Evaluation of a cancer patient navigation program ("Onkolotse") in terms of hospitalization rates, resource use and healthcare costs: rationale and design of a randomized, controlled study.

BACKGROUND: Concepts for the nursing and care of cancer patients through a "navigation service" have attracted much interest. However, there is still room for improvement in terms of their funding and coverage. The Saxon Cancer Society designed a prospective, randomized, multicenter, longitudinal study with a view to determining the positive effects of a cancer patient navigator program. The objective of this ongoing study is to evaluate the impact of the cancer patient navigation program on cancer patients and cost bearers in Germany. METHODS: The study population in this evaluation comprises cancer patients with gastric carcinoma, pancreatic carcinoma, colorectal cancer, melanoma or gynecological cancer who have been hospitalized at least once at one of the study centers as well as their relatives, outpatient and inpatient physicians, and cancer nurses. It is planned to randomize 340 cancer patients (stomach, colonic/rectal cancer, gynecological cancer, melanoma) at five centers to an intervention group (care by patient navigators based on standardized operating procedures) or a control group in a one-to-one ratio. The primary target parameter is the number of hospitalizations within the 12-month intervention period. The participants are asked to complete various questionnaires on patient-related outcomes at baseline and at 3 and 12 months (SF 36, HADS, PAM 13, and others). Data on drug therapy, utilization of health services, and medical expenses will also be analyzed. DISCUSSION: For the first time, the study will provide data on the effectiveness of a patient support program in cancer care in Germany from a randomized trial with a high level of evidence. TRIAL REGISTRATION: The study has been registered under DRKS00013199 in the German Clinical Trials Register.

High-resolution melting analysis of the TPMT gene: a study in the Polish population.

The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines.

A simple method of investigating mutations in CHEK2 by DHPLC: a study of the German populations of Saxony, Saxony-Anhalt, and Thuringia.

Allele variants of the CHEK2 gene have been found to be associated with several types of cancer, including cancer of the breast, prostate, lung, and ovary. In the Polish population, three founder mutations of CHEK2 have been identified: I157T, 444+1G>A (formerly IVS2+1G>A), and 1100delC. The aim of our study was to establish a simple method to identify founder CHEK2 mutations and determine the prevalence of these changes in the population of Eastern Germany (Saxony, Saxony-Anhalt, and Thuringia). We drew up denaturing high-performance liquid chromatography (DHPLC) conditions for analysis of intron 2 and exon 3 for two mutations (444+1G>A, I157T) and exon 10 for mutation 1100delC. We tested 251 patients and controls. Mutations show a similar frequency in the general population of Eastern Germany as in neighboring Poland (4.95% vs. 4.8% for the missense mutation I157T and 0.99% vs. 0.5% for the truncating mutations 444+1G>A and 1100delC). Investigation of these mutations by DHPLC is highly sensitive and less time-consuming compared to restriction fragment length polymorphism or allele-specific oligonucleotide polymerase chain reaction. It can be easily integrated into diagnostic testing.

New mutation in the CYLD gene within a family with Brooke-Spiegler syndrome.

Brooke-Spiegler syndrome is a rare, autosomal dominant disease characterized by multiple skin appendage tumors caused by various mutations in the CYLD gene on chromosome 16q12-q13. We describe a family, in which we performed a molecular-genetic examination and found a new mutation in exon 19 in the CYLD gene leading to a frameshift. It is important to be aware of this syndrome and its pathogenesis as its phenotypic features can vary so that apparently different diseases are caused by the same genetic defect. In addition, there may be malignant transformation of the generally benign tumors, so that a timely diagnosis is essential for appropriate monitoring and therapy.

Evaluation of IP-RP-HPLC for length determination of the trinucleotide repeat fragments in Huntington's disease.

Expansion of an unstable trinucleotide (CAG)(n) repeat region within exon 1 of the gene IT15 causes autosomal, dominantly inherited Huntington's disease (HD). The number of CAG-repeats varies from 6 to 35 in normal individuals, whereas in affected patients the expanded allele contains 40 or more CAG-repeats. Thus, exact determination of both alleles of the gene (normal and expanded) on the molecular level is of great importance for clinical diagnosis and prognosis regarding the course of the disease. In our study, we optimized and evaluated a highly sensitive, automated, and economical molecular method for length characterization of the trinucleotide fragment expansion such as (CAG)(n) repeat region based on ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). We found that IP-RP-HPLC can be used for exact fragment length measuring between 60-280 bp as a sensitive and advantageous alternative method to conventional techniques.

Contralateral breast cancer risk in BRCA1 and BRCA2 mutation carriers.

PURPOSE: To estimate the risk for contralateral breast cancer in members of BRCA1- and BRCA2-positive families and to determine predictive risk factors. PATIENTS AND METHODS: A retrospective, multicenter, cohort study was performed from 1996 until 2008 and comprised 2,020 women with unilateral breast cancer (index patients, n = 978; relatives, n = 1.42) from 978 families who had a BRCA1 or BRCA2 mutation. Cox regression analysis was applied to assess the association of age at first breast cancer with time from first to contralateral breast cancer, stratified by the affected BRCA gene. RESULTS: The cumulative risk for contralateral breast cancer 25 years after first breast cancer was 47.4% (95% CI, 38.8% to 56.0%) for patients from families with BRCA1 or BRCA2 mutations. Members of families with BRCA1 mutations had a 1.6-fold (95% CI, 1.2-fold to 2.3-fold) higher risk of contralateral breast cancer than members of families with BRCA2 mutations. Younger age at first breast cancer was associated with a significantly higher risk of contralateral breast cancer in patients with BRCA1 mutation, and a trend was observed in patients with BRCA2 mutation. After 25 years, 62.9% (95% CI, 50.4% to 75.4%) of patients with BRCA1 mutation who were younger than 40 years of age at first breast cancer developed contralateral breast cancer, compared with only 19.6% (95% CI, 5.3% to 33.9%) of those who were older than 50 years of age at first breast cancer. CONCLUSION: Contralateral breast cancer risk depends on age at first breast cancer and on the affected BRCA gene, and this risk should be considered in treatment planning.

New analysis method of myotonic dystrophy 1 based on quantitative fluorescent polymerase chain reaction.

Molecular genetic testing of myotonic dystrophy type 1 (DM1) is based on the identification and determination of a cytosine-thymine-guanine (CTG) repeat expansion in the DMPK gene. This is usually done by Southern blot analysis-a time-consuming and very laborious technique requiring high molecular weight DNA. The aim of our study was to develop a highly sensitive, rapid, and cost-effective molecular analysis characterizing the CTG repeat region of the DMPK gene based on a two-step polymerase chain reaction (PCR) protocol. (1) For the detection of alleles of up to 100 repeats, a quantitative fluorescent (QF) amplification with primers flanking the repeat region of the DM1 locus and two reference genes (PAX2 and DHCR7) for standardization was used. By this method it was possible to identify both homozygous and heterozygous DM1 alleles. (2) Long PCR was only performed if a single wild-type allele was detected that gave a QF-PCR signal of only half intensity compared to a homozygous sample. The results obtained using combined QF and Long PCR are highly accurate compared with Southern blot analysis. We conclude that our new rapid analysis is reliable for genetic testing of DM1 patients.

New mutations of EXT1 and EXT2 genes in German patients with Multiple Osteochondromas.

Mutations in either the EXT1 or EXT2 genes lead to Multiple Osteochondromas (MO), an autosomal dominantly inherited disorder. This is a report on clinical findings and results of molecular analyses of both genes in 23 German patients affected by MO. Mutation screening was performed by using denaturing high performance liquid chromatography (dHPLC) and automated sequencing. In 17 of 23 patients novel pathogenic mutations have been identified; eleven in the EXT1 and six in the EXT2 gene. Five patients were carriers of recurrent mutations in the EXT2 gene (p.Asp227Asn, p.Gln172X, p.Gln258X) and one patient had no detectable mutation. To demonstrate their pathogenic effect on transcription, two complex mutations in EXT1 and EXT2 and three splice site mutations were characterized by mRNA investigations. The results obtained provide evidence for different aberrant splice effects - usage of new cryptic splice sites and exon skipping. Our study extends the mutational spectrum and understanding of pathogenic effects of mutations in EXT1 and EXT2.

No association of TGFB1 L10P genotypes and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a multi-center cohort study.

BACKGROUND: The transforming growth factor beta-1 gene (TGFB1) is a plausible candidate for breast cancer susceptibility. The L10P variant of TGFB1 is associated with higher circulating levels and secretion of TGF-beta, and recent large-scale studies suggest strongly that this variant is associated with breast cancer risk in the general population. METHODS: To evaluate whether TGFB1 L10P also modifies the risk of breast cancer in BRCA1 or BRCA2 mutation carriers, we undertook a multi-center study of 3,442 BRCA1 and 2,095 BRCA2 mutation carriers. RESULTS: We found no evidence of association between TGFB1 L10P and breast cancer risk in either BRCA1 or BRCA2 mutation carriers. The per-allele HR for the L10P variant was 1.01 (95%CI: 0.92-1.11) in BRCA1 carriers and 0.92 (95%CI: 0.81-1.04) in BRCA2 mutation carriers. CONCLUSIONS: These results do not support the hypothesis that TGFB1 L10P genotypes modify the risk of breast cancer in BRCA1 or BRCA2 mutation carriers.

Surgical outcome after phototherapeutic keratectomy in patients with TGFBI-linked corneal dystrophies in relation to molecular genetic findings.

PURPOSE: To evaluate the correlation between surgical outcome after phototherapeutic keratectomy in patients with autosomal dominant transforming growth factor, beta-induced (TGFBI)-linked corneal dystrophies (CD) and molecular genetic findings regarding the TGFBI gene. METHODS: Twelve patients were examined to investigate genotype by direct sequencing of the TGFBI gene. Twenty eyes of 12 patients were treated with phototherapeutic keratektomy (PTK) to remove superficial corneal opacifications and to decrease recurrent erosions. Surgical outcome, including visual improvement, recurrence of opacifications, postoperative complications, and additional therapeutic proceedings were reported and compared with the molecular genetic results. RESULTS: Four different missense mutations were identified within the coding region of the TGFBI gene: Arg124Cys in one eye, Arg555Trp in nine eyes, Arg124His in four eyes and Gly623Arg in six eyes. In all eyes the PTK was successful without clinically significant recurrent opacifications after a mean follow-up time of 17.6 months (min 3 months, max 42 months). The best corrected visual acuity (BCVA) improved with an average increase of 3.1 lines (minimum 2 lines, maximum 5 lines). In one eye (Arg124Cys), we observed delayed wound healing and a delayed increase in BCVA, in two eyes we performed an Epilasik to correct remaining hyperopia, and in four eyes we fitted rigid gas-permeable tricurve contact lenses to correct the remaining irregular astigmatism. CONCLUSIONS: The variable genotypes in patients with TGFBI-linked corneal dystrophies lead to significantly different results after surgical treatment. The Gly623Arg mutation seems to be an optimum genotype on which to perform PTK even in older patients. It is essential to determine the genotype in order to standardize the PTK treatment and to evaluate the success in TGFBI-linked corneal dystrophies.

Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein.

BACKGROUND: Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS. METHODS: The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF. RESULTS: The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter. CONCLUSION: The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

Macular corneal dystrophy: mutational spectrum in German patients, novel mutations and therapeutic options.

INTRODUCTION: The objective of this study was to investigate genotype-phenotype correlations, the consequences for surgical treatment, and the therapeutical options in patients with macular corneal dystrophy (MCD). MATERIAL AND METHODS: We investigated MCD genotype by using polymerase chain reaction followed by direct sequencing in one family and four patients with MCD. Results were confirmed by restriction analysis. Clinical phenotypes, histopathological findings, and therapeutical proceedings of each patient were reported and compared with the molecular genetic results. RESULTS: Five mutations, four missense mutations, and one frameshift mutation, from which three were novel, and one single-nucleotide polymorphism, were identified within the coding region of the CHST6 gene. In three patients, two with a homozygous mutation within the start codon (Met1Leu) and one with a heterozygous mutation (Leu200Arg) and a polymorphism (Arg162Gly), with irregular corneal surface and recurrent erosions a phototherapeutic keratectomy lead to a transient success. An additional fitting of rigid gas permeable contact lenses in one patient could further improve irregular astigmatism. In two patients, one with a frameshift mutation (1734_1735delTG; Arg211Gln) and one with two compound heterozygous mutations (Leu200Arg; Leu173Phe) and an additional polymorphism (Arg162Gly) a penetrating keratoplasty improved BCVA without any recurrence of the opacities within the follow-up time. DISCUSSION: Different genotypes imply several phenotypes, which influence therapeutical proceedings in MCD patients. Our study shows the wide range of diagnostic findings and therapeutical options in patients suffering from macular corneal dystrophy depending on the genotype.

Different phenotypes including gynecological cancer in three female patients with Peutz-Jeghers syndrome and mutations in the STK11 gene.

BACKGROUND: Peutz-Jeghers syndrome (PJS), a rare hereditary disorder, is characterized by the occurrence of gastrointestinal hamartomatous polyps associated with mucocutaneous pigmentation. Patients are at an increased cancer risk not only for gastrointestinal but also for extraintestinal neoplasms. PATIENTS AND RESULTS: We report on the clinical and molecular findings in 3 young female patients with PJS; 2 of them suffered from severe gynecological cancer. One patient died at the age of 29 years of an incurable mucin-producing cervical adenocarcinoma. Another patient had a papillary serous carcinoma of the ovary. In all patients, we identified corresponding mutations in the STK11 gene, 2 of them novel. CONCLUSION: PJS should be considered in differential diagnosis in young women with gynecological malignancies. Identification of STK11 mutations in patients and their relatives can help to improve the clinical management.

Prenatal diagnosis and genetic counseling in a case of spina bifida in a family with Waardenburg syndrome type I.

OBJECTIVE: Waardenburg syndrome type I (WS I) is an autosomal dominant inherited disorder with an incidence of 1:45,000 in Europe. Mutations within the PAX3 gene are responsible for the clinical phenotype ranging from mild facial features to severe malformations detectable in prenatal diagnosis. METHODS: Here, we report a four-generation family with several affected members showing various symptoms of WS I. We diagnosed the syndrome first in a pregnant young woman; she was referred because of a spina bifida in prenatal diagnosis. We performed clinical genetic investigations and molecular genetic analysis in all available family members. RESULTS: The phenotype displays a wide intra-familial clinical variability of pigmentary disturbances, facial anomalies and developmental defects. Molecular studies identified a novel splice site mutation within the PAX3 gene in intron 5 in all affected family members, but in none of the unaffected relatives. CONCLUSIONS: This case demonstrates the prenatal diagnosis of spina bifida in a fetus which leads to the initial diagnosis of WS I. Further studies could identify a private splice site mutation within the PAX3 gene responsible for the phenotype in this family.

Expanding the spectrum of TBX5 mutations in Holt-Oram syndrome: detection of two intragenic deletions by quantitative real time PCR, and report of eight novel point mutations.

Mutations in the gene TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant disorder characterized by anterior (i.e., radial ray) upper limb malformations and congenital heart defects and/or cardiac conduction anomalies. The detection rate for TBX5 mutations in HOS patients has been given as 30-35% in most reports. However, a detection rate of 74% was reported when strict clinical inclusion criteria for HOS were applied prior to TBX5 analysis. Still, in a significant proportion of typical HOS cases no mutation can be found within the TBX5 coding region and flanking intronic sequences. One explanation could be that large but submicroscopic deletions of TBX5 could cause HOS, yet only one such TBX5 deletion has been reported to date. We developed a quantitative Real Time PCR strategy to detect large, submicroscopic deletions in TBX5. Using this assay, we screened a total of 102 TBX5 mutation negative patients and discovered two novel intragenic deletions. One deletion of 7756 bp removes exon 6 and a considerable part of the neighboring intronic sequences, and the other of 3695 bp removes exon 9 with the stop codon and the 3'UTR completely as well as a part of the preceding intron 8. We conclude that quantitative Real Time PCR is a reliable method to detect submicroscopic deletions within TBX5. However, such deletions explain only approximately 2% of the TBX5 mutational spectrum in HOS cases. In addition, we also present eight novel TBX5 mutations (three nonsense, one splice mutation, four short deletions) as detected by direct sequencing in 21 families not previously analyzed for mutations.