Distal muscle weakness and optic atrophy without central nervous system involvement in a patient with a homozygous missense mutation in the C19ORF12-gene.

Variants of the C19ORF12-gene have been described in patients with spastic paraplegia type 43 and in patients with mitochondrial membrane protein-associated neurodegeneration (MPAN), a subtype of neurodegeneration associated with brain iron accumulation (NBIA). In both subtypes optic atrophy and neuropathy have been frequently described. This case report describes a patient with bilateral optic atrophy and severe distal muscle weakness based on motor neuropathy without involvement of the central nervous system. Exome sequencing revealed a homozygous pathogenic missense variant (c.187G>C;p.Ala63Pro) of the C19ORF12-gene while iron deposits were absent on repeat MR-imaging of the brain, thus showing that peripheral neuropathy and optic neuropathy can be the sole manifestations of the C19ORF12-related disease spectrum whereby iron accumulation in the brain may be absent.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

Clinical and biological implications of ancestral and non-ancestral IDH1 and IDH2 mutations in myeloid neoplasms.

Mutations in isocitrate dehydrogenase 1/2 (IDH1/2(MT)) are drivers of a variety of myeloid neoplasms. As they yield the same oncometabolite, D-2-hydroxyglutarate, they are often treated as equivalent, and pooled. We studied the validity of this approach and found IDH1/2 mutations in 179 of 2119 myeloid neoplasms (8%). Cross-sectionally, the frequencies of these mutations increased from lower- to higher risk disease, thus suggesting a role in clinical progression. Variant allelic frequencies indicated that IDH1(MT) and IDH2(MT) are ancestral in up to 14/74 (19%) vs 34/99 (34%; P=0.027) of cases, respectively, illustrating the pathogenic role of these lesions in myeloid neoplasms. IDH1/2(MT) was associated with poor overall survival, particularly in lower risk myelodysplastic syndromes. Ancestral IDH1(MT) cases were associated with a worse prognosis than subclonal IDH1(MT) cases, whereas the position of IDH2(MT) within clonal hierarchy did not impact survival. This may relate to distinct mutational spectra with more DNMT3A and NPM1 mutations associated with IDH1(MT) cases, and more ASXL1, SRSF2, RUNX1, STAG2 mutations associated with IDH2(MT) cases. Our data demonstrate important clinical and biological differences between IDH1(MT) and IDH2(MT) myeloid neoplasms. These mutations should be considered separately as their differences could have implications for diagnosis, prognosis and treatment with IDH1/2(MT) inhibitors of IDH1/2(MT) patients.

AKT1(E17K) in human solid tumours.

The serine-threonine kinase AKT1 is a central player in the oncogenic pathway controlled by PI3K. Recently, a somatic mutation in AKT1 (E17K) has been detected in breast, colorectal, lung and ovarian cancers. The E17K change results in constitutive AKT1 activation and induces leukaemia in mice. We determined the occurrence of the E17K variant in a panel of 764 tumour samples. These included breast, lung, ovarian, colorectal and pancreatic carcinomas as well as melanomas and glioblastomas. Despite the fact that these tumours are known to bear alterations in genes involved in the PI3K signalling pathway, AKT1(E17K) was detected only in breast (16/273), colorectal (1/88) and lung (1/155) cancers. Within the neoplasms of breast origin, the AKT1(E17K) variant was mutually exclusive with respect to the PIK3CA(E454K or H1047R) alleles and was present only in ductal and lobular histotypes. Our results, showing that AKT1 mutations seem to occur in a tissue-specific fashion have basic and clinical implications. First, the activity of mutated AKT1 in oncogenic PI3K signalling could be strictly dependent on the cell and tissue milieu. Second, therapeutic efforts aimed at selective targeting the AKT1(E17K) variant could be effective mainly in specific cancer types.

[Subtle skin lesions in the midline as an indication of a neurodermal closing defect]. / Subtiele huidafwijkingen in de mediaanlijn als aanduiding van een neurodermaal sluitingsdefect.

Three patients, aged 2, 2.5 and 24 years, presented with a skin lesion in the median or paramedian area at the nose, in the lumbar region and between the shoulders, respectively. The first patient suffered from recurrent infection, the skin lesion of the second patient was a coincidental finding, and the third patient had cosmetic problems. Although there were no signs of neurological deficits, neurodermal closing defects were found by MRI. Two patients underwent surgery to prevent infection and neurological complaints in the future. It is stressed that even if there are no neurological signs or infections, congenital dermal lesions situated in the midline should be considered as possible neural tube defects and therefore analysed by MRI. Ifa neurodermal dysraphism is found, patients should be referred to a neurosurgical centre. A good clinical assessment of the neural lesion, clinical signs and age will determine whether surgical resection is indicated to prevent or resolve neurological problems, infections or cosmetic complaints.

Direct comparison of the sensitivity of enzyme histochemical and immunohistochemical methods: cathepsin B expression in human colorectal mucosa.

Immunohistochemical localization of the proteinase cathepsin B has been compared directly with localization of cathepsin B activity with a catalytic (enzyme) histochemical method. The 2 approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is active or not whereas the catalytic method visualizes the functionally active enzyme only. Sensitivity of both approaches to localize low amounts of enzyme protein or activity has never been compared. In the present study, we show that cathepsin B protein has a wider distribution pattern than cathepsin B activity in human colorectal mucosa, which means that inactive cathepsin B protein is present. With respect to sensitivity of the methods, it is shown that cathepsin B protein could only be demonstrated properly when strong signal amplification was applied by using Nanogold with silver enhancement, whereas activity could be demonstrated with a simple and direct fluorogenic histochemical assay. It is concluded that catalytic histochemical methods are relatively simple methods for the localization of activity of enzymes in tissues and cells and that their sensitivity is high in comparison with immunohistochemical methods.

Comparative localization of cathepsin B protein and activity in colorectal cancer.

Cathepsin B is a lysosomal cysteine proteinase that may participate in cancer progression. We compared localization of its protein and activity during progression of human colorectal cancer. In adenomas and carcinomas, protein expression and, particularly, activity were elevated compared with those in normal colorectal mucosa. In normal mucosa, cathepsin B protein expression was moderate in stroma and variable in epithelium, whereas activity was mainly present in distinct areas of stroma directly underneath the surface of the colon and in epithelium at the surface of the colon. Stroma in adenomas and carcinomas contained moderate to high protein levels but little activity except for areas of angiogenesis, inflammation, and necrosis, in which activity was high. In adenomas and the majority of well-differentiated carcinomas and moderately differentiated carcinomas, cathepsin B protein and activity were found in granular form in the epithelium, close to the basement membrane. Protein and activity levels were low and diffusely distributed in cancer cells in the remainder of the well-differentiated and moderately differentiated carcinomas and in all poorly differentiated carcinomas. Invasive fronts in most cancers contained moderate protein levels but high activity. We conclude that (a) activity localization is essential to understand the role of cathepsin B in cancer progression, and (b) cathepsin B activity in human colon is associated with invasion of cancer cells, endothelial cells, and inflammatory cells, and in cell death, both apoptotic and necrotic.