Death domain-associated protein (DAXX) expression is associated with poor survival in metastatic high-grade serous carcinoma.

The objective of this study was to analyze the expression and clinical role of mitosis regulators α-thalassemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) in metastatic high-grade serous carcinoma (HGSC). ATRX and DAXX protein expression by immunohistochemistry was analyzed in 400 HGSC effusions. DAXX expression was additionally studied in 15 cancer cell lines, including 4 ovarian carcinoma lines, and in 81 of the 400 HGSC effusions using Western blotting. ATRX and DAXX were expressed in HGSC cells in 386/400 (96%) and 348/400 (87%) effusions, respectively. Western blotting showed DAXX expression in all 15 cell lines and in 70/81 (86%) HGSC effusions. DAXX expression by immunohistochemistry was higher in pleural compared to peritoneal effusions (p = 0.006) and in post-chemotherapy compared to pre-chemotherapy effusions (p = 0.004), and its expression was significantly associated with poor overall survival in univariate of the entire cohort (p = 0.014), as well as analysis limited to chemo-naïve effusions tapped at diagnosis (p = 0.038). The former association retained its prognostic role in Cox multivariate survival analysis (p = 0.011). ATRX expression was unrelated to clinicopathologic parameters or survival. In conclusion, DAXX is associated with disease progression and could be a prognostic marker in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Telomere length alterations and ATRX/DAXX loss in pituitary adenomas.

Telomeres are nucleoprotein complexes located at the termini of eukaryotic chromosomes that prevent exonucleolytic degradation and end-to-end chromosomal fusions. Cancers often have critically shortened, dysfunctional telomeres contributing to genomic instability. Telomere shortening has been reported in a wide range of precancerous lesions and invasive carcinomas. However, the role of telomere alterations, including the presence of alternative lengthening of telomeres (ALT), has not been studied in pituitary adenomas. Telomere length and the presence of ALT were assessed directly at the single cell level using a telomere-specific fluorescence in situ hybridization assay in tissue microarrays. Tumors were characterized as either ALT-positive or having short, normal, or long telomere lengths and then these categories were compared with clinicopathological characteristics. ATRX and DAXX expression was studied through immunohistochemistry. We characterized a discovery set of 106 pituitary adenomas including both functional and nonfunctional subsets (88 primary, 18 recurrent). Telomere lengths were estimated and we observed 64 (59.4%) cases with short, 39 (36.8%) cases with normal, and 0 (0%) cases with long telomeres. We did not observe significant differences in the clinicopathological characteristics of the group with abnormally shortened telomeres compared to the group with normal telomeres. However, three pituitary adenomas were identified as ALT-positive of which two were recurrent tumors. Two of these three ALT-positive cases had alterations in either of the chromatin remodeling proteins, ATRX and DAXX, which are routinely altered in other ALT-positive tumor subtypes. In a second cohort of 32 recurrent pituitary adenomas from 22 patients, we found that the tumors from 36% of patients (n = 8) were ALT-positive. This study demonstrates that short telomere lengths are prevalent in pituitary adenomas and that ALT-positive pituitary adenomas are enriched in recurrent disease.

The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.

All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative "C-circle" assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.

Performance of DAXX Immunohistochemistry as a Screen for DAXX Mutations in Pancreatic Neuroendocrine Tumors.

OBJECTIVES: DAXX immunohistochemistry (IHC) is often used as a surrogate for sequencing. We aimed to elucidate the sensitivity of IHC for DAXX mutation. METHODS: All pancreatic neuroendocrine tumors (PanNETs) with DAXX mutations detected by sequencing and a subset of DAXX wild-type PanNETs were analyzed for DAXX expression by IHC. RESULTS: Of 154 PanNETs with MSK-IMPACT testing, 36 (30%) harbored DAXX mutations. DAXX mutations were associated with TSC2 mutations (46% vs 10%, P < 0.0001), tended to co-occur with MEN1 mutations (63% vs 49%, P = 0.11), and tended to be mutually exclusive with ATRX mutations (11% vs 25%, P = 0.053). Of 27 available DAXX mutant PanNETs, 23 lost DAXX expression (85.2%). All 4 DAXX mutants with retained expression harbored DAXX mutations within the SUMO-interacting motif of the last exon. Telomere-specific fluorescence in situ hybridization demonstrated alternative lengthening of telomeres in all 4 cases. Of 20 PanNETs with wild-type DAXX, 19 retained DAXX IHC expression (95%). CONCLUSIONS: The sensitivity and specificity of IHC for DAXX mutation are 85% and 95%, respectively. Last exon DAXX mutant PanNETs often show alternative lengthening of telomeres despite retained DAXX expression, likely due to escape of nonmediated decay.

Clinicopathological analysis of ATRX, DAXX and NOTCH receptor expression in angiosarcomas.

AIMS: Multiple genetic alterations, including alternative lengthening of telomeres (ALT) and NOTCH mutations, have been described in angiosarcoma. Loss of α-thalassaemia/mental retardation syndrome X-linked (ATRX) and death domain-associated protein 6 (DAXX) expression is frequently associated with the ALT phenotype. Additionally, inhibition of NOTCH signalling induces the development of malignant vascular tumours in mice, indicating a tumour suppressive role of the NOTCH pathway in the pathogenesis of angiosarcoma. The aim of this study was to evaluate the immunohistochemical expression of ATRX, DAXX and NOTCH receptors (NOTCH1 and NOTCH2) in a large cohort of angiosarcomas, and study their clinicopathological and prognostic significance. METHODS AND RESULTS: One hundred and forty cases of angiosarcoma were stained for ATRX, DAXX, NOTCH1 and NOTCH2. ATRX loss (<10% labelling) was seen in seven of 118 (6%) cases, and was more frequent in deep soft tissue tumours than in other body sites (P = 0.004). Angiosarcomas with ATRX loss were associated with worse event-free survival than angiosarcomas with retained ATRX expression (P = 0.003). DAXX was retained in all specimens examined. Decreased NOTCH1 expression (≤1+ intensity) was seen in 29 of 123 (24%) cases, and was associated with a cutaneous site of origin (P = 0.013) and advanced disease (P = 0.026). NOTCH2 expression was decreased in 16 of 103 (16%) cases, was associated with visceral tumours (P = 0.001), and correlated with worse disease-specific survival (P = 0.033). CONCLUSIONS: ATRX, NOTCH1 and NOTCH2 expression varies in angiosarcomas and shows significant correlations with site of origin and poor clinical outcome, thus highlighting the biological heterogeneity within this tumour type.

A simple algorithmic approach using histology and immunohistochemistry for the current classification of adult diffuse glioma in a resource-limited set-up.

AIMS: The WHO 2016 classification of diffuse gliomas combines histological and molecular parameters for diagnosis. However, in view of cost constraints for molecular testing, an economical working formula is essential to reach a meaningful diagnosis in a resource-limited setting. The aim of this study was to establish a practical algorithmic approach using histology and immunohistochemistry (IHC) in the classification of diffuse gliomas in such a set-up. METHODS: Diffuse gliomas of WHO grade II and III diagnosed in our institute in the year 2016 were analysed for histological and IHC features, using the markers isocitrate dehydrogenase 1 (IDH1R132H) and α thalassemia/mental retardation syndrome X-linked gene (ATRX). Fluorescence in situ hybridisation (FISH) for 1p/19q co-deletion was performed when requested. RESULTS: 449 diffuse gliomas (grades II/III) were included in the study. Integrating histology and IHC features, as per the WHO 2016 guidelines, we derived the following groups: Astrocytoma, IDH-mutant (A,IDH-mt, 37.2%); astrocytoma, not otherwise specified (A,NOS, 12.7%); oligoastrocytoma, NOS (OA,NOS, 4.5%); and oligodendroglioma, NOS (ODG,NOS, 45.6%). FISH was performed in a subset of ODG,NOS, OA,NOS and A,NOS gliomas. This revealed 1p/19q co-deletion in all cases of ODG,NOS, 15.8% of OA,NOS and 37.5% of A,NOS. Sequencing for rare IDH 1/2 mutations was not carried out in this study. CONCLUSION: In a resource-limited set-up, histology with IHC (IDH1(R132H) and ATRX) form the baseline to reasonably derive four histomolecular subgroups of diffuse glioma. Of these, we recommend, OA,NOS and IDH1(R132H)-non-mt ODG,NOS to be our priority for performing 1p/19q co-deletion studies in comparison to IDH-mt ODG,NOS, and it would not be mandatory for astrocytoma. Sequencing for rare IDH mutations is advised for A,NOS and OA,NOS groups, but not for the IDH1(R132H)-non-mutant diffuse gliomas with 1p/19q co-deletion.