Anti-B-Cell Maturation Antigen BiTE Molecule AMG 420 Induces Responses in Multiple Myeloma.

PURPOSE: The anti-B-cell maturation antigen BiTE molecule AMG 420 was assessed in patients with relapsed/refractory multiple myeloma. PATIENTS AND METHODS: In this first-in-human study, up to 10 cycles of AMG 420 were given (4-week infusions/6-week cycles). Patients had progression after ≥ 2 lines of prior therapy and no extramedullary disease. Minimal residual disease (MRD) response was defined as < 1 tumor cell/104 bone marrow cells by flow cytometry. RESULTS: Forty-two patients received AMG 420 at 0.2-800 µg/d. Median age was 65 years, and median disease duration was 5.2 years. Median exposure was 1 cycle (range, 1-10 cycles) and 7 cycles (range, 1-10 cycles) for responders. Patients discontinued for disease progression (n = 25), adverse events (AEs; n = 7), death (n = 4), completion of 10 cycles (n = 3), and consent withdrawal (n = 1). Two patients remain on treatment. There were 2 nontreatment-related deaths from AEs, influenza/aspergillosis and adenovirus-related hepatitis. Serious AEs (n = 20; 48%) included infections (n = 14) and polyneuropathy (n = 2); treatment-related serious AEs included 2 grade 3 polyneuropathies and 1 grade 3 edema. There were no grade ≥ 3 CNS toxicities or anti-AMG 420 antibodies. In this study, 800 µg/d was considered to not be tolerable because of 1 instance each of grade 3 cytokine release syndrome and grade 3 polyneuropathy, both of which resolved. The overall response rate was 31% (n = 13 of 42). At the maximum tolerated dose (MTD) of 400 µg/d, the response rate was 70% (n = 7 of 10). Of these, five patients experienced MRD-negative complete responses, and 1 had a partial response, and 1 had a very good partial response; all 7 patients responded during the first cycle, and some responses lasted > 1 year. CONCLUSION: In this study of AMG 420 in patients with relapsed/refractory multiple myeloma, the response rate was 70%, including 50% MRD-negative complete responses, at 400 µg/d, the MTD for this study.

Phase I dose-escalation study of volasertib in pediatric patients with acute leukemia or advanced solid tumors.

BACKGROUND: Volasertib induces mitotic arrest and apoptosis by targeting Polo-like kinases. In this phase I dose-escalation study, the maximum tolerated dose (MTD), pharmacokinetics (PK), and preliminary efficacy of volasertib were determined in pediatric patients. METHODS: Patients aged 2 to <18 years with relapsed/refractory acute leukemia/advanced solid tumors (ST) without available effective treatments were enrolled-cohort C1 (aged 2 to <12 years); cohort C2 (aged 12 to <18 years). The patients received volasertib intravenously (starting dose: 200 mg/m2 body surface area on day 1, every 14 days). The primary endpoint was the pediatric MTD for further development. RESULTS: Twenty-two patients received treatment (C1: leukemia, n = 4; ST, n = 8; C2: leukemia, n = 3; ST, n = 7). No dose-limiting toxicities (DLTs) occurred up to 300 mg/m2 volasertib in C1; two patients in C2, at 250 mg/m2 volasertib, had DLTs in cycle 1, one of which led to death; therefore, the MTD of volasertib in C2 was 200 mg/m2 . The most common grade 3/4 adverse events (all patients) were febrile neutropenia, thrombocytopenia, and neutropenia (41% each). Stable disease (SD) was the best objective response (leukemia, n = 5; ST, n = 2); the duration of SD was short in all patients, except in one with an ST. PK profiles were generally comparable across dose groups and were consistent with those in adults. CONCLUSION: The pediatric MTD/dose for further development was identified. There were no unexpected safety or PK findings; limited antitumor/antileukemic activity was demonstrated.

A 3'UTR polymorphism modulates mRNA stability of the oncogene and drug target Polo-like Kinase 1.

BACKGROUND: The Polo-like Kinase 1 (PLK1) protein regulates cell cycle progression and is overexpressed in many malignant tissues. Overexpression is associated with poor prognosis in several cancer entities, whereby expression of PLK1 shows high inter-individual variability. Although PLK1 is extensively studied, not much is known about the genetic variability of the PLK1 gene. The function of PLK1 and the expression of the corresponding gene could be influenced by genomic variations. Hence, we investigated the gene for functional polymorphisms. Such polymorphisms could be useful to investigate whether PLK1 alters the risk for and the course of cancer and they could have an impact on the response to PLK1 inhibitors. METHODS: The coding region, the 5' and 3'UTRs and the regulatory regions of PLK1 were systematically sequenced. We determined the allele frequencies and genotype distributions of putatively functional SNPs in 120 Caucasians and analyzed the linkage and haplotype structure using Haploview. The functional analysis included electrophoretic mobility shift assay (EMSA) for detected variants of the silencer and promoter regions and reporter assays for a 3'UTR polymorphism. RESULTS: Four putatively functional polymorphisms were detected and further analyzed, one in the silencer region (rs57973275), one in the core promoter region (rs16972787), one in intron 3 (rs40076) and one polymorphism in the 3'untranslated region (3'UTR) of PLK1 (rs27770). Alleles of rs27770 display different secondary mRNA structures and showed a distinct allele-dependent difference in mRNA stability with a significantly higher reporter activity of the A allele (p < 0.01). CONCLUSION: The present study provides evidence that at least one genomic variant of PLK1 has functional properties and influences expression of PLK1. This suggests polymorphisms of the PLK1 gene as an interesting target for further studies that might affect cancer risk, tumor progression as well as the response to PLK1 inhibitors.

Effect of ADRB2 polymorphisms on the efficacy of salmeterol and tiotropium in preventing COPD exacerbations: a prespecified substudy of the POET-COPD trial.

BACKGROUND: The effect of ß2-adrenergic receptor (ADRB2) polymorphisms on the treatment response to longacting bronchodilators in chronic obstructive pulmonary disease (COPD) is unclear. We aimed to establish whether ADRB2 polymorphisms differentially affected COPD exacerbation outcomes in response to tiotropium versus salmeterol. METHODS: We did a prespecified analysis of the ADRB2 polymorphisms Arg16Gly and Gln27Glu within the 1 year randomised, double-blind, double-dummy, parallel-group Prevention Of Exacerbations with Tiotropium in COPD (POET-COPD) trial, comparing the effects of treatment with tiotropium or salmeterol on exacerbations in 7376 patients with COPD. One blood sample was collected for pharmacogenetic testing from each patient who elected to participate in the substudy. Random assignment of patients to treatment groups was not stratified according to genotypes. Genomic DNA was extracted from whole-blood specimens and samples were genotyped for the two SNPs, rs1042713 (Arg16Gly) and rs1042714 (Gln27Glu). All assays were done in technical duplicates and 10% of samples that were randomly chosen were repeated as technical duplicates in a second independent genotyping process. Our primary endpoint was the risk of a first exacerbation of COPD based on time to first exacerbation data. An exacerbation of COPD was defined as the increase or new onset of more than one symptom of COPD (cough, sputum, wheezing, dyspnoea, or chest tightness), with at least one of the symptoms lasting for 3 days or more and needing treatment with antibiotics or systemic glucocorticoids (moderate exacerbations), or admission to hospital (severe exacerbations). POET-COPD is registered with ClinicalTrials.gov, number NCT00563381. FINDINGS: 5125 patients gave informed consent for genotyping. The distributions of ADRB2 genotypes were well matched among groups. Polymorphisms at aminoacid 27 did not affect exacerbation outcomes. In the salmeterol group, patients with Arg16Arg genotype had a significantly reduced exacerbation risk compared with patients with Arg16Gly (p=0·0130) and Gly16Gly (p=0·0018) genotypes (proportion of patients with at least one exacerbation was 32·3% in Arg16Arg, 39·8% in Arg16Gly, and 42·1% in Gly16Gly). By contrast, exacerbation risk was not modified by polymorphisms at aminoacid 16 in the tiotropium group. The effect of the Arg16Gly polymorphism on treatment response to salmeterol was dependent on the use of inhaled corticosteroids (ICS). In patients untreated with ICS at baseline, Arg16Gly and Arg16Arg genotypes were associated with significantly prolonged time to first exacerbation compared with Gly16Gly (vs Arg16Gly p=0·0164; Arg16Arg p=0·0316; proportion of patients with at least one exacerbation was 28·3% in Arg16Arg, 31·6% in Arg16Gly, and 39·2% in Gly16Gly), whereas in patients on ICS at baseline, only the Arg16Arg genotype was associated with significantly prolonged time to first exacerbation compared with Gly16Gly (p=0·0198; not Arg16Gly p=0·64; proportion of patients with at least one exacerbation was 35·9% in Arg16Arg, 46·7% in Arg16Gly, and 44·8% in Gly16Gly). The respiratory disorders, in particular worsening of COPD, were the most common serious adverse events. INTERPRETATION: Patients with the Arg16Arg genotype had better exacerbation outcomes in response to salmeterol than Gly16Gly and Arg16Gly genotypes, suggesting a potential differential Arg16Gly genotype effect on treatment response to longacting ß-agonists (LABAs). However, the use of ADRB2 polymorphisms for predicting LABA treatment response is still limited and further prospective validation will be needed to advance the mechanistic understanding of ß-adrenergic polymorphisms and their association with clinical features of COPD. FUNDING: Boehringer Ingelheim and Pfizer.

The CYP1A1 Ile462Val polymorphism and platinum resistance of epithelial ovarian neoplasms.

The role of estrogens in ovarian carcinogenesis and progression of ovarian cancer is unclear. Cytochrome P450 is involved in estrogen metabolism, and polymorphisms have been associated with functional changes and risk for ovarian cancer. In this study, we investigated the impact of the CYP1A1 Ile462Val polymorphism upon tumor risk and disease progression in ovarian cancer patients. One hundred and eleven ovarian cancer patients who had been treated at the University Hospital of Essen between 1999 and 2007 and 119 age-matched healthy female controls were enrolled in this study. Genotyping was performed using PCR-RFLP. The distribution of genotypes was statistically significant different between ovarian cancer patients and healthy controls. We observed a significant association of the Ile allele with ovarian cancer (OR 2.6, 95% CI 1.5-4.7, p = 0.001). Clinical parameters such as overall survival, FIGO stage, grading, and age at diagnosis did not differ significantly. We observed a statistically significant association between the 462Val allele and platinum resistance, which was defined as a time interval < 6 months to disease progression after administration of a platinum-based primary chemotherapy (OR 5.9, 95% CI 1.5-23.2, p = 0.005). We observed a significant association between the presence of the 462Ile allele with ovarian cancer. While there is uncertainty about the potential involvement of CYP1A1 in the metabolism of platinum-containing agents, our findings suggest an association between the 462Val allele and the development of platinum resistance in ovarian tumors. If confirmed in a larger, independent collective, our findings would have important relevance with respect to the clinical consequences for the primary chemotherapy of ovarian cancer patients.

Association of the GNAS1 T393C polymorphism with tumor stage and survival in gastric cancer.

AIM: To analyze the impact of the GNAS1 T393C polymorphism on prognosis and histopathology of gastric cancer. METHODS: Genomic DNA was extracted from paraffin-embedded tissues of 122 patients with primary gastric carcinoma and from the blood of 820 healthy white individuals. Allelic discrimination was performed by quantitative real-time polymerase chain reaction. Genotyping was correlated with histopathologic parameters and with overall survival according to the Kaplan-Meier approach and with multivariate analysis by multiple stepwise regression. RESULTS: Thirty-nine (32%) patients displayed a CC genotype, 57 (46.7%) a CT genotype and 26 (21.3%) a TT genotype. The frequency of the C allele (fC) in the patient group was 0.55, which was not significantly different from that of healthy blood donors. The distribution was compatible with the Hardy-Weinberg equilibrium. Analysis of clinicopathological parameters did not show any significant correlation of the T393C genotype with gender (P = 0.50), differentiation (P = 0.29), pT-category (P = 0.19), pN-category (P = 0.30), pM-category (P = 0.25), R-category (P = 0.95), the classifications according to WHO (P = 0.34), Laurén (P = 0.16), Goseki (P = 1.00) and Ming (P = 0.74). Dichotomization between C+ (CC+CT) and C-genotypes (TT), however, revealed significantly more advanced tumor stages (P = 0.023) and lower survival rates (P = 0.043) for C allele carriers. CONCLUSION: The present study provides strong evidence to suggest that the GNAS1 T393C allele carrier status influences tumor progression and survival in gastric cancer with higher tumor stages and a worse outcome for C allele carriers.

Association of GNB4 intron-1 haplotypes with survival in patients with UICC stage III and IV colorectal carcinoma.

BACKGROUND: Polymorphisms in genes encoding subunits of heterotrimeric G proteins have been repeatedly associated with the course of cancer. As previously shown, intron 1 of GNB4 harbours distinct haplotype blocks and block 1 is associated with survival and disease progression in urothelial bladder cancer. This study investigated whether haplotype block 2 is associated with survival in colorectal cancer patients. PATIENTS AND METHODS: The haplotype tagging polymorphism of GNB4 haplotype block 2 was genotyped in 136 colorectal cancer patients and associated with demographic and clinical data and survival. RESULTS: Haplotype block 2 is associated with survival in colorectal cancer patients. Patients with advanced tumour stages carrying the 2*1 haplotype revealed decreased survival (HR 2.04, 95% CI 0.91-3.69). In multivariate analysis, the diplotypes were independent prognostic factors. CONCLUSION: Intron-1 haplotypes of GNB4 might be predictive markers for survival of patients with advanced colorectal cancer, thus influencing the clinical management of these patients.

The haplotype of two FSHR polymorphisms in ovarian cancer--a potential role of ethnology in risk modification.

OBJECTIVES: The precise role of gonadotropins in the carcinogenesis of epithelial ovarian cancer remains uncertain. Recently, the haplotype of two single nucleotide polymorphisms, Thr307Ala (rs6165) and Asn680Ser (rs6166), has been described as a risk factor for ovarian cancer in Chinese women. In this study we investigated the impact of this haplotype regarding the risk to develop ovarian cancer as well as possible effects upon the clinical course in a Caucasian patient sample. SUBJECTS AND METHODS: Determination of genotypes in 115 patients with primary epithelial ovarian cancer and 115 age-matched controls was performed by Pyrosequencing for Thr307Ala and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for Asn680Ser. RESULTS: Analysis of the genotypes revealed almost complete linkage disequilibrium of both SNPs. The distribution of genotypes was not statistically significant different between ovarian cancer patients and age-matched controls. Clinical parameters such as overall survival, CA12-5 elevation at primary diagnosis, age at diagnosis, FIGO stage, grading, and platinum resistance were not statistically significantly different regarding genotypes. CONCLUSIONS: We could not confirm the FSHR Ala307-Ser680 haplotype as a risk factor for epithelial ovarian cancer in Caucasian women. Hence, the modification of tumor risk may be affected by the ethnology of the patient collective. We could not find any associations of clinical parameters or course of the disease with the different genotypes.

DNA repair gene XRCC1 polymorphisms and outcome of renal cell carcinoma in Caucasian patients.

BACKGROUND: The human X-ray repair cross-complementing group 1 (XRCC1) enzyme plays an important role in response to DNA damage. Two common polymorphisms in XRCC1, Arg194Trp and Arg399Gln, have been repeatedly associated with risk for and outcome of numerous types of cancer treated with radio- and chemotherapy. Recently, a Japanese study suggested these polymorphisms both as risk factors and outcome predictors in renal cell carcinoma (RCC). PATIENTS AND METHODS: In the present study, 142 Caucasian patients suffering from RCC were genotyped and analyzed for tumor-related and overall survival and time to metastasis and progression. RESULTS: Analyses revealed absence of the pre-described risk haplotype (194Trp/399Gln) as well as a lack of a statistically significant difference between the different endpoints and genotypes and diplotypes, respectively. CONCLUSION: We conclude that in Caucasian patients, XRCC1 polymorphisms do not influence the outcome of RCC.

Characterization of intron-1 haplotypes of the G protein beta 4 subunit gene--association with survival and progression in patients with urothelial bladder carcinoma.

PURPOSE: Polymorphisms in genes encoding subunits of heterotrimeric G proteins have been repeatedly associated with various cancers. As G beta gamma signaling is presumed to be involved in proliferation and invasion processes, we analyzed genetic variations in regulatory regions of GNB4, which encodes the G beta 4 subunit, for their potential influence on cancer progression. EXPERIMENTAL DESIGN: We characterized the promoter of GNB4 and screened the promoter as well as exon 1 and intron 1 for single nucleotide polymorphisms by sequencing 100 healthy controls. Following a haplotype analysis, we determined the functional impact upon gene expression of the defined haplotypes by reporter assays, electrophoretic mobility shift assay and western blot. In addition, these haplotypes were tested for their relation to the disease course of urothelial bladder cancer. RESULTS: Whereas the promoter of GNB4 revealed no polymorphisms, 33 single nucleotide polymorphisms located in exon 1 and intron 1 were identified and together with a common exon-4 polymorphism implemented in haplotype analysis, which resulted in the determination of distinct haplotype blocks. Reporter activity was haplotype-dependently different (P=0.001). 1*1/1*1 showed increased G beta 4 protein (P=0.003), and bladder cancer patients carrying this diplotype displayed more progressive disease (P=0.046) and a significantly increased mortality (P=0.046). In multivariate analysis,the diplotypes were independent prognostic factors for survival and progression. CONCLUSION: Intron-1 haplotypes of GNB4 may, thus, serve as predictive markers for progression and survival of patients suffering from bladder cancer.

Factor V Leiden mutation is associated with improved 30-day survival in patients with acute respiratory distress syndrome.

OBJECTIVE: Activation of coagulation and inflammation are parts of the innate host response to infection that may contribute to organ dysfunction and death when control of these systems is compromised. Thus, functional single nucleotide polymorphisms within candidate genes of the inflammation and coagulation cascade are possible factors which may influence severity and/or mortality in acute respiratory distress syndrome. The aim of this study was to investigate whether the factor V Leiden mutation (Arg506Gln) is associated with altered severity and/or mortality in acute respiratory distress syndrome. DESIGN: Retrospective cohort, genetic association study. SETTING: Tertiary care intensive care unit. PATIENTS: Adults (white Germans) with acute respiratory distress syndrome (n = 106). INTERVENTIONS: Genotyping for the factor V Leiden mutation. MEASUREMENTS AND MAIN RESULTS: Using Kaplan-Meier estimates to compare outcome, 30-day survival was significantly associated with the factor V Leiden mutation (p = .049). Thirty-day survival rates were 100% for Arg/Gln (n = 7) genotypes but only 58% for Arg/Arg (n = 99) genotypes, respectively. CONCLUSION: We show for the first time that a heterozygous factor V Leiden genotype is associated with improved 30-day survival in patients with acute respiratory distress syndrome.

Comparison of manual and automated nucleic acid extraction from whole-blood samples.

Nucleic acid extraction and purification from whole blood is a routine application in many laboratories. Automation of this procedure promises standardized sample treatment, a low error rate, and avoidance of contamination. The performance of the BioRobot M48 (Qiagen) and the manual QIAmp DNA Blood Mini Kit (Qiagen) was compared for the extraction of DNA from whole blood. The concentration and purity of the extracted DNAs were determined by spectrophotometry. Analytical sensitivity was assessed by common PCR and genotyping techniques. The quantity and quality of the generated DNAs were slightly higher using the manual extraction method. The results of downstream applications were comparable to each other. Amplification of high-molecular-weight PCR fragments, genotyping by restriction digest, and pyrosequencing were successful for all samples. No cross-contamination could be detected. While automated DNA extraction requires significantly less hands-on time, it is slightly more expensive than the manual extraction method.

No association of the NFKB1 insertion/deletion promoter polymorphism with survival in colorectal and renal cell carcinoma as well as disease progression in B-cell chronic lymphocytic leukemia.

PURPOSE: Nuclear factor-kappaB (NF-kappaB) is an inducible transcription factor that plays a major role in the regulation of genes involved in immune and inflammatory response. Activation of NF-kappaB has also been associated with apoptosis and proliferation, thereby potentially also controlling oncogenesis. A functional insertion/deletion polymorphism has been identified in the promoter region of NFKB1, which apparently controls the transcription of NF-kappaB. The purpose of this study was, therefore, to investigate associations of the -94ins/delATTG polymorphism with susceptibility and survival of patients with different types of cancer. EXPERIMENTAL DESIGN: Genotype distributions in patients with colorectal carcinoma (n=139), B-cell chronic lymphocytic leukemia (n=72) and clear cell renal cell carcinoma (n=140), and in controls (n=307) were analyzed by pyrosequencing and compared with each other as well as associated with clinico-pathological parameters and demographic data. RESULTS: No statistically significant differences in both allele and genotype frequencies were observed between patients and healthy controls. Likewise, no association between -94ins/delATTG alleles and survival or disease progression was noticed. CONCLUSION: These results suggest that the NFKB1 promoter polymorphism has no effect on risk and course of disease in the three cancer entities that were analyzed.

Active succinate dehydrogenase (SDH) and lack of SDHD mutations in sporadic paragangliomas.

BACKGROUND: Paragangliomas are benign, slow-growing tumours of the head and neck region. The candidate gene for familial and some sporadic paragangliomas, SDHD (succinate dehydrogenase, subunit D), has been mapped to the PGL1 locus in 11q23.3. MATERIALS AND METHODS: Normal and tumour DNA of 17 patients with sporadic paragangliomas were analysed by sequencing (SDHD, SDHB and SDHC genes), fluorescence in situ hybridisation (FISH). In addition, loss of heterozygosity (LOH) and succinate dehydrogenase (SDH) enzyme activity assays were performed. RESULTS AND CONCLUSION: Only two patients from our collective showed SDH gene mutations, one in SDHD and one in SDHB, respectively. Moreover, SDH activity detected in 5/8 patients confirmed the fact that SDH inactivation is not a major event in sporadic paragangliomas. LOH and FISH analysis demonstrated a frequent loss of regions within chromosome 11, indicating that additional genes in 11q may play a role in tumour genesis of sporadic paragangliomas.

GJB2 mutations in patients with non-syndromic hearing loss from Northeastern Hungary.

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, c.35delG, accounts for the majority of mutations in Caucasian patients with HI. In the present study we screened 500 healthy control individuals and a group of patients with HI from Northeastern Hungary for GJB2 mutations. The patients' group consisted of 102 familial from 28 families and 92 non-familial cases. The most common mutation in the Hungarian population is the c.35delG, followed by the c.71G>A (p.W24X) mutation. 34.3% of the patients in the familial group were homozygous, and 17.6% heterozygous for 35delG. In the non-familial group the respective values were 37% and 18% (allele frequency: 46.2%). In the general population an allele frequency of 2.4% was determined. Several patients were identified with additional, already described or new GJB2 mutations, mostly in heterozygous state. The mutation c.380G>A (p.R127H) was formerly found only in heterozygous state and its disease relation was controversial. We demonstrated the presence of this mutation in a family with three homozygous patients and 4 heterozygous unaffected family members, a clear indication of recessively inherited HI. Furthermore, we provided evidence for the pathogenic role of two new mutations, c.51C>A (p.S17Y) and c.177G>T (p.G59V), detected in the present study. In the latter case the pattern of inheritance might be dominant. Our results confirm the importance of GJB2 mutations in the Hungarian population displaying mutation frequencies that are comparable with those in the Mediterranean area.

Chromosome 11 monosomy in conjunction with a mutated SDHD initiation codon in nonfamilial paraganglioma cases.

Paragangliomas of the head and neck region are a group of rare, usually benign, slow-growing tumors developing from paraganglionic chemoreceptors in most patients. Mutations in a subunit of the mitochondrial enzyme II complex (succinate dehydrogenase [SDHD]) were shown to be responsible for the formation of paragangliomas. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), was observed recently. We analyzed DNA derived from tumor sections of three unrelated paraganglioma patients (one case with multiple paragangliomas, two cases with single tumors; all of them sporadic cases) for mutations in the SDHD gene by direct sequencing. Microsatellite-based LOH was performed, and events of chromosomal loss were validated by fluorescence in situ hybridization (FISH) on paraffin-embedded tumor and normal tissue by using centromeric satellite DNA. Sequence analysis revealed mutations in SDHD exon 1 in all patients, affecting the initiation codon (M1V). Another alteration was detected in exon 2 but was lacking in tumor DNA and therefore classified as polymorphism (H50R). LOH and FISH analyses demonstrated partial/total monosomy for chromosome 11 in the tumor samples tested. A common genetic mechanism appears to be the pathophysiologic basis for sporadic tumor development because the proposed two-hit model comprising both LOH and point mutation is manifest in our patients. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors in one of the patients.