loss of 13q14 as a sole abnormality (excluding copy number changes arising at known sites of normal variation) was associated with an overall favorable outcome, but specimens with loss of both loci (MIR15A/16-1 and RB1) versus one locus (MIR15A/16-1) did not display significantly different outcomes. As expected loss of 17p13 associated with shorter TTFT and OS, and was observed at higher levels in treated specimens. A similar result was observed for 11q22 loss but not in the second dataset, perhaps due to the relatively short follow-up time. Importantly, four additional copy number changes (gain of 2p, 3q, and 8q, and loss of 8p) were found to associate with shorter TTFT and/or OS, and also occurred at higher frequency in treated specimens. Notably, all but one specimen exhibiting two of these CNAs, were Rai Stage 0-II. After multiple comparisons correction, gain of 2p and 3q, and loss of 8p remained significantly associated with an unfavorable outcome. Gain of 2p25.3-p15 was observed exclusively in unmutated IGHV specimens. Loss of 18p and gain of 17q24 were not considered further for testing due to low frequency or lower frequency in treated specimens (data not shown). Uniquely, these data demonstrate in low-intermediate risk CLL cohorts the prognostic value of genomic gain/loss at multiple sites and support implementation of array-CGH into a clinical setting for risk stratification in CLL where genomic gain or loss of multiple clinically relevant genomic regions can be assessed simultaneously.Dataset 1 Untreated n = 81TTFT p-valueOS p-valueTreated n = 38Dataset 2 n = 169TTFT p-valueOS p-valueTreated n = 28Median TTFT87.6 mo24.1 moMedian OS117.7 mo37.2 moRai Stage02577I-II4248III-IV51na943Unmutated IGHV46% (n=80)0.00030.000438% (n=163)0.0020.04413q14 loss (sole abnormality)52.5%0.038‡0.087‡33.7%0.144‡0.008‡MIR15A/16-1, RB127.5%0.770.33711.2%0.0111MIR15A/16-125.0%22.5%11q22 loss (ATM)12.3%0.1250.00923.7%8.3%0.3930.97714.3%17p13 loss (TP53)2.5%0.0100.01215.8%4.7%0.006<.000110.7%2p25.3-p15 gain6.2%0.002<.000110.5%3.0%0.7020.02510.7%8q24 gain2.5%0.2380.0147.9%4.1%0.5640.0070.0%3q26-q27 gain2.5%<.0001<.00015.3%3.0%0.850<.00017.1%8p23-p21 loss2.5%0.0020.01610.5%1.2%1<.00017.1%Unless otherwise noted, all values associated with shorter times‡Associated with longer time na not available Disclosures:Houldsworth:Cancer Genetics, Inc.: Employment. Guttapalli:Cancer Genetics, Inc.: Employment. Ma:Cancer Genetics, Inc.: Employment. Chen:Cancer Genetics, Inc.: Employment. Patil:Cancer Genetics, Inc.: Consultancy.