Diagnostic Molecular Pathology最新文献

More than 90% of congenital adrenal hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macroconversion or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbor rare mutations that do not originate from the pseudogene. A complete list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.imm.Ki.se/CYPalleles/cyp21.htm). In this report, we describe clinical and genetic findings regarding an Italian woman suffering from a classic salt-wasting form of CAH due to a severe 21-hydroxylase deficiency. A complex genetic family study was performed including a prenatal diagnosis. The patient was found to be heterozygous for p.I172N (exon 4), p.E238del (exon 6), p.M239K (exon 6), and p.F306insT (exon 7) mutations and homozygous for p.I236N (exon 6) and p.V237E (exon 6) mutations. The deletion of glutamic acid 238 is a new mutation not reported before in the literature. CYP21A2 genotyping has become a valuable complement to biochemical CAH investigation. We highlight the contribution of molecular genetic advancements to the clinical management of patients with 21-hydroxylase deficiency.

Introduction: Cutaneous squamous cell carcinoma (SCC) is one of the most commonly diagnosed nonmelanoma skin cancers. Occasionally, the diagnosis can be challenging as there are many simulating preneoplastic and reactive squamous lesions. One of the most difficult lesions to differentiate from SCC is pseudoepitheliomatous hyperplasia (PEH). The objective of our study is to differentiate cutaneous SCC from PEH using gene expression microarrays and examine the enriched molecular pathways and genes.

Design: DNA microarray studies were performed on formalin-fixed and paraffin-embedded blocks of the skin: 10 cases of SCCs and 10 cases of PEHs using the U133 plus 2.0 array.

Results: A total of 703 differentially expressed genes were identified between SCCs and PEHs (>2-fold change, P<0.05) including multiple upregulated S100 calcium-binding proteins and downregulated homeobox genes. Functional analysis of these genes suggests that oxidative phosphorylation, mitochondrial dysfunction and the polyamine regulation pathways are involved in the pathogenesis of SCC.

Conclusions: The distinctive gene expression profile of SCC and PEH offers the ability to use DNA microarrays to distinguish between them by an objective molecular measure. The molecular pathways and differentially expressed genes provide an insight into the pathogenesis of SCCs and may serve as future targets for therapeutic intervention.

Morphologic "special types" of breast carcinomas have been recognized for many years, and their molecular and genetic properties have not been specifically studied until recently. Lobular carcinoma lacks functional E-cadherin expression but shares molecular similarities with low-grade invasive ductal carcinomas. Papillary carcinoma is relatively rare, and molecular features are just being elucidated. We report a case of concurrent invasive lobular and papillary carcinoma, the latter with extensive nodal involvement. Multiplex screening for activating point mutations identified different point mutations in the distinct morphologic components: lobular PIK3CA H1047R, papillary; PIK3CA Q546P, and IDH1 R132H. These molecular data favor coincidental "collision tumors" over clonal evolution. The IDH1 R132H point mutation is common in gliomas and acute myelogenous leukemia, but this has not been previously reported in breast carcinoma. The characterization of activating point mutations in morphologic special types of breast carcinoma may suggest avenues amenable to targeted therapy.

The increasing knowledge about genetic alterations and molecular biomarkers in cancer initiation and progression opens new possibilities for the treatment of various types of cancer. This requires the inclusion of sensitive, and preferably multiplex, methods for the detection of molecular genetic alterations in the toolbox of classic pathology. Multiplex ligation-dependent probe amplification (MLPA) is a multiplex polymerase chain reaction-based method that can detect changes in the gene copy number status, DNA methylation, and point mutations simultaneously. MLPA probes recognize target sequences of only 50 to 100 nucleotides in length. This makes it possible to use MLPA even on highly fragmented DNA, and allows the detection of small deletions encompassing only a single exon. MLPA is a reliable, cost-effective, and robust method that can be performed using a standard thermocycler and capillary electrophoresis equipment, generating results within 24 hours with a short hands-on working time. Up to 50 different genomic locations can be tested in a single reaction, which can be sufficient to detect those genetic alterations that are of diagnostic and prognostic significance in a certain tumor entity. In the last years, MLPA has been used successfully in tumor diagnostics and in cancer research. This review gives an overview on the collected experience of MLPA applications on tumor DNA, about the advantages but also potential pitfalls and limitations of this technique.

The most common genetic aberration in follicular lymphoma (FL) is the t(14;18)(q32;q21) translocation that juxtaposes the antiapoptotic BCL2 gene with the promoter of the immunoglobulin heavy-chain (IgH) gene, which is the molecular hallmark of FL, whereas a subset of cases harbor translocations involving the BCL6 gene locus. To date, there has been no integrated analysis based on grade, phenotype, and genotype from large numbers of FL cases in a representative Chinese population. In this study, we graded 98 FL cases; fluorescence in situ hybridization was used to determine the BCL2 and BCL6 translocation statuses, and these were compared with morphologic and immunohistochemical parameters. The expressions of the 4 antigens were B-cell leukemia/lymphoma (BCL)-2(88.8%), BCL-6(80.6%), CD10(62.2%), and Ki67(50.0%), respectively. The frequencies of BCL2 and BCL6 translocations were 58.5% and 16.3%. In conclusion, the incidence of IgH/BCL2-positive FL in Chinese patients is relatively lower compared with that in western countries. BCL2 translocation strongly correlated with CD10 and Ki67 expression. Our data confirm the presence of a relationship between the translocation status and the FL histologic grade. All BCL6 translocations occurred in high-grade FL, and this suggests that FL carrying BCL6 translocation probably constitute a special biological subtype.

Wilms tumor gene 1 (WT1) expression has been suggested as an applicable minimal residual disease marker in acute myeloid leukemia (AML). We evaluated the use of this marker in 43 adult AML patients. Quantitative assessment of WT1 gene transcripts was performed using real-time quantitative-polymerase chain reaction assay. Samples from both the peripheral blood and the bone marrow were analyzed at diagnosis and during follow-up. A strong correlation was observed between WT1 normalized with 2 different control genes (β-actin and ABL1, P<0.001). WT1 mRNA level at diagnosis was of no prognostic relevance (P>0.05). A≥1-log reduction in WT1 expression in bone marrow samples taken <1 month after diagnosis significantly correlated with an improved overall survival (P=0.004) and freedom from relapse (P=0.010) when β-actin was used as control gene. Furthermore, a reduction in WT1 expression by ≥2 logs in peripheral blood samples taken at a later time point significantly correlated with a better outcome for overall survival (P=0.004) and freedom from relapse (P=0.012). This result was achieved when normalizing against both β-actin and ABL1. These results therefore suggest that WT1 gene expression can provide useful information for minimal residual disease detection in adult AML patients and that combined use of control genes can give more informative results.

Purpose: Non-small cell lung cancer (NSCLC) occurs most frequently in individuals older than 60 years of age. Currently, no biological indicators associated with NSCLC in younger patients (30 to 60 y) have been identified. To explore epigenetic influences, promoter methylation of selected tumor suppressor genes was analyzed in early-stage NSCLC patients ranging in age from 30 to 87 years at diagnosis.

Experimental design: The analysis was performed on formalin-fixed tumor tissue from 193 surgically treated NSCLC patients (127, older than 60 y; 66, 60 y and younger). Methylation was quantified in p16, MGMT, DAPK, RASSF1, CDH1, LET7-3-a, NORE1(RASSF5), and PTEN promoters by pyrosequencing. p16 protein expression was assessed by immunohistochemistry (IHC). Outcome, defined by time to recurrence and overall survival, was evaluated by Kaplan-Meier analysis.

Results: Promoter methylation levels were generally higher in patients older than 60 years of age than in patients 60 years or younger at diagnosis. Of the genes tested, methylation levels of the p16 promoter showed age-related differences. Although p16 promoter methylation was significantly lower using cut-points of 50 years or younger and 40 years or younger (P=0.001 to 0.012, respectively), p16 protein expression increased with age. Patients 60 years or younger with p16 promoter hypermethylation had a significantly shortened time to recurrence (P=0.002) and a shortened survival time (P=0.011). No effect of p16 hypermethylation was seen in patients older than 60 years.

Conclusions: p16 promoter hypermethylation was associated with a worse outcome in patients with age at diagnosis of 60 years or younger, but was not associated with the outcome in the older than 60-year age group. Overall, these data support methylation-dependent and methylation-independent age-related regulation of p16 expression with differential effects on the outcome after surgical resection for early-stage NSCLC.

Ataxia with oculomotor apraxia type 2 (AOA2) is a recently described autosomal recessive cerebellar ataxia caused by mutations in the SETX gene. It is a rare monogenic disease characterized by progressive cerebellar ataxia, oculomotor apraxia, axonal sensorimotor neuropathy, and an elevated serum α-fetoprotein level. To date, >100 AOA2 patients have been described and 75 different mutations in the SETX gene have been identified. We report here the clinical and genetic findings of 13 AOA2 patients from 5 unrelated Tunisian consanguineous families. DNA was collected from probands and available family members, and the 24 SETX exons were screened by direct sequencing. Four different homozygous SETX gene mutations were identified. The missense mutation 915G>T [W305C] has been described previously in Algeria. The 3 other SETX mutations are novel, including a missense mutation c.7231C>T [R 2380 W], a nonsense mutation c.6475 C>T [R2098X], and a deletion c.7180-7183delAAAA [D2332fsX2343]. More extensive screening by molecular genetic analysis of SETX in patients with Friedreich ataxia-like phenotype may show that AOA2 is more common in Tunisia than previously thought.

The molecular profiling of brain tumors, including testing for MGMT promoter methylation and chromosome 1p/19q deletion, can provide both diagnostic and prognostic information that may guide treatment. Isocitrate dehydrogenase (IDH) mutation testing is a recent addition to this armamentarium of molecular pathology tools that similarly provides both diagnostic (eg, glioma vs. gliosis) and prognostic information. Herein, we describe a pyrosequencing-based approach to IDH1 and IDH2 mutation testing and its application to 139 neoplastic and non-neoplastic central nervous system specimens. Several technical issues encountered in the development of the assay, particularly with regard to the optimization of the sequencing reaction, are described. Mutations in IDH1 codon 132 or IDH2 codon 172 were identified in 31.2% of all screened cases and 46.2% of screened World Health Organization grade I to IV gliomas (n=93), with mutations arising exclusively in grade II to IV oligodendroglial, astrocytic, or mixed oligoastrocytic neoplasms. Examination of the relationship between the mutation status and other pertinent variables demonstrated a significant male predominance among IDH1-mutated gliomas, most notably in grade III to IV astrocytic neoplasms. A significant association between IDH1/IDH2 mutation and 1p/19q deletion was also seen (Kendall τ coefficient=0.26, P=0.018), although several cases with 1p/19q deletion were IDH1/IDH2 wild type.