Molecular and immunophenotypic characterization of anal squamous cell carcinoma reveals distinct clinicopathologic groups associated with HPV and TP53 mutation status.

Squamous cell carcinoma (SqCC) is the most common malignancy of the anal canal, where it is strongly associated with HPV infection. Characteristic genomic alterations have been identified in anal SqCC, but their clinical significance and correlation with HPV status, pathologic features, and immunohistochemical markers are not well established. We examined the molecular and clinicopathologic features of 96 HPV-positive and 20 HPV-negative anal SqCC. HPV types included 89 with HPV16, 2 combined HPV16/HPV18, and 5 HPV33. HPV-positive cases demonstrated frequent mutations or amplifications in PIK3CA (30%; p = 0.027) or FBXW7 mutations (10%). HPV-negativity was associated with frequent TP53 (53%; p = 0.00001) and CDKN2A (21%; p = 0.0045) mutations. P16 immunohistochemistry was positive in all HPV-positive cases and 3/20 HPV-negative cases (p < 0.0001; sensitivity: 100%; specificity: 85%) and was associated with basaloid morphology (p = 0.0031). Aberrant p53 immunohistochemical staining was 100% sensitive and specific for TP53 mutation (p < 0.0001). By the Kaplan-Meier method, HPV-negativity, aberrant p53 staining, and TP53 mutation were associated with inferior overall survival (OS) (p < 0.0001, p = 0.0103, p = 0.0103, respectively) and inferior recurrence-free survival (p = 0.133, p = 0.0064, and p = 0.0064, respectively). TP53/p53 status stratified survival probability by HPV status (p = 0.013), with HPV-negative/aberrant p53 staining associated with the worst OS, HPV-positive/wild-type p53 with best OS, and HPV-positive/aberrant p53 or HPV-negative/wild-type p53 with intermediate OS. On multivariate analysis HPV status (p = 0.0063), patient age (p = 0.0054), T stage (p = 0.039), and lymph node involvement (p = 0.044) were independently associated with OS. PD-L1 expression (CPS ≥ 1) was seen in 30% of HPV-positive and 40% of HPV-negative cases, and PD-L1 positivity was associated with a trend toward inferior OS within the HPV-negative group (p = 0.064). Our findings suggest that anal SqCC can be subclassified into clinically, pathologically, and molecularly distinct groups based on HPV and TP53 mutation status, and p16 and p53 immunohistochemistry represent a clinically useful method of predicting these prognostic groups.

Next-Generation Sequencing to Detect Deletion of RB1 and ERBB4 Genes in Chromophobe Renal Cell Carcinoma: A Potential Role in Distinguishing Chromophobe Renal Cell Carcinoma from Renal Oncocytoma.

Overlapping morphologic, immunohistochemical, and ultrastructural features make it difficult to diagnose chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Because ChRCC is a malignant tumor, whereas RO is a tumor with benign behavior, it is important to distinguish these two entities. We aimed to identify genetic markers that distinguish ChRCC from RO by using next-generation sequencing (NGS). NGS for hotspot mutations or gene copy number changes was performed on 12 renal neoplasms, including seven ChRCC and five RO cases. Matched normal tissues from the same patients were used to exclude germline variants. Rare hotspot mutations were found in cancer-critical genes (TP53 and PIK3CA) in ChRCC but not RO. The NGS gene copy number analysis revealed multiple abnormalities. The two most common deletions were tumor-suppressor genes RB1 and ERBB4 in ChRCC but not RO. Fluorescence in situ hybridization was performed on 65 cases (ChRCC, n = 33; RO, n = 32) to verify hemizygous deletion of RB1 (17/33, 52%) or ERBB4 (11/33, 33%) in ChRCC, but not in RO (0/32, 0%). In total, ChRCCs (23/33, 70%) carry either a hemizygous deletion of RB1 or ERBB4. The combined use of RB1 and ERBB4 fluorescence in situ hybridization to detect deletion of these genes may offer a highly sensitive and specific assay to distinguish ChRCC from RO.

Immunohistochemical loss of 5-hydroxymethylcytosine expression in acute myeloid leukaemia: relationship to somatic gene mutations affecting epigenetic pathways.

AIMS: Genes affecting epigenetic pathways are frequently mutated in myeloid malignancies, including acute myeloid leukaemia (AML). The genes encoding TET2, IDH1 and IDH2 are among the most commonly mutated genes, and cause defective conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), impairing demethylation of DNA, and presumably serving as driver mutations in leukaemogenesis. The aim of this study was to correlate 5hmC immunohistochemical loss with the mutation status of genes involved in epigenetic pathways in AML. METHODS AND RESULTS: Immunohistochemical staining with an anti-5hmC antibody was performed on 41 decalcified, formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with AML. Archived DNA was subjected to next-generation sequencing for analysis of a panel of genes, including TET2, IDH1, IDH2, WT1 and DNMT3A. TET2, IDH1, IDH2, WT1 and DNMT3A mutations were found in 46% (19/41) of the cases. Ten of 15 cases (67%) with TET2, IDH1, IDH2 or WT1 mutations showed deficient 5hmC staining, whereas nine of 26 cases (35%) without a mutation in these genes showed loss of 5hmC. It is of note that all four cases with TET2 mutations showed deficient 5hmC staining. CONCLUSIONS: Overall, somatic mutations in TET2, IDH1, IDH2, WT1 and DNMT3A were common in our cohort of AML cases. Immunohistochemical staining for 5hmC was lost in the majority of cases harbouring mutations in these genes, reflecting the proposed relationship between dysfunctional epigenetic pathways and leukaemogenesis.

Mutually exclusive extracellular signal-regulated kinase pathway mutations are present in different stages of multi-focal pulmonary Langerhans cell histiocytosis supporting clonal nature of the disease.

AIMS: Pulmonary Langerhans cell histiocytosis (PLCH) is an idiopathic cigarette smoking-related disorder of the lung. Molecular changes in cellular or fibrotic stages of PLCH have not been investigated. We studied the prevalence of extracellular signal-regulated kinase (ERK) pathway mutations in different PLCH stages and other non-PLCH smoking-related lung diseases. METHODS AND RESULTS: The cohort included 28 PLCH with cellular (n = 10), mixed cellular/fibrotic (n = 4) and fibrotic histology (n = 14). Seven cases had concurrent multi-focal/multi-lobar tumours. Respiratory bronchiolitis interstitial lung disease (RB-ILD, n = 2), desquamative interstitial pneumonia (DIP, n = 4) and mixed RB-ILD/DIP (n = 2) were included for comparison. BRAF(V) (600E) immunohistochemistry, next-generation sequencing (NGS) and peptide nucleic acid (PNA) clamp polymerase chain reaction (PCR) with high analytical sensitivity (<0.1-0.2%) were used to analyse RAS, BRAF and MAP2K1 genes. Of 26 cases with gene mutation data, BRAF(V) (600E) was identified in eight of 12 (67%) cellular cases and in one of 14 (7%) fibrotic cases. MAP2K1 or KRAS mutations were observed in four of 14 (29%) fibrotic cases and three of the 12 (25%) cellular cases. Multi-focal/multi-lobar specimens carried identical BRAF (n = 5) or non-hotspot MAP2K1 (n = 2) mutations. The other smoking-related disorders were negative for mutations. Patients with cellular lesions or BRAF mutation were significantly younger than patients with fibrotic or BRAF wild-type PLCH. CONCLUSION: The presence of identical but mutually exclusive ERK pathway mutations in multi-focal PLCH supports a neoplastic/clonal origin for this disease. Patient age and mutation type differed between cellular and fibrotic histology and may indicate a natural progression or a mutation-specific pathogenicity.

Complete Spontaneous Regression of Merkel Cell Carcinoma After Biopsy: A Case Report and Review of the Literature.

Merkel cell carcinoma (MCC) is a rare primary cutaneous neuroendocrine tumor that typically occurs on the head and neck of the elderly and follows an aggressive clinical course. Merkel cell polyomavirus (MCPyV) has been identified in up to 80% of cases and has been shown to participate in MCC tumorigenesis. Complete spontaneous regression of MCC has been rarely reported in the literature. We describe a case of a 79-year-old man that presented with a rapidly growing, 3-cm mass on the left jaw. An incisional biopsy revealed MCC. Additional health issues were discovered in the preoperative workup of this patient which delayed treatment. One month after the biopsy, the lesion showed clinical regression in the absence of treatment. Wide excision of the biopsy site with sentinel lymph node dissection revealed no evidence of MCC 2 months later. The tumor cells in the patient's biopsy specimen were negative for MCPyV by polymerase chain reaction and immunohistochemistry (CM2B4 antibody, Santa Cruz, CA). The exact mechanism for complete spontaneous regression in MCC is unknown. To our knowledge, only 2 previous studies evaluated the presence of MCPyV by polymerase chain reaction in MCC with spontaneous regression. Whether the presence or absence of MCPyV correlates with spontaneous regression warrants further investigation.

Molecular Profiling of Syringocystadenocarcinoma Papilliferum Reveals RAS-Activating Mutations.

CONTEXT.­: Syringocystadenocarcinoma papilliferum (SCACP) is a rare adnexal carcinoma and the malignant counterpart of syringocystadenoma papilliferum (SCAP), which is commonly located on the head and neck and may arise in association with a nevus sebaceus. RAS mutations have been identified in both SCAP and nevus sebaceus. OBJECTIVE.­: To evaluate the clinicopathologic and molecular features of SCACPs, which have not been previously explored. DESIGN.­: We obtained 11 SCACPs from 6 institutions and reviewed the clinicopathologic features. We also performed molecular profiling using next-generation sequencing. RESULTS.­: The cohort comprised 6 women and 5 men with ages ranging from 29 to 96 years (mean, 73.6 years). The neoplasms occurred on the head and neck (n = 8; 73%) and extremities (n = 3; 27%). Three tumors possibly arose in a nevus sebaceus. A total of 4 cases showed at least carcinoma in situ (adenocarcinoma, n = 3; squamous cell carcinoma [SCC], n = 1), and 7 cases were invasive (SCC, n = 5; mixed adenocarcinoma + SCC, n = 2). A total of 8 of 11 cases (73%) had hot spot mutations consisting of HRAS (n = 4), KRAS (n = 1), BRAF (n = 1), TP53 (n = 4), ATM (n = 2), FLT3 (n = 1), CDKN2A (n = 1), and PTEN (n = 1). All 4 cases with HRAS mutations occurred on the head and neck, whereas the KRAS mutation occurred on the extremity. CONCLUSIONS.­: RAS-activating mutations were detected in 50% of the cases, of which most (80%) involved HRAS and occurred on the head and neck, which shows overlapping features with SCAP, supporting that a subset may arise as a result of malignant transformation and likely an early oncogenic event.

Frameshift Mutations in Leukemia-Associated Genes Correlate With Superior Outcomes in Patients Undergoing Allogeneic Stem Cell Transplant for De Novo Acute Myeloid Leukemia.

Background: Allogeneic stem cell transplant (allo-SCT) is a mainstay of treatment for acute myeloid leukemia (AML). Its success depends largely on response of donor T lymphocytes against leukemia cells, known as graft-vs-leukemia (GvL) effect. A key potential driver of GvL is immune response to mutation-derived neoantigens. Previous studies in solid tumors have demonstrated enhanced immunogenicity of frameshift (FS)-derived peptides vs. those from non-synonymous single nucleotide variants (SNVs). We therefore hypothesized that AML cases bearing FS mutations in leukemia-associated genes would be more immunogenic than those with only other types of mutations (non-FS), and thus benefit more from allo-SCT via more robust GvL. Methods: We identified AML patients who had undergone allo-SCT between 2010 and 2022 and had next-generation sequencing data available on diagnostic specimens using a 42-gene hot spot panel. We compared the impact of tumor mutations present at diagnosis on overall survival and relapse-free survival based on FS versus non-FS status. Results: Ninety-five AML allo-SCT patients were identified. We observed superior relapse-free survival (P = 0.038, hazard ratio (HR): 0.24) and borderline superior overall survival (P = 0.058, HR: 0.55) post-transplant in de novo AML patients, who had at least one FS mutation (other than NPM1) in one of the 42 assessed genes versus those with only non-FS mutations. Conclusions: Our findings suggest that FS-mutated AML cases may benefit more from allo-SCT than those with only non-FS mutations, possibly due to increased generation of immunogenic neoepitopes. If validated in an expanded study, incorporation of somatic FS mutation status in AML could improve patient selection algorithms for bone marrow transplant and thereby lead to superior outcomes.

Is it a primary or metastatic melanocytic neoplasm of the central nervous system?: A molecular based approach.

Primary melanocytic neoplasms of the central nervous system (CNS) are uncommon and must be distinguished from metastatic lesions as patients with metastatic disease carry a worse prognosis. Therefore, tools to aid in the diagnosis of a primary CNS melanocytic neoplasm would be of clinical utility. Primary CNS melanocytic neoplasms, including uveal melanomas have frequent mutations in GNAQ and GNA11, but are rare in cutaneous and mucosal melanomas. Additionally, primary uveal melanomas often exhibit monosomy 3 conferring an elevated risk of metastasis. We present a 63 year-old male with a melanocytic neoplasm in the thoracic spinal cord. Molecular studies revealed the tumor contained a GNAQ mutation and four-color fluorescent in situ hybridization (FISH) composed of chromosome enumeration probes for 3, 7, 17 and a locus specific probe for 9p21/CDKN2A yielded a normal result (i.e. two copies per cell), favoring a primary versus metastatic melanocytic neoplasm of the CNS. We report a case in which the combination of mutational analysis and FISH aided in identifying the origin of the neoplasm.

Mass-Forming Gastric Heterotopia of the Rectum: A Series of 3 Cases from a Single Tertiary Health Center.

BACKGROUND Gastric heterotopia is a benign entity found throughout the gastrointestinal tract but is rarely identified in the rectum. Since 1939, only 94 cases have ever been identified, and it can present as a mass formation with symptomatology that mimics colorectal malignancy. In some instances, malignancy has been shown to arise within rectal gastric heterotopia. Here, we present 3 cases from the past 20-year period of rectal gastric heterotopia at a single tertiary institution. CASE REPORT A 25-year-old man (case 1), a 58-year-old woman (case 2), and a 33-year-old man (case 3) were found to have polypoid mass-like lesions greater than 1.0 cm within the rectum. Following biopsy, pathology showed gastric oxyntic mucosa flanked by colorectal mucosa, thus indicating gastric heterotopia. Presenting symptoms from all patients consisted of unspecified anal pain, hematochezia, or a combination of both. All patients were treated with endoscopic mucosal resection (EMR), which provided relief of symptoms and confirmed no evidence of invasive malignancy. CONCLUSIONS Rectal gastric heterotopia can mimic malignancy and in very rare instances can harbor high-grade dysplasia as well as invasive carcinoma. EMR seems to be a definitive treatment that offers relief to patient symptomatology and reassurance that any dysplasia is identified and removed.

Diagnostic value of a comprehensive, urothelial carcinoma-specific next-generation sequencing panel in urine cytology and bladder tumor specimens.

BACKGROUND: Urine cytology can reliably diagnose high-grade urothelial carcinoma (HGUC) but not low-grade urothelial carcinoma (LGUC), and a more sensitive test is needed. Previously, a pilot study highlighted the possible diagnostic utility of next-generation sequencing (NGS) in identifying both LGUC and HGUC in urine cytology specimens. METHODS: Twenty-eight urine ThinPrep cytology specimens and preceding or subsequent bladder tumor biopsy/resection specimens obtained within 3 months were included in the study (LGUC, n = 15; HGUC, n = 13). A customized, bladder-specific NGS panel was performed; it covered 69 frequently mutated or altered genes in urothelial carcinoma (UC) that were reported by The Cancer Genome Atlas and the Catalogue of Somatic Mutations in Cancer. RESULTS: The sequencing results were compared between the urine cytology specimens and the corresponding bladder tumor biopsies/resections. TP53 was the most frequently identified mutation in HGUC cases (11 of 13 [85%]). PIK3CA and KDM6A were the most frequently identified mutations in LGUC: they occurred in 7 of 15 cases (47%) and in 6 of 15 cases (40%), respectively. Additional frequent mutations identified in the panel included ARID1A (n = 5), EP300 (n = 4), LRP1B (n = 3), ERBB2 (n = 2), STAG2 (n = 2), FGFR3 (n = 3), MLL (n = 2), MLL3 (n = 2), CREBBP1 (n = 1), RB1 (n = 1), and FAT4 (n = 1). Overall, the concordance between the cytology and surgical specimens was 75%. The sensitivity and specificity for identifying mutations in urine cytology specimens were 84% and 100%, respectively. CONCLUSIONS: A bladder-specific NGS panel increases the sensitivity and specificity of urine cytology's diagnostic utility in both low- and high-grade tumors and may serve as a noninvasive surveillance method in the follow-up of patients with UC harboring known mutations.

Mutational Profile Using Next-Generation Sequencing May Aid in the Diagnosis and Treatment of Urachal Adenocarcinoma.

Objectives. The rare urachal adenocarcinoma (UAC) of the bladder has striking morphologic and immunohistochemical overlap with colorectal adenocarcinoma (CAC) and bladder adenocarcinoma (BAC). To date, the mutational status in UAC and BAC has not been well investigated. Methods. We retrospectively evaluated 34 UACs (mucinous, n = 9; intestinal, n = 3; signet ring cell, n = 1; not otherwise specified, n = 21) and 4 BACs (n = 4). Next-generation sequencing analysis of 50 cancer "hotspot" gene mutations using the Ampliseq Cancer Hotspot Panel v2 was performed. Two UAC cases did not have adequate DNA quality with poor sequencing coverage and were excluded from the study. Results. RAS mutations were identified in 16 of 32 (50%) UACs (15 KRAS; 1 NRAS) and none of the BACs (0%). TP53 mutations were found in both UACs (18/32; 56%) and BACs (4/4; 100%). GNAS (n = 4), SMAD4 (n = 3), and BRAF (n = 1) mutations were only found in UACs. In contrast, APC (n = 2) mutations were only found in BACs. The mucinous subtype of UAC contained a SMAD4 mutation in 33% of cases (3/9), which was not identified in any other subtype (0/23; 0%) (P = .0169). The only BRAF mutation was identified in the single signet ring cell subtype of UAC. There were no other differences in the mutation profile when comparing histologic subtypes of UAC. Conclusions. In summary, UAC and BAC have overlapping but distinct mutation profiles and these differences may aid in separating these 2 entities. Next-generation sequencing to identify therapeutic targets or resistance markers may aid treatment decisions.

Heterogeneous mutational profile and prognosis conferred by TP53 mutations in appendiceal mucinous neoplasms.

The eighth edition of American Joint Committee on Cancer (AJCC) advocates a 3-tier grading system for appendiceal mucinous tumors. The mutational profile for each tumor grade and the impact of TP53 mutation on survival are unknown. We classified appendiceal mucinous tumors into 3 grades based on the eighth edition of American Joint Committee on Cancer: 21 G1 low-grade mucinous neoplasms, 21 G2 appendiceal adenocarcinomas, and 26 G3 signet ring cell carcinomas. Mutation profiles were obtained using next-generation sequencing. The impact of TP53 on prognosis was investigated by multivariable analysis. Most G1 tumors harbor KRAS/GNAS mutations with TP53 and SMAD4 in a small subset of cases. G2 and G3 tumors show a more complex mutation pattern carrying PIK3CA, BRAF, or TP53 mutations in addition to KRAS/GNAS. PTEN mutations were detected exclusively in G2 tumors. The prevalence of KRAS and GNAS mutations is significantly lower in G3 tumors relative to G1/G2, whereas TP53, PIK3CA, or BRAF mutations are common. Mutations in NRAS, IDH2, CDH1, RB1, CTNNB1, CDKN2A, PTPN11, and KIT genes were observed in single cases. Patients with TP53-mutated disseminated G2 and G3 tumors had worse progression-free survival than did those with wild-type TP53 tumors (P = .0315). A trend toward worse overall survival was observed in TP53-mutated G3 tumors (P = .102). p53 expression correlated with mutation status. We demonstrate a distinct but overlapping pattern of gene mutations in each grade of appendiceal mucinous tumors and the independent impact of TP53 mutation on progression-free survival but not overall survival.

Somatic molecular analysis augments cytologic evaluation of pancreatic cyst fluids as a diagnostic tool.

Objective: Better tools are needed for early diagnosis and classification of pancreatic cystic lesions (PCL) to trigger intervention before neoplastic precursor lesions progress to adenocarcinoma. We evaluated the capacity of molecular analysis to improve the accuracy of cytologic diagnosis for PCL with an emphasis on non-diagnostic/negative specimens. Design: In a span of 7 years, at a tertiary care hospital, 318 PCL endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) were evaluated by cytologic examination and molecular analysis. Mucinous PCL were identified based on a clinical algorithm and 46 surgical resections were used to verify this approach. The mutation allele frequency (MAF) of commonly altered genes (BRAF, CDKN2A, CTNNB1, GNAS, RAS, PIK3CA, PTEN, SMAD4, TP53 and VHL) was evaluated for their ability to identify and grade mucinous PCL. Results: Cytology showed a diagnostic sensitivity of 43.5% for mucinous PCL due in part to the impact of non-diagnostic (28.8%) and negative (50.5%) specimens. Incorporating an algorithmic approach or molecular analysis markedly increased the accuracy of cytologic evaluation. Detection of mucinous PCL by molecular analysis was 93.3% based on the detection of KRAS and/or GNAS gene mutations (p = 0.0001). Additional genes provided a marginal improvement in sensitivity but were associated with cyst type (e.g. VHL) and grade (e.g. SMAD4). In the surgical cohort, molecular analysis and the proposed algorithm showed comparable sensitivity (88.9% vs. 100%). Conclusions: Incorporating somatic molecular analysis in the cytologic evaluation of EUS-FNA increases diagnostic accuracy for detection, classification and grading of PCL. This approach has the potential to improve patient management.

IRS2 mutations linked to invasion in pleomorphic invasive lobular carcinoma.

Pleomorphic invasive lobular carcinoma (PILC) is an aggressive variant of invasive lobular breast cancer that is associated with poor clinical outcomes. Limited molecular data are available to explain the mechanistic basis for PILC behavior. To address this issue, targeted sequencing was performed to identify molecular alterations that define PILC. This sequencing analysis identified genes that distinguish PILC from classic ILC and invasive ductal carcinoma by the incidence of their genomic changes. In particular, insulin receptor substrate 2 (IRS2) is recurrently mutated in PILC, and pathway analysis reveals a role for the insulin receptor (IR)/insulin-like growth factor-1 receptor (IGF1R)/IRS2 signaling pathway in PILC. IRS2 mutations identified in PILC enhance invasion, revealing a role for this signaling adaptor in the aggressive nature of PILC.

Somatic mutations in histiocytic sarcoma identified by next generation sequencing.

Histiocytic sarcoma is a rare malignant neoplasm of presumed hematopoietic origin showing morphologic and immunophenotypic evidence of histiocytic differentiation. Somatic mutation importance in the pathogenesis or disease progression of histiocytic sarcoma was largely unknown. To identify somatic mutations in histiocytic sarcoma, we studied 5 histiocytic sarcomas [3 female and 2 male patients; mean age 54.8 (20-72), anatomic sites include lymph node, uterus, and pleura] and matched normal tissues from each patient as germ line controls. Somatic mutations in 50 "Hotspot" oncogenes and tumor suppressor genes were examined using next generation sequencing. Three (out of five) histiocytic sarcoma cases carried somatic mutations in BRAF. Among them, G464V [variant frequency (VF) of 43.6 %] and G466R (VF of 29.6 %) located at the P loop potentially interfere with the hydrophobic interaction between P and activating loops and ultimately activation of BRAF. Also detected was BRAF somatic mutation N581S (VF of 7.4 %), which was located at the catalytic loop of BRAF kinase domain: its role in modifying kinase activity was unclear. A similar mutational analysis was also performed on nine acute monocytic/monoblastic leukemia cases, which did not identify any BRAF somatic mutations. Our study detected several BRAF mutations in histiocytic sarcomas, which may be important in understanding the tumorigenesis of this rare neoplasm and providing mechanisms for potential therapeutical opportunities.

Detection of homozygous and heterozygous SMN deletions of spinal muscular atrophy in a single assay with multiplex ligation-dependent probe amplification.

OBJECTIVE: Spinal muscular atrophy(SMA), an autosomal recessive neuromuscular degeneration of the anterior horn cells of the spinal cord and brain stem, results in one of the most common diseases with muscle fatigue and atrophy. Most SMA cases including all the types are due to the homozygous deletion of at least exon 7 within the survival motor neuron 1 (SMN-1) gene. Although a "golden standard" assay (PCR with mismatch primer followed by enzyme digestion) is very reliable for the identification of homozygous SMN-1 deletion, the carrier detection of heterozygous SMN-1 deletion remains a challenge. METHODS: Some PCR-based gene dosage assays or multiplex PCR allow for the determination of the copy number of SMN-1 gene to identify heterozygous deletion, but these procedures are often time consuming and available on a limited clinical basis. Recently developed MLPA (multiplex ligation-dependent probe amplification) is an efficient procedure that can accurately analyze relative quantification to establish the copy number of the SMN gene. We performed a validation for simultaneous detection of homozygous SMN-1 deletions of SMA patients and heterozygous SMN-1 deletions of SMA carriers in a simple assay using a MLPA-SMA assay specific reagent. RESULTS: Six out of 20 patients with SMA were found to have homozygous SMN-1 deletion, confirmed by the PCR/digestion assay. All 4 parents of the children with SMA had heterozygous SMN-1 deletion, confirmed by an independent relative quantitative analysis. CONCLUSION: MLPA provides a simple, rapid and accurate method of simultaneously detecting homozygous deletions and heterozygous deletions in a single assay for both SMN-1 and SMN-2 genes.

The utility of MYC and FLT4 in the diagnosis and treatment of postradiation atypical vascular lesion and angiosarcoma of the breast.

Atypical vascular lesions (AVLs) and angiosarcomas (ASs) are well-recognized complications of radiotherapy for breast cancer. Early diagnosis may be challenging, particularly on small biopsies, and the treatment options are limited. Recently, MYC and sometimes FLT4 gene amplification has been reported in AS, but not in AVL, and FLT4 may be a target for therapy. We evaluated the MYC/FLT4 status in AVL and AS by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), determined its utility in differentiating these 2 entities in small biopsies, and ascertained the value of FLT4 as a potential marker for targeted therapy. Thirty-five vascular neoplasms were reviewed from 21 breast cancer patients who received radiotherapy (AVL, n = 18; AS, n = 17). MYC expression by IHC and/or gene amplification by FISH were identified in 13 (77%) of 17 ASs, but none of the AVL cases. Four patients had biopsies with follow-up excisions, of which 1 showed the morphology of an AVL on biopsy and AS on excision, both positive for MYC. Of 17 ASs, 3 (18%) showed strong and diffuse 3+ cytoplasmic FLT4 staining by IHC and FLT4 gene amplification by FISH. All 3 cases were coamplified for the MYC gene. Focal and weak FLT4 cytoplasmic immunoreactivity was present in 2 (12%) of 17 AVL cases and 12 (70%) of 17 AS cases. Although MYC is a valuable ancillary tool in distinguishing AS from AVL, FLT4 IHC may be used to screen for patients with FLT4 gene amplification who might benefit from targeted therapy, as only diffuse strong FLT4 immunoreactivity correlated with FLT4 gene amplification.

BRAF/KRAS gene sequencing of sebaceous neoplasms after mismatch repair protein analysis.

Sebaceous neoplasms are cutaneous markers for the autosomal-dominant Muir-Torre syndrome (MTS). This phenotypic variant of Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. Microsatellite instability or loss of protein expression suggests a mutation or promoter hypermethylation in 1 of the MMR genes. BRAF gene sequencing may help to distinguish between patients with sporadic and LS-associated colorectal carcinomas with loss of MLH1 expression. LS-associated carcinomas are virtually negative for BRAF mutations, but a subset harbors KRAS mutations. The aim of our study was to test sebaceous neoplasms for V600E BRAF or KRAS mutations to determine if these mutations are associated with somatic or germline MMR defects, analogous to colorectal carcinomas. Over a 4-year period, 32 cases comprising 21 sebaceous adenomas, 3 sebaceomas, and 8 sebaceous carcinomas with sufficient material for testing were collected. MMR immunohistochemistry showed that 7 neoplasms had combined loss of MLH1-PMS2, 16 neoplasms had combined loss of MSH2-MSH6, 2 neoplasms had solitary loss of MSH6, and 7 sebaceous neoplasms had intact protein expression. BRAF/KRAS testing revealed all sebaceous neoplasms contained a wild-type BRAF gene. Two (15%) of 13 patients with MTS were found to harbor a KRAS mutation and loss of MLH1 expression. We conclude that a V600E BRAF mutation may not be helpful in distinguishing sporadic from MTS-associated sebaceous neoplasms. Further studies are needed to determine if KRAS mutations are restricted to patients with MTS or are also present in sporadic sebaceous neoplasms.

Development of a focused oligonucleotide-array comparative genomic hybridization chip for clinical diagnosis of genomic imbalance.

BACKGROUND: Submicroscopic genomic imbalance underlies well-defined microdeletion and microduplication syndromes and contributes to general developmental disorders such as mental retardation and autism. Array comparative genomic hybridization (CGH) complements routine cytogenetic methods such as karyotyping and fluorescence in situ hybridization (FISH) for the detection of genomic imbalance. Oligonucleotide arrays in particular offer advantages in ease of manufacturing, but standard arrays for single-nucleotide polymorphism genotyping or linkage analysis offer variable coverage in clinically relevant regions. We report the design and validation of a focused oligonucleotide-array CGH assay for clinical laboratory diagnosis of genomic imbalance. METHODS: We selected >10 000 60-mer oligonucleotide features from Agilent's eArray probe library to interrogate all subtelomeric and pericentromeric regions and 95 additional clinically relevant regions for a total of 179 loci. Sensitivity and specificity were measured for 105 patient samples, including 51 with known genomic-imbalance events, as detected by bacterial artificial chromosome-based array CGH, FISH, or multiplex ligation-dependent probe amplification. RESULTS: Focused array CGH detected all known regions of genomic imbalance in 51 validation samples with 100% concordance and an excellent signal-to-noise ratio. The mean SD among log(2) ratios of all noncontrol features without copy number alteration was 0.062 (median, 0.055). Clinical testing of another 211 samples from individuals with developmental delay, unexplained mental retardation, dysmorphic features, or multiple congenital anomalies revealed genomic imbalance in 25 samples (11.9%). CONCLUSIONS: This focused oligonucleotide-array CGH assay, a flexible, robust method for clinically diagnosing genetic disorders associated with genomic imbalance, offers appreciable advantages over currently available platforms.