[Colorectal Carcinoma with Suspected Lynch Syndrome: A Multidisciplinary Algorithm]. / Kolorektales Karzinom bei V. a. Lynch-Syndrom: ein interdisziplinärer Algorithmus.

Lynch syndrome is the most frequent hereditary cancer syndrome, accounting for approximately 3-5 % of all colorectal cancers. In addition, it is the most frequent predisposing hereditary cause of endometrial cancer and is also associated with gastric cancer, ovarian cancer, cancer of the urinary tract as well as several other cancers. In clinical practise Lynch syndrome is frequently not detected and many clinicians admit uncertainties regarding diagnostic procedures. Also, counselling of patients is considered difficult regarding therapeutic - especially prophylactic surgical and chemopreventive options and recommendations. Based on a review of available literature we discuss optimized strategies for improved detection of suspected Lynch syndrome patients. The aim of this review is to establish a clinical algorithm of how to proceed on a diagnostic level and to discuss surgical options at the time of a colorectal cancer. In order to identify patients with Lynch syndrome, family history should be ascertained and evaluated in regards to fulfilment of the Amsterdam-II- and/or the revised Bethesda criteria. Subsequently immunohistochemical staining for the mismatch-repair-genes, BRAF testing for MLH1 loss of expression, as well as testing for microsatellite instability in some, followed by genetic counselling and mutation analysis when indicated, is recommended. Pathological identification of suspected Lynch syndrome is readily feasible and straightforward. However, the need of performing these analyses in the tumor biopsy at the time of (gastroenterological) diagnosis of CRC neoplasia is essential, in order to offer patients the option of a prophylactically extended surgery and - as recommended in the German S3 guidelines - to discuss the option of a merely prophylactical hysterectomy and oophorectomy (if postmenopausal) in women. Close cooperation between gastroenterologists, pathologists and surgeons is warranted, so that patients may benefit from options of extended or prophylactically extended surgery at the time of diagnosis of a colorectal primary. Patients nowadays must be involved in informed decision-making regarding prophylactic or extended prophylactic surgery at the time of a colorectal primary. To date, however, limitations in daily clinical practise, the failure to assess family history and the lack of awareness of this important hereditary syndrome is the major asset leading to severe underdiagnosis and putting to risk the indexpatients themselves and their families to (metachronous) CRC and the associated extracolonic cancers. If at all tumors of patients fulfilling Bethesda criteria will be analysed for MSI in the surgical specimen and therefore Lynch syndrome patients are not given the opportunity to opt for extended surgery. In clinical experience the postoperative MSI-analysis is inconsistently performed - even if the Bethesda criteria are fulfilled - and in case of suspected Lynch syndrome genetically counselling is not consistently recommended. Therefore affected cancer patients are left unaware of their increased genetic risk and in average 3 high-risk gene carriers per family miss the opportunity to actively engage in the recommended screening program.

Shared Copy Number Variation in Simultaneous Nephroblastoma and Neuroblastoma due to Fanconi Anemia.

Concurrent emergence of nephroblastoma (Wilms Tumor; WT) and neuroblastoma (NB) is rare and mostly observed in patients with severe subtypes of Fanconi anemia (FA) with or without VACTER-L association (VL). We investigated the hypothesis that early consequences of genomic instability result in shared regions with copy number variation in different precursor cells that originate distinct embryonal tumors. We observed a newborn girl with FA and VL (aplasia of the thumbs, cloacal atresia (urogenital sinus), tethered cord at L3/L4, muscular ventricular septum defect, and horseshoe-kidney with a single ureter) who simultaneously acquired an epithelial-type WT in the left portion of the kidney and a poorly differentiated adrenal NB in infancy. A novel homozygous germline frameshift mutation in PALB2 (c.1676_c1677delAAinsG) leading to protein truncation (pGln526ArgfsX1) inherited from consanguineous parents formed the genetic basis of FA-N. Spontaneous and induced chromosomal instability was detected in the majority of cells analyzed from peripheral lymphocytes, bone marrow, and cultured fibroblasts. Bone marrow cells also showed complex chromosome rearrangements consistent with the myelodysplastic syndrome at 11 months of age. Array-comparative genomic hybridization analyses of both WT and NB showed shared gains or amplifications within the chromosomal regions 11p15.5 and 17q21.31-q25.3, including genes that are reportedly implicated in tumor development such as IGF2, H19, WT2, BIRC5, and HRAS.

Comprehensive array CGH of normal karyotype myelodysplastic syndromes reveals hidden recurrent and individual genomic copy number alterations with prognostic relevance.

About 40% of patients with myelodysplastic syndromes (MDSs) present with a normal karyotype, and they are facing different courses of disease. To advance the biological understanding and to find molecular prognostic markers, we performed a high-resolution oligonucleotide array study of 107 MDS patients (French American British) with a normal karyotype and clinical follow-up through the Duesseldorf MDS registry. Recurrent hidden deletions overlapping with known cytogenetic aberrations or sites of known tumor-associated genes were identified in 4q24 (TET2, 2x), 5q31.2 (2x), 7q22.1 (3x) and 21q22.12 (RUNX1, 2x). One patient with a 7q22.1 deletion had an additional 5q31.2 deletion of the acute myeloid leukemia/MDS region, the smallest deletion identified so far and including the putative tumor suppressor (ts) genes, EGR1 and CTNNA1. One TET2 deletion was homozygous and one heterozygous, with a missense mutation in the remaining allele, further supporting its role as a ts gene. Besides these recurrent alterations, additional individual imbalances were found in 34 cases; in total, 42/107 (39%) cases had genomic imbalances. These patients had an inferior survival as compared with the rest of the patients (P=0.002). This study emphasizes the heterogeneity of MDS, but points to interesting genes that may have diagnostic and prognostic impact.

A novel transcript of the LMO2 gene, LMO2-c, is regulated by GATA-1 and PU.1 and encodes an antagonist of LMO2.

Ectopic expression of LIM-only protein 2 (LMO2) in T-cells, as a result of chromosomal translocations or retroviral insertion, plays an important role in the onset of T-cell leukemias. Two transcripts of LMO2 gene (LMO2-a and LMO2-b) have been reported to encode a same 158-amino-acid protein. We have previously reported a novel transcript of human LMO2 gene (LMO2-c) encoding a 151-amino-acid protein, and defined its promoter region. In the present study, we investigated the regulation of the LMO2-c expression and the functions of LMO2-c. We found that LMO2-c expression is regulated by the cooperation of two essential hematopoietic transcription factors GATA-1 and PU.1 in various hematopoietic cell lines, suggesting an important functional role for LMO2-c in the hematopoietic system. More importantly, we demonstrated that LMO2-c acts as an antagonist of LMO2-a/b binding to its partners, therefore blocking the transactivation of LMO2-a/b on its target genes. These findings provide novel evidence to the functions of LMO2 gene in the hematopoietic system and leukemia.

Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders.

Congenital nephrotic syndrome is clinically and genetically heterogeneous. The majority of cases can be attributed to mutations in the genes NPHS1, NPHS2, and WT1. By homozygosity mapping in a consanguineous family with isolated congenital nephrotic syndrome, we identified a potential candidate region on chromosome 3p. The LAMB2 gene, which was recently reported as mutated in Pierson syndrome (microcoria-congenital nephrosis syndrome; OMIM #609049), was located in the linkage interval. Sequencing of all coding exons of LAMB2 revealed a novel homozygous missense mutation (R246Q) in both affected children. A different mutation at this codon (R246W), which is highly conserved through evolution, has recently been reported as causing Pierson syndrome. Subsequent LAMB2 mutational screening in six additional families with congenital nephrotic syndrome revealed compound heterozygosity for two novel missense mutations in one family with additional nonspecific ocular anomalies. These findings demonstrate that the spectrum of LAMB2-associated disorders is broader than previously anticipated and includes congenital nephrotic syndrome without eye anomalies or with minor ocular changes different from those observed in Pierson syndrome. This phenotypic variability likely reflects specific genotypes. We conclude that mutational analysis in LAMB2 should be considered in congenital nephrotic syndrome, if no mutations are found in NPHS1, NPHS2, or WT1.

STK11 status and intussusception risk in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is caused by germline STK11 mutations and characterised by gastrointestinal polyposis. Although small bowel intussusception is a recognised complication of PJS, risk varies between patients. OBJECTIVE: To analyse the time to onset of intussusception in a large series of PJS probands. METHODS: STK11 mutation status was evaluated in 225 PJS probands and medical histories of the patients reviewed. RESULTS: 135 (60%) of the probands possessed a germline STK11 mutation; 109 (48%) probands had a history of intussusception at a median age of 15.0 years but with wide variability (range 3.7 to 45.4 years). Median time to onset of intussusception was not significantly different between those with identified mutations and those with no mutation detected, at 14.7 years and 16.4 years, respectively (log-rank test of difference, chi(2) = 0.58, with 1df; p = 0.45). Similarly no differences were observed between patient groups on the basis of the type or site of STK11 mutation. CONCLUSIONS: The risk of intussusception in PJS is not influenced by STK11 mutation status.

Survival in nephroblastoma treated according to the trial and study SIOP-9/GPOH with respect to relapse and morbidity.

BACKGROUND: Recent Wilms' tumor (WT) trials and studies have tried to determine the minimal therapy needed for cure. The goal was survival without morbidity. PATIENTS AND METHODS: From January 1989 to March 1994 the German Society of Pediatric Oncology and Hematology registered 440 patients (median age 2.9 years; 231 male, 209 female) with WTs (preoperative chemotherapy 362) for therapy according to the International Society of Pediatric Oncology Trial and Study 9. Therapy for relapse depended on site of relapse and therapy already received. Follow-up included inquiries for morbidity. Prognostic factors for relapse and death were evaluated. RESULTS: Five-year survival of WTs was 89.5%; 98.2% (385 of 392) of survivors had a follow-up of 5 years (range 0.8-12.6; median 8). In non-anaplastic WTs, young age (<2 years) was of significance (P = 0.026) for a better survival. Non-anaplastic WTs (407 patients) had a 5-year survival of 92.3%, versus 48.5% in anaplastic WTs (33 patients), and a 5-year relapse-free survival of 87.6% versus 42.4%. Survival after relapse was significantly worse for anaplastic than for non-anaplastic WTs (residual 3-year survival 11.8% versus 54.3%; P <0.0001). In preoperatively treated WTs, anaplasia was a strong prognostic factor for death [relative risk (RR) 4.7], followed by poor response to preoperative therapy (RR 3.6), stage IV (RR 3.2) and abdominal stage III (RR 2.2). Low abdominal stages (

Germline Wilms tumor suppressor gene (WT1) mutation leading to isolated genital malformation without Wilms tumor or nephropathy.

Mutations of the Wilms tumor suppressor gene (WT1 ) have been described only in patients with syndromes associated with urogenital malformation and Wilms tumor or nephropathy. We present a male patient with an isolated genital malformation caused by a WT1 mutation.

Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1.

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator involved in differentiation and the regulation of cell growth. WT1 is subject to alternative splicing, one isoform including a 17-amino acid region that is specific to mammals. The function of this 17-amino acid insertion is not clear, however. Here, we describe a transcriptional activation domain in WT1 that is specific to the WT1 splice isoform that contains the 17-amino acid insertion. We show that the function of this domain in transcriptional activation is dependent on a specific interaction with the prostate apoptosis response factor par4. A mutation in WT1 found in Wilms' tumor disturbs the interaction with par4 and disrupts the function of the activation domain. Analysis of WT1 derivatives in cells treated to induce par4 expression showed a strong correlation between the transcription function of the WT1 17-amino acid insertion and the ability of WT1 to regulate cell survival and proliferation. Our results provide a molecular mechanism by which alternative splicing of WT1 can regulate cell growth in development and disease.

A novel post-transcriptional splicing form of the acute T cell leukemia proto-oncogene Lmo2.

Lmo2 is a T cell leukemia-related proto-oncogene, which belongs to the LIM protein family. Previous work has established its key role in yolk sac erythropoiesis and adult hematopoiesis, and it is also necessary for regulating angiogenesis. It has been demonstrated that this gene encodes a protein of 158 amino acids, consisting of two tandem cysteine-rich LIM domains, but the detailed mechanism of its transcriptional regulation remains to be elucidated. To further investigate the mechanism of transcriptional regulation of Lmo2, we combined SMART PCR technology with 5'RACE and found a novel post-transcriptional splicing form of Lmo2 in adult human kidney. This alternative transcript contains only two exons, encoding a smaller protein of 151 amino acids. Interestingly, it shares the same reading frame as the original Lmo2, but differs in 7 amino acids at the N-terminus. A genomic DNA fragment (from -294 nts to +180 nts) containing the putative promoter region has been inserted into the luciferase reporter gene vector pGL3-basic and showed stable promoter activity when transfected into COS7. RT-PCR analysis revealed that this variant transcript was expressed widely in human tissues and cell lines, suggesting its potential basic functional importance.

Evidence for susceptibility genes to familial Wilms tumour in addition to WT1, FWT1 and FWT2.

Three loci have been implicated in familial Wilms tumour: WT1 located on chromosome 11p13, FWT1 on 17q12-q21, and FWT2 on 19q13. Two out of 19 Wilms tumour families evaluated showed strong evidence against linkage at all three loci. Both of these families contained at least three cases of Wilms tumour indicating that they were highly likely to be due to genetic susceptibility and therefore that one or more additional familial Wilms tumour susceptibility genes remain to be found.

The size of the CAG repeat in exon 1 of the androgen receptor gene shows no significant relationship to impaired spermatogenesis in an infertile Caucasoid sample of German origin.

The androgen receptor (AR) gene, located on the X-chromosome at Xq11-12, contains in exon 1 a polymorphic CAG repeat which codes for a polyglutamine tract. Contractions of the CAG repeat are said to be related to prostate cancer. In contrast, sizeable expansion of the CAG repeat can cause spinal and bulbar muscular atrophy (SBMA). In infertile patients of Chinese origin and in a Melbourne multinational population impaired sperm production has been postulated to be related to moderate expansions of the polyglutamine tract. In a study of a Swedish population of infertile patients these findings could not be corroborated. The aim of our investigation was to examine the correlation between the length of the CAG repeat and impaired sperm production in an infertile Caucasoid patient sample of German ethnic origin. We found no statistically significant relationship between the size of the CAG repeat or polyglutamine tract and idiopathic impaired sperm production in the population studied. The variability of the results by various investigators may be attributed to different ethnic origins and hence different genetic modifiers of the populations studied and/or to the high probability that these infertile males may represent a heterogeneous group with respect to the causes of defective spermatogenesis.

Analysis of native WT1 protein from frozen human kidney and Wilms' tumors.

The Wilms' tumor susceptibility gene, WT1, is altered in a subset of Wilms' tumors and encodes a transcription factor with four zinc fingers. Here we describe the isolation of native WT1 protein from frozen normal human kidney and Wilms' tumor samples. Through size exclusion chromatography and Western blot analysis we determined the elution pattern of WT1. The majority of WT1 from adult kidney and Wilms' tumor specimens was found to elute at a size of approximately 120 kDa, consistent with a WT1 homodimer and some WT1 protein was also found in a higher molecular weight complex. In 14 week fetal kidney the majority of the WT1 protein eluted at a size of 80 kDa, suggesting that at this developmental stage the WT1 protein is not present as a homodimer. The identity of complexing partners can now be studied using this approach.

Bilateral Wilms tumor in a boy with severe hypospadias and cryptochidism due to a heterozygous mutation in the WT1 gene.

Mutations in the WT1 gene causing Wilms tumors were first reported in WAGR syndrome (Wilms tumor, Aniridia, Genitourinary malformation, mental Retardation) and Denys Drash syndrome (pseudohermaphroditism, Wilms tumor, nephropathy), but only in a few patients with hypospadias and cryptorchidism without other signs of Denys Drash (DDS) or WAGR syndrome WT1 mutations were identified. We report a boy, who was born in 1989 with hypospadias and bilateral cryptorchidism. Previous karyotyping and endocrine studies had ruled out any known cause of male pseudohermaphroditism. Subsequently, he developed a bilateral Wilms tumor, which was detected by palpation at the age of 15 months during a routine visit by the general pediatrician. Because of its extensive size, surgery and chemotherapy were needed for treatment. Analysis of the WT1 gene was performed 5 y after diagnosis and revealed a C to T transition in one allele generating a stop codon at codon 362 and subsequently leading to a truncated protein with loss of its ability to bind to DNA. No signs of DDS or WAGR syndrome are present in the boy. The work up of this patient and the so far known few comparable cases from the literature lead to the conclusion that in newborns with severe urogenital malformations not due to known chromosomal or endocrine disorders mutational screening of the WT1 gene should be performed, to evaluate the high risk of developing a Wilms tumor. We favor mutational screening in these patients as an easy tool for investigation, because in the future it will probably decrease the necessity of frequent control visits in patients without a WT1 mutation.

Genomic structure, alternative transcripts and chromosome location of the human LIM domain binding protein 1 gene LDB1.

By protein interaction screening using a radioactive LMO2 protein probe we have isolated a LIM domain binding protein. The gene shows high homology to independently isolated genes from mouse, Xenopus and Drosophila called Ldb1/Nli/Clim-2, Xldb1 and Chip, respectively. The human and mouse genes differ by only two amino acids, suggesting that the gene that we have isolated is the human homologue. Here we describe the genomic organization, alternative transcript forms and the chromosome mapping of the human gene LDB1 (alias NLI). The gene is spread over at least 12 kb and has 11 exons. Preceding the described ATG initiation site in the mouse a highly conserved region between mouse, chicken and human was detected with a second possible in frame initiation site coding for further 36 amino acids. An alternative splice site adding six nucleotides corresponding to the addition of two amino acids at the end of exon 10 was found. The gene was mapped to chromosome 10q24-->q25 by in situ hybridization, a region frequently deleted in many types of cancer. Fine mapping with a radiation hybrid panel localized the gene in the interval between the markers D10S603 and D10S540.