Thomas W. Prior

Feasibility of Screening for Lynch Syndrome Among Patients With Colorectal Cancer

PURPOSE Identifying individuals with Lynch syndrome (LS) is highly beneficial. However, it is unclear whether microsatellite instability (MSI) or immunohistochemistry (IHC) should be used as the screening test and whether screening should target all patients with colorectal cancer (CRC) or those in high-risk subgroups. PATIENTS AND METHODS MSI testing and IHC for the four mismatch repair proteins was performed on 500 tumors from unselected patients with CRC. If either MSI or IHC was abnormal, complete mutation analysis for the mismatch repair genes was performed. RESULTS Among the 500 patients, 18 patients (3.6%) had LS. All 18 patients detected with LS (100%) had MSI-high tumors; 17 (94%) of 18 patients with LS were correctly predicted by IHC. Of the 18 probands, only eight patients (44%) were diagnosed at age younger than 50 years, and only 13 patients (72%) met the revised Bethesda guidelines. When these results were added to data on 1,066 previously studied patients, the entire study cohort (N = 1,566) showed an overall prevalence of 44 of 1,566 patients (2.8%; 95% CI, 2.1% to 3.8%) for LS. For each proband, on average, three additional family members carried MMR mutations. CONCLUSION One of every 35 patients with CRC has LS, and each has at least three relatives with LS; all of whom can benefit from increased cancer surveillance. For screening, IHC is almost equally sensitive as MSI, but IHC is more readily available and helps to direct gene testing. Limiting tumor analysis to patients who fulfill Bethesda criteria would fail to identify 28% (or one in four) cases of LS.

Identification of Proximal Spinal Muscular Atrophy Carriers and Patients by Analysis of SMNT and SMNC Gene Copy Number

The survival motor neuron (SMN) transcript is encoded by two genes, SMNT and SMNC. The autosomal recessive proximal spinal muscular atrophy that maps to 5q12 is caused by mutations in the SMNT gene. The SMNT gene can be distinguished from the SMNC gene by base-pair changes in exons 7 and 8. SMNT exon 7 is not detected in approximately 95% of SMA cases due to either deletion or sequence-conversion events. Small mutations in SMNT now have been identified in some of the remaining nondeletion patients. However, there is no reliable quantitative assay for SMNT, to distinguish SMA compound heterozygotes from non-5q SMA-like cases (phenocopies) and to accurately determine carrier status. We have developed a quantitative PCR assay for the determination of SMNT and SMNC gene-copy number. This report demonstrates how risk estimates for the diagnosis and detection of SMA carriers can be modified by the accurate determination of SMNT copy number.

Myoblast Transfer in the Treatment of Duchenne's Muscular Dystrophy

BACKGROUND Myoblast transfer has been proposed as a technique to replace dystrophin, the skeletal-muscle protein that is deficient in Duchennes muscular dystrophy. Donor myoblasts injected into muscles of affected patients can fuse with host muscle fibers, thus contributing their nuclei, which are potentially capable of replacing deficient gene products. Previous controlled trials involving a single transfer of myoblasts have been unsuccessful. METHODS We injected donor muscle cells once a month for six months to the biceps brachii muscles of one arm of each of 12 boys with Duchennes muscular dystrophy. The opposite arms served as sham-injected controls. In each procedure 110 million cells donated by fathers or brothers were transferred. The patients were randomly assigned to receive either cyclosporine or placebo. Strength was measured by quantitative isometric muscle testing. Six months after the final myoblast transfer, the presence of dystrophin was assessed with the use of peptide antibodies specific to the deleted exons of the dystrophin gene. RESULTS There was no significant difference in muscle strength between arms injected with myoblasts and sham-injected arms. In one patient, 10.3 percent of muscle fibers expressed donor-derived dystrophin after myoblast transfer. Three other patients also had a low level of donor dystrophin (< 1 percent); eight had none. CONCLUSIONS Myoblasts transferred once a month for six months failed to improve strength in patients with Duchennes muscular dystrophy. The value of exon-specific peptide antibodies in the interpretation of myoblast-transfer results was demonstrated in a patient with Duchennes muscular dystrophy who had a high percentage of donor-derived dystrophin. Specific variables affecting the efficiency of myoblast transfer need to be identified in order to improve upon this technique.

Screening for Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer) among Endometrial Cancer Patients

Endometrial cancer is the most common cancer in women with Lynch syndrome. The identification of individuals with Lynch syndrome is desirable because they can benefit from increased cancer surveillance. The purpose of this study was to determine the feasibility and desirability of molecular screening for Lynch syndrome in all endometrial cancer patients. Unselected endometrial cancer patients (N = 543) were studied. All tumors underwent microsatellite instability (MSI) testing. Patients with MSI-positive tumors underwent testing for germ line mutations in MLH1, MSH2, MSH6, and PMS2. Of 543 tumors studied, 118 (21.7%) were MSI positive (98 of 118 MSI high and 20 of 118 MSI low). All 118 patients with MSI-positive tumors had mutation testing, and nine of them had deleterious germ line mutations (one MLH1, three MSH2, and five MSH6). In addition, one case with an MSI-negative tumor had abnormal MSH6 immunohistochemical staining and was subsequently found to have a mutation in MSH6. Immunohistochemical staining was consistent with the mutation result in all seven truncating mutation-positive cases but was not consistent in two of the three missense mutation cases. We conclude that in central Ohio, at least 1.8% (95% confidence interval, 0.9-3.5%) of newly diagnosed endometrial cancer patients had Lynch syndrome. Seven of the 10 Lynch syndrome patients did not meet any published criteria for hereditary nonpolyposis colorectal cancer, and six of them were diagnosed at age >50. Studying all endometrial cancer patients for Lynch syndrome using a combination of MSI and immunohistochemistry for molecular prescreening followed by gene sequencing and deletion analysis is feasible and may be desirable.

Phase II Clinical Trial of Sorafenib in Metastatic Medullary Thyroid Cancer

PURPOSE Mutations in the RET proto-oncogene and vascular endothelial growth factor receptor (VEGFR) activity are critical in the pathogenesis of medullary thyroid cancer (MTC). Sorafenib, a multikinase inhibitor targeting Ret and VEGFR, showed antitumor activity in preclinical studies of MTC. PATIENTS AND METHODS In this phase II trial of sorafenib in patients with advanced MTC, the primary end point was objective response. Secondary end points included toxicity assessment and response correlation with tumor markers, functional imaging, and RET mutations. Using a two-stage design, 16 or 25 patients were to be enrolled onto arms A (hereditary) and B (sporadic). Patients received sorafenib 400 mg orally twice daily. RESULTS Of 16 patients treated in arm B, one achieved partial response (PR; 6.3%; 95% CI, 0.2% to 30.2%), 14 had stable disease (SD; 87.5%; 95% CI, 61.7% to 99.5%), and one was nonevaluable. In a post hoc analysis of 10 arm B patients with progressive disease (PD) before study, one patient had PR of 21+ months, four patients had SD >or= 15 months, four patients had SD <or= 6 months, and one patient had clinical PD. Median progression-free survival was 17.9 months. Arm A was prematurely terminated because of slow accrual. Common adverse events (AEs) included diarrhea, hand-foot-skin reaction, rash, and hypertension. Although serious AEs were rare, one death was seen. Tumor markers decreased in the majority of patients, and RET mutations were detected in 10 of 12 sporadic MTCs analyzed. CONCLUSION Sorafenib is reasonably well tolerated, with suggestion of clinical benefit for patients with sporadic MTC. Caution should be taken because of the rare but fatal toxicity potentially associated with sorafenib.

Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines.

Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.

Natural history of denervation in SMA: Relation to age, SMN2 copy number, and function

Denervation was assessed in 89 spinal muscular atrophy (SMA) 1, 2, and 3 subjects via motor unit number estimation (MUNE) and maximum compound motor action potential amplitude (CMAP) studies, and results correlated with SMN2 copy, age, and function. MUNE and maximum CMAP values were distinct among SMA subtypes (p < 0.05). Changes in MUNE and maximum CMAP values over time were dependent on age, SMA type, and SMN2 copy number. SMN2 copy number less than 3 correlated with lower MUNE and maximum CMAP values (p < 0.0001) and worse functional outcomes. As SMN2 copy number increases, so does functional status (p < 0.0001). Change in MUNE longitudinally over the time intervals examined in this study was not statistically significant for any SMA cohort. However, a decline in maximum CMAP over time was apparent in SMA2 subjects (p = 0.049). Age‐dependent decline in MUNE and maximum CMAP was apparent in both SMA 1 (p < 0.0001) and SMA 2 (p < 0.0001) subjects, with age as an independent factor regardless of type. Maximum CMAP at the time of the initial assessment was most predictive of functional outcome (p < 0.0001). Prospective longitudinal studies in four prenatally diagnosed infants demonstrated significant progressive denervation in association with symptomatic onset or functional decline. These data highlight the potential value of such measures in increasing our understanding of pathophysiological factors involved in denervation in SMA. Ann Neurol 2005;57:704–712

Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2

Purpose: This study describes SMN1 deletion frequency, carrier studies, and the effect of the modifying SMN2 gene on the spinal muscular atrophy (SMA) phenotype. A novel allele-specific intragenic mutation panel increases the sensitivity of SMN1 testing.Methods: From 1995 to 2001, 610 patients were tested for SMN1 deletions and 399 relatives of probands have been tested for carrier status. SMN2 copy number was compared between 52 type I and 90 type III patients, and between type I and type III patients with chimeric SMN genes. A fluorescent allele-specific polymerase chain reaction (PCR) -based strategy detected intragenic mutations in potential compound heterozygotes and was used on 366 patients.Results: Less than half of the patients tested were homozygously deleted for SMN1. A PCR-based panel detected the seven most common intragenic mutations. SMN2 copy number was significantly different between mild and severely affected patients.Conclusions: SMN1 molecular testing is essential for the diagnosis of SMA and allows for accurate carrier testing. Screening for intragenic mutations in SMN1 increases the sensitivity of diagnostic testing. Finally, SMN2 copy number is conclusively shown to ameliorate the phenotype and provide valuable prognostic information.

The prevalence of PTEN mutations in a clinical pediatric cohort with autism spectrum disorders, developmental delay, and macrocephaly

Purpose: To define the prevalence of PTEN mutations in a clinical cohort of pediatric subjects with autism spectrum disorders (ASDs), developmental delay/mental retardation (DD/MR), and/or macrocephaly and to assess genotype–phenotype correlations.Methods: Medical records of patients who had clinical PTEN gene sequencing ordered through our institution between January 1, 2005 and December 31, 2007 were abstracted to confirm genetic test results and medical diagnoses. Phenotypic information related to the diagnoses, prenatal history, early developmental milestones, physical characteristics, and family history for those with a confirmed PTEN mutation was also recorded.Results: One hundred fourteen patients were tested during this time period for indications of ASDs (N = 60), DD/MR (N = 49), or macrocephaly only (N = 5). Eleven mutations were identified: five in patients with ASDs and six in those with DD/MR, resulting in a prevalence of 8.3% and 12.2% in these respective clinical populations. All individuals with a PTEN mutation had significant macrocephaly (>2.0 SD)Conclusions: These data illustrate that PTEN gene sequencing has a high diagnostic yield when performed in a selected population of individuals with ASDs or DD/MR and macrocephaly. Germline mutations in PTEN are an important, identifiable etiology among these patients.

Confirmation study of PTEN mutations among individuals with autism or developmental delays/mental retardation and macrocephaly.

There is a strong genetic component to autism spectrum disorders (ASD), but due to significant genetic heterogeneity, individual genetic abnormalities contribute a small percentage to the overall total. Previous studies have demonstrated PTEN mutations in a sizable proportion of individuals with ASD or mental retardation/developmental delays (MR/DD) and macrocephaly that do not have features of Cowden or Bannayan–Riley–Ruvalcaba syndrome. This study was performed to confirm our previous results. We reviewed the charts of individuals who had PTEN clinical sequencing performed at our institution from January 2008 to July 2009. There were 93 subjects tested from our institution during that period. PTEN mutations were found in 2/39 (5.1%) ASD patients and 2/51 (3.9%) MR/DD patients. Three additional patients without mutations had no diagnostic information. Multiple relatives of individuals with a PTEN mutation had macrocephaly, MR, or early onset cancer (breast, renal, and prostate). Of those relatives tested, all had the familial PTEN mutation. None of the affected relatives had previously been diagnosed with Cowden or Bannayan–Riley–Ruvalcaba syndrome. We noted in our previous study several adult relatives without any findings who carried a mutation. Combined with data from our previous cohort, we have found PTEN mutations in 7/99 (7.1%) of individuals with ASD and 8/100 (8.0%) of individuals with MR/DD, all of whom had macrocephaly. We recommend testing for mutations in PTEN for individuals with ASD or MR/DD and macrocephaly. If mutations are found, other family members should be offered testing and the adults offered cancer screening if they have a PTEN mutation.