Reporting of Diagnostic Cytogenetic Results.

This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a useful reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding, FISH, or chromosomal microarray). Multi-specimen usage macros are included that can be applied to two or more specimen types.

CLK2 Is an Oncogenic Kinase and Splicing Regulator in Breast Cancer.

Genetically activated kinases have been attractive therapeutic targets in cancer due to the relative ease of developing tumor-specific treatment strategies for them. To discover novel putative oncogenic kinases, we identified 26 genes commonly amplified and overexpressed in breast cancer and subjected them to a lentiviral shRNA cell viability screen in a panel of breast cancer cell lines. Here, we report that CLK2, a kinase that phosphorylates SR proteins involved in splicing, acts as an oncogene in breast cancer. Deregulated alternative splicing patterns are commonly observed in human cancers but the underlying mechanisms and functional relevance are still largely unknown. CLK2 is amplified and overexpressed in a significant fraction of breast tumors. Downregulation of CLK2 inhibits breast cancer growth in cell culture and in xenograft models and it enhances cell migration and invasion. Loss of CLK2 in luminal breast cancer cells leads to the upregulation of epithelial-to-mesenchymal transition (EMT)-related genes and a switch to mesenchymal splice variants of several genes, including ENAH (MENA). These results imply that therapeutic targeting of CLK2 may be used to modulate EMT splicing patterns and to inhibit breast tumor growth.

Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments.

Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin-fixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 µm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50-µm tissue sections. To interpret FISH results using 4 to 6 µm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single-cell suspension generally gives an interpretable result.

Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-rearranged lung adenocarcinomas.

INTRODUCTION: ALK gene rearrangements occur in approximately 5% of lung adenocarcinomas (ACAs), leading to anaplastic lymphoma kinase (ALK) overexpression and predicting response to targeted therapy. Fluorescence in situ hybridization (FISH) is the standard procedure for detection of ALK rearrangements in lung ACA but requires specialized equipment and expertise. Immunohistochemistry (IHC) for ALK protein overexpression is a promising screening modality, with reports of newer antibodies showing excellent sensitivity and specificity for ALK-rearranged lung ACA. METHODS: In this study, we analyzed ALK IHC (5A4 clone) in 186 cases from our clinical service and compared it with ALK FISH and EGFR and KRAS mutation status. RESULTS: Twelve cases had concordant ALK protein overexpression and ALK rearrangement by FISH. Three ALK-rearranged cases lacked ALK protein expression. Of these discrepant cases, one had a coexisting EGFR mutation and a subtle atypical ALK rearrangement manifested as a break in the 5' centromeric portion of the FISH probe. One case had a concurrent BRAF mutation. Follow-up testing on a metastasis revealed absence of the ALK rearrangement, with persistent BRAF mutation. In one ALK-rearranged protein negative case, very limited tissue remained for ALK IHC, raising the possibility of false negativity because of protein expression heterogeneity. Importantly, ALK protein expression was detected in one case initially thought not to have an ALK rearrangement. In this case, FISH was falsely negative because of interference by benign reactive nuclei. After correcting for these cases, ALK IHC was 93% sensitive and 100% specific as compared with FISH. CONCLUSIONS: ALK IHC improves the detection of ALK rearrangements when used together with FISH, and its use in lung ACA genetic testing algorithms should be considered.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Single-cell DNA and FISH analysis for application to preimplantation genetic diagnosis.

Preimplantation genetic testing, which includes preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS), is a form of a very early prenatal testing. The goal of this method is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole-genome amplification by primer-extension preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single-cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with support protocols for probe preparation and probe validation, which is recommended as a preliminary step before performing any PGD or PGS FISH analysis.

Regulation of in situ to invasive breast carcinoma transition.

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.

Integrative genomic approaches identify IKBKE as a breast cancer oncogene.

The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery.

Maternally inherited duplication of chromosome 7, dup(7)(p11.2p12), associated with mild cognitive deficit without features of Silver-Russell syndrome.

We report on a familial duplication in the short arm of chromosome 7, dup(7)(p11.2p12), present in three generations. The duplication was identified by GTG-banding and fluorescence in situ hybridization (FISH) with a whole chromosome 7 DNA painting probe that verified that the duplicated material originated from chromosome 7. The multicolor banding (mBAND) was used to refine the breakpoint assignment. The duplication identified in the proband was also present in her son and mother. All three carriers have mild cognitive deficiencies. Interstitial duplications of the short arm of chromosome 7, although relatively uncommon, have been described in association with a variety of clinical features, including mental retardation of varying severity. Duplication of the p11.2p13 region on chromosome 7 was reported in association with Silver-Russell syndrome (SRS), and an overlapping dup(7)(p11.2p14.1)dn was described in an individual with autistic disorder. Furthermore, a potentially overlapping maternally transmitted inverted duplication, dup(7)(p13p12.2), was reported in patients with cognitive delay. These observations and the phenotype of our duplication carriers suggest that partial trisomy of the proximal 7p region causes cognitive deficiency. The maternal origin of the duplication is of special interest in light of genomic imprinting and implication of the 7p11-p13 region in the SRS etiology. Locus-specific FISH targeting a growth factor receptor binding protein 10 (GRB10), the strong candidate for SRS residing at 7p12.2, showed that it is not duplicated in our patients. Our study helps refine the SRS critical region on 7p and extends our understanding of the clinical manifestations associated with 7p duplications.

Molecular definition of breast tumor heterogeneity.

Cells with distinct phenotypes including stem-cell-like properties have been proposed to exist in normal human mammary epithelium and breast carcinomas, but their detailed molecular characteristics and clinical significance are unclear. We determined gene expression and genetic profiles of cells purified from cancerous and normal breast tissue using markers previously associated with stem-cell-like properties. CD24+ and CD44+ cells from individual tumors were clonally related but not always identical. CD44+ cell-specific genes included many known stem-cell markers and correlated with decreased patient survival. The TGF-beta pathway was specifically active in CD44+ cancer cells, where its inhibition induced a more epithelial phenotype. Our data suggest prognostic relevance of CD44+ cells and therapeutic targeting of distinct tumor cell populations.

Preparation of cells from formalin-fixed, paraffin-embedded tissue for use in fluorescence in situ hybridization (FISH) experiments.

Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin-fixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 microm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50 microm tissue sections. To interpret FISH results using 4 to 6 microm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single cell suspension generally gives an interpretable result.

Validation of DNA probes for preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) R1.

BACKGROUND: Preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) is being employed increasingly by medical centers and private companies. Validation of any clinical assay, particularly one with novel applications such as PGD by FISH, is of critical importance in the clinical setting. This importance is recognized by both the College of American Pathologists (CAP) and the American College of Medical Genetics (ACMG), who recommend validation of FISH assays in the clinical setting. Validation of FISH assays for PGD is especially significant, as only one or two cells (blastomeres) will be available for testing of a given embryo. METHODS: We have developed validation protocols for a variety of FISH assays, including sex identification, structural chromosomal aneusomy, and aneuploidy screening with the Vysis, Inc., PGT probe panel. RESULTS: Our validation results show good individual performance of commercially available probes, and decreasing overall efficiency as the number of probes included in an assay increases.

Combined cDNA array comparative genomic hybridization and serial analysis of gene expression analysis of breast tumor progression.

To identify genetic changes involved in the progression of breast carcinoma, we did cDNA array comparative genomic hybridization (CGH) on a panel of breast tumors, including 10 ductal carcinoma in situ (DCIS), 18 invasive breast carcinomas, and two lymph node metastases. We identified 49 minimal commonly amplified regions (MCRs) that included known (1q, 8q24, 11q13, 17q21-q23, and 20q13) and several uncharacterized (12p13 and 16p13) regional copy number gains. With the exception of the 17q21 (ERBB2) amplicon, the overall frequency of copy number alterations was higher in invasive tumors than that in DCIS, with several of them present only in invasive cancer. Amplification of candidate loci was confirmed by quantitative PCR in breast carcinomas and cell lines. To identify putative targets of amplicons, we developed a method combining array CGH and serial analysis of gene expression (SAGE) data to correlate copy number and expression levels for each gene within MCRs. Using this approach, we were able to distinguish a few candidate targets from a set of coamplified genes. Analysis of the 12p13-p12 amplicon identified four putative targets: TEL/ETV6, H2AFJ, EPS8, and KRAS2. The amplification of all four candidates was confirmed by quantitative PCR and fluorescence in situ hybridization, but only H2AFJ and EPS8 were overexpressed in breast tumors with 12p13 amplification compared with a panel of normal mammary epithelial cells. These results show the power of combined array CGH and SAGE analysis for the identification of candidate amplicon targets and identify H2AFJ and EPS8 as novel putative oncogenes in breast cancer.

Complete hydatidiform mole retaining a chromosome 11 of maternal origin: molecular genetic analysis of a case.

Hydatidiform moles are pregnancies characterized by abnormal development of both embryonic and extraembryonic tissues and are associated with the misexpression of imprinted genes. The vast majority of complete hydatidiform moles are diploid and androgenetic, whereas partial hydatidiform moles are triploid, with an extra set of chromosomes of paternal origin. Here, we present an unusual complete mole that showed strong expression of two imprinted, maternally transcribed genes, CDKN1C (encoding p57(KIP2)) and PHLDA2 (TSSC3/IPL), both part of a large imprinted gene domain on chromosome 11. Using microsatellite genotyping and fluorescent in situ hybridization, we show that this paradoxical gene expression was due to retention of a maternal copy of chromosome 11 in addition to the two paternal copies normally present in complete moles. These findings demonstrate that, despite being predominantly androgenetic, some complete moles contain small amounts of DNA of maternal origin. Furthermore, these results provide an explanation for rare false negatives that can arise when p57(KIP2) is used as a diagnostic marker for complete moles.

Mutation of perinatal myosin heavy chain associated with a Carney complex variant.

BACKGROUND: Familial cardiac myxomas occur in the hereditary syndrome Carney complex. Although PRKAR1A mutations can cause the Carney complex, the disorder is genetically heterogeneous. To identify the cause of a Carney complex variant associated with distal arthrogryposis (the trismus-pseudocamptodactyly syndrome), we performed clinical and genetic studies. METHODS: A large family with familial cardiac myxomas and the trismus-pseudocamptodactyly syndrome (Family 1) was identified and clinically evaluated along with two families with trismus and pseudocamptodactyly. Genetic linkage analyses were performed with the use of microsatellite polymorphisms to determine a locus for this Carney complex variant. Positional cloning and mutational analyses of candidate genes were performed to identify the genetic cause of disease in the family with the Carney complex as well as in the families with the trismus-pseudocamptodactyly syndrome. RESULTS: Clinical evaluations demonstrated that the Carney complex cosegregated with the trismus-pseudocamptodactyly syndrome in Family 1, and genetic analyses demonstrated linkage of the disease to chromosome 17p12-p13.1 (maximum multipoint lod score, 4.39). Sequence analysis revealed a missense mutation (Arg674Gln) in the perinatal myosin heavy-chain gene (MYH8). The same mutation was also found in the two families with the trismus-pseudocamptodactyly syndrome. Arg674 is highly conserved evolutionarily, localizes to the actin-binding domain of the perinatal myosin head, and is close to the ATP-binding site. We identified nonsynonymous MYH8 polymorphisms in patients with cardiac myxoma syndromes but without arthrogryposis. CONCLUSIONS: We describe a novel heart-hand syndrome involving familial cardiac myxomas and distal arthrogryposis and demonstrate that these disorders are caused by a founder mutation in the MYH8 gene. Our findings demonstrate novel roles for perinatal myosin in both the development of skeletal muscle and cardiac tumorigenesis.

Identification of a locus for maturity-onset diabetes of the young on chromosome 8p23.

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal dominant inheritance and a young onset. Six MODY genes have been discovered to date. To identify additional MODY loci, we conducted a genome scan in 21 extended U.S. families (15 white and 6 from minorities, for a total of 237 individuals) in which MODY was not caused by known MODY genes. Seven chromosomal regions (1q42, 2q24, 2q37, 4p13, 8p23, 11p15, and 19q12) had a parametric heterogeneity logarithm of odds (HLOD) > or =1.00 or a nonparametric logarithm of odds (LOD) > or =0.59 (P < or = 0.05) in the initial screen. After typing additional markers at these loci to reduce the spacing to 2-3 cM, significant linkage was detected on 8p23 (HLOD = 3.37 at D8S1130 and nonparametric LOD = 3.66; P = 2 x 10(-5) at D8S265), where a 4.7-Mb inversion polymorphism is located. Thirty percent of the families (6 of 21) were linked with this region. Another linkage peak on chromosome 2q37 with an HLOD of 1.96 at D2S345/D2S2968 accounted for diabetes in an additional 25% of families (5 of 21). All 6 minority families were among the 11 families linked to these loci. None of the other loci followed up had an HLOD exceeding 1.50. In summary, we have identified a MODY locus on 8p23 that accounts for diabetes in a substantial proportion of MODY cases unlinked to known MODY genes. Another novel MODY locus may be present on 2q37. Cloning these new MODY genes may offer insights to disease pathways that are relevant to the cause of common type 2 diabetes.