Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical-risk profiles. Presentation, age-dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609?620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected.

Early occurrence of lung adenocarcinoma and breast cancer after radiotherapy of a chest wall sarcoma in a patient with a de novo germline mutation in TP53

We report a 26-year-old female patient who was diagnosed within 4 years with chest sarcoma, lung adenocarcinoma, and breast cancer. While her family history was unremarkable, DNA sequencing of TP53 revealed a germline de novo non-sense mutation in exon 6 p.Arg213X. One year later, she further developed a contralateral ductal carcinoma in situ, and 18 months later a jaw osteosarcoma. This case illustrates the therapeutic pitfalls in the care of a young cancer patient with TP53 de novo germline mutations and the complications related to her first-line therapy. Suggestion is made to use the less stringent Chompret criteria for germline TP53 mutation screening. Our observation underlines the possibly negative effect of radiotherapy in generating second tumors in patients with a TP53 mutation. We also present a review of six previously reported cases, comparing their cancer phenotypes with those generally produced by TP53 mutations.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome

Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.

Origin and prognostic value of circulating KRAS mutations in lung cancer patients

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

Germline NF1 mutational spectra and loss-of-heterozygosity analyses in patients with pheochromocytoma and neurofibromatosis type 1

BACKGROUND: Neurofibromatosis type 1 (NF1) is a pheochromocytoma-associated syndrome. Because of the low prevalence of pheochromocytoma in NF1, we ascertained subjects by pheochromocytoma that also had NF1 in the hope of describing the germline NF1 mutational spectra of NF1-related pheochromocytoma. MATERIALS AND METHODS: An international registry for NF1-pheochromocytomas was established. Mutation scanning was performed using denaturing HPLC for intragenic variation and quantitative PCR for large deletions. Loss-of-heterozygosity analysis using markers in and around NF1 was performed. RESULTS: There were 37 eligible subjects (ages 14-70 yr). Of 21 patients with corresponding tumor available, 67% showed somatic loss of the nonmutated allele at the NF1 locus vs. 0 of 12 sporadic tumors (P = 0.0002). Overall, 86% of the 37 patients had exonic or splice site mutations, 14% large deletions or duplications; 79% of the mutations are novel. The cysteine-serine rich domain (CSR) was affected in 35% but the RAS GTPase activating protein domain (RGD) in only 13%. There did not appear to be an association between any clinical features, particularly pheochromocytoma presentation and severity, and NF1 mutation genotype. CONCLUSIONS: The germline NF1 mutational spectra comprise intragenic mutations and deletions in individuals with pheochromocytoma and NF1. NF1 mutations tended to cluster in the CSR over the RAS-GAP domain, suggesting that CSR plays a more prominent role in individuals with NF1-pheochromocytoma than in NF1 individuals without this tumor. Loss-of-heterozygosity of NF1 markers in NF1-related pheochromocytoma was significantly more frequent than in sporadic pheochromocytoma, providing further molecular evidence that pheochromocytoma is a true component of NF1.

Somatic CEBPA mutations are a frequent second event in families with germline CEBPA mutations and familial acute myeloid leukemia

PURPOSE: The transcription factor CCAAT/enhancer binding protein-alpha (CEBPA) is crucial for normal myeloid differentiation. Mutations in the CEBPA gene are found in subsets of patients with acute myeloid leukemia (AML). Recently, three families were reported in whom several family members had germline CEBPA mutations and subsequently developed AML. Whereas familial AML is considered a rare event, the frequency of CEBPA germline mutations in AML is not known. PATIENTS AND METHODS: In this study, we screened 187 consecutive AML patients for CEBPA mutations at diagnosis. We detected 18 patients (9.6%) with CEBPA mutations. We then analyzed remission samples and constitutive DNA from these patients. RESULTS: We found that two (11.1%) of 18 AML patients with CEBPA mutations carried a germline N-terminal frameshift CEBPA mutation. Interestingly, additional members in the families of both of these patients have been affected by AML, and the germline CEBPA mutations were also observed in these patients. Additional somatic mutations in AML patients with germline CEBPA mutations in the two families comprised in-frame C-terminal CEBPA mutations in two patients, two nonsilent CEBPA point mutations in one patient, and monosomy 7 in one patient. CONCLUSION: This study shows, for the first time to our knowledge, that germline CEBPA mutations are frequently observed among AML patients with CEBPA mutations. Including the families with germline CEBPA mutations reported previously, additional somatic CEBPA mutations represent a frequent second event in AML with germline CEBPA mutations. Our data strongly indicate that germline CEBPA mutations predispose to AML and that additional somatic CEBPA mutations contribute to the development of the disease.

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

BACKGROUND: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. OBJECTIVE: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. METHODS AND RESULTS: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. CONCLUSIONS: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.

An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis

BACKGROUND: Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series. METHODS: Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing. FINDINGS: SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively. INTERPRETATION: Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome. FUNDING: The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Santé et de la Recherche Médicale, and a PHRC grant COMETE 3 for the COMETE network.

Neoadjuvant chemoradiotherapy with or without panitumumab in patients with wild-type KRAS, locally advanced rectal cancer (LARC): a randomized, multicenter, phase II trial SAKK 41/07

BACKGROUND We conducted a randomized, phase II, multicenter study to evaluate the anti-epidermal growth factor receptor (EGFR) mAb panitumumab (P) in combination with chemoradiotherapy (CRT) with standard-dose capecitabine as neoadjuvant treatment for wild-type KRAS locally advanced rectal cancer (LARC). PATIENTS AND METHODS Patients with wild-type KRAS, T3-4 and/or N+ LARC were randomly assigned to receive CRT with or without P (6 mg/kg). The primary end-point was pathological near-complete or complete tumor response (pNC/CR), defined as grade 3 (pNCR) or 4 (pCR) histological regression by Dworak classification (DC). RESULTS Forty of 68 patients were randomly assigned to P + CRT and 28 to CRT. pNC/CR was achieved in 21 patients (53%) treated with P + CRT [95% confidence interval (CI) 36%-69%] versus 9 patients (32%) treated with CRT alone (95% CI: 16%-52%). pCR was achieved in 4 (10%) and 5 (18%) patients, and pNCR in 17 (43%) and 4 (14%) patients. In immunohistochemical analysis, most DC 3 cells were not apoptotic. The most common grade ≥3 toxic effects in the P + CRT/CRT arm were diarrhea (10%/6%) and anastomotic leakage (15%/4%). CONCLUSIONS The addition of panitumumab to neoadjuvant CRT in patients with KRAS wild-type LARC resulted in a high pNC/CR rate, mostly grade 3 DC. The results of both treatment arms exceeded prespecified thresholds. The addition of panitumumab increased toxicity.