Loss of ATRX and DAXX in pancreatic neuroendocrine tumors: Association with recurrence risk, cellular phenotype, and heterogeneity.

Pancreatic neuroendocrine tumors (PanNETs) comprise a heterogeneous group of neoplasms in terms of biological behavior. This study aims to develop a practical algorithm based on emerging biomarkers, including chromatin-remodeling molecules DAXX/ATRX/H3K36me3, in conjunction with established prognostic factors, such as WHO grade and size. In immunohistochemical analyses, 18 of the 111 (16.2%) primary PanNETs showed DAXX or ATRX loss in a mutually exclusive manner. DAXX/ATRX loss was significantly correlated with higher recurrence risk and better predicted postoperative recurrence than WHO grade. We proposed a novel algorithm for stratifying patients with resectable PanNET into three groups according to recurrence risk: (A) WHO Grade 1 and ≤2 cm (very low-risk); for the others, (B) retained DAXX/ATRX (low-risk) and (C) DAXX/ATRX complete/heterogeneous loss (high-risk). Furthermore, we elucidated the intratumoral heterogeneities of PanNETs. Among cases with DAXX or ATRX loss, nine cases demonstrated heterogeneous loss of expression of DAXX/ATRX/H3K36me3. The majority of cases with DAXX/ATRX loss, either homogeneous or heterogeneous loss, showed uniform α-cell-like phenotype (ARX1+/PDX1-). In cases of metastatic or recurrent tumors, the expression pattern was identical to that observed in at least part of the primary tumor. In some instances, the expression pattern differed among different metastatic or recurrent tumors of the same patient. In summary, we propose a clinically useful and practical algorithm for postoperative recurrence risk stratification in PanNETs, by combining DAXX/ATRX status with WHO grade and size. Moreover, our findings highlighted the frequent spatiotemporal heterogeneity of chromatin-remodeling molecule expression in PanNETs with an α-cell phenotype, offering insights into tumorigenesis.

H3K27me3 immunostaining is diagnostic and prognostic in diffuse gliomas with oligodendroglial or mixed oligoastrocytic morphology.

Oligodendroglioma is defined by IDH mutation and 1p/19q codeletion. The latter is mutually exclusive to ATRX immunohistochemical loss and has been recently associated with the loss of H3K27me3 immunostaining. We aimed to assess the diagnostic and prognostic value of H3K27me3 immuno-expression in diffuse gliomas with oligodendroglial or mixed oligoastrocytic morphology. H3K27me3 immunostaining was performed in 69 diffuse gliomas with oligodendroglial (n = 62) or oligoastrocytic (n = 7) morphology. The integration with routinely assessed IDH mutations, ATRX immunostaining, and 1p/19q codeletion classified these cases as 60 oligodendroglial and 9 astrocytic. H3K27me3 was lost in 58/60 oligodendrogliomas with retained (n = 47) or non-conclusive (n = 11) ATRX staining, 3/6 IDH-mutant astrocytomas with ATRX loss, and 3/3 IDH-wt astrocytomas. H3K27me3 was retained in 2/60 oligodendrogliomas with retained ATRX, and in 3/6 IDH-mutant astrocytomas, two of which had lost and one retained ATRX. The combination of H3K27me3 and ATRX immunostainings with IDH mutational status correctly classified 55/69 (80%) cases. In IDH-mutant gliomas, ATRX loss indicates astrocytic phenotype, while ATRX retention and H3K27me3 loss identify oligodendroglial phenotype. Only 14 (20%) IDH-mutant cases with retained ATRX and H3K27me3 or inconclusive ATRX immunostaining would have requested 1p/19q codeletion testing to be classified. Furthermore, H3K27me3 retention was associated with significantly shorter relapse-free survival (P < 0.0001), independently from IDH mutation or 1p/19q codeletion (P < 0.005). Our data suggest that adding H3K27me3 immunostaining to the diagnostic workflow of diffuse gliomas with oligodendroglial or mixed morphology is useful for drastically reducing the number of cases requiring 1p/19q codeletion testing and providing relevant prognostic information.

Loss of ATRX expression predicts worse prognosis in pulmonary carcinoid tumors.

Loss of alpha thalassemia/mental retardation syndrome X-linked (ATRX), a chromatin regulator, is associated with worse prognosis in pancreatic neuroendocrine tumors. We investigated ATRX expression in pulmonary carcinoid tumors (PCT) and its diagnostic and prognostic role in these patients. Resected PCTs (1997-2017) were reviewed. Tumors were staged according to 8th UICC/AJCC system. ATRX nuclear expression was recorded independently by 2 reviewers. A cutoff of ≤5% of nuclear ATRX expression was statistically established as loss of expression. One-hundred-fifteen patients (72 women [63%]; median age of 60.5 years [interquartile range, 50.8-71.5]) harbored 69 (60%) typical and 46 (40%) atypical PCTs. Median tumor size was 2.3 cm (interquartile range, 1.6-3.8 cm). Loss of ATRX expression was associated with atypical PCTs (OR 7.4 [95% CI, 2.6-23, P < .001]), when adjusted for lymphovascular invasion and perineural invasion. ATRX expression predicted atypical PCT with sensitivity of 37% (95% CI, 24%-52%), specificity of 92% (95% CI, 86%-98%), AUC of 0.62 (95% CI, 0.52-0.72). Loss of ATRX expression was associated with shorter disease-specific survival (HR = 11, 95% CI, 1.8-68, P = .01), after adjusting for lymphovascular invasion and presence of metastatic disease at time of diagnosis. Interobserver agreement on ATRX expression by two reviewers was substantial (κ = 0.72 [95% CI, 0.60-0.80]). ATRX expression is more commonly lost in atypical than in typical PCT, and is associated with more aggressive tumor characteristics and shorter disease-specific survival.

Immunohistochemistry for ATRX Can Miss ATRX Mutations: Lessons From Neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood with a median age of presentation of 17 months. A common theme in high-risk neuroblastoma is maintenance of telomeres, one mechanism for which involves alternate lengthening of telomeres (ALT) associated with ATRX gene mutations. Mutations are believed to result in loss of ATRX protein, and therefore immunohistochemistry is used to detect mutations. We screened 133 cases of neuroblastoma by ATRX immunohistochemistry, and found 9 cases with partial to total absence of ATRX. Sequencing for ATRX mutations detected a mutation in 1 of 9 cases, suggesting immunostaining was not reliable for detecting mutations. To correlate immunostaining with ALT, fluorescence in situ hybridization (FISH) for ALT was performed in 6 of these cases and 5 (from 4 patients) showed ALT, implying impaired ATRX protein function, despite the failure to identify a mutation. Two other cases with large deletions in the ATRX gene showed diffusely positive staining for ATRX protein but showed ALT by FISH. Four of the 6 patients with ALT-positive tumors were over 5 years old. Therefore, 29 additional patients 5 years old and above with ATRX-positive tumors were screened for ALT by FISH and 6 additional cases with ALT were detected, bringing the total to 29% (10/34) of children 5 years old and above, 70% of which showed positive ATRX immunohistochemistry. Patients with ATRX mutations in neuroblastoma tend to have a more chronic and progressive course of disease. Screening neuroblastoma tumors at diagnosis for ATRX mutations may help identify patients who might benefit from personalized therapy directed against ALT. However, relaying on negative immunohistochemistry for ATRX protein to identify ALT in neuroblastoma may miss a significant proportion of patients. The addition of FISH for ALT as part of the diagnostic workup, especially for older children (5 y old and above), would help ensure that patients are correctly identified for anti-ALT therapy.

Perfusion MRI grading diffuse gliomas: Impact of permeability parameters on molecular biomarkers and survival.

BACKGROUND AND PURPOSE: Our objectives were: (1) compare dynamic susceptibility-weighted (DSC) and dynamic contrast-enhanced (DCE) permeability parameters, (2) evaluate diagnostic accuracy of DSC and DCE discriminating high- and low-grade tumors, (3) analyze relationship of permeability parameters with overall (OS) and progression-free survival (PFS) and (4) assess differences in high-grade tumors classified according to molecular biomarkers. MATERIALS AND METHODS: 49 patients with histologically proved diffuse gliomas underwent DSC and DCE imaging. Parametric maps of cerebral blood volume (CBV), CBV-leakage corrected, volume transfer coefficient (Ktrans), fractional volume of the extravascular extracellular space (EES) (Ve), fractional blood plasma volume (Vp) and rate constant between EES and blood plasma (Kep) were calculated. High-grade gliomas were also classified according to isocitrate dehydrogenase (IDH), alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-dna-methyltransferase promoter methylation (MGMT) status. RESULTS: There is correlation between parameters leakage, Ktrans and Vp. ROC curve analysis showed significance in both Ktrans and Ve for glioma grading. Threshold value of 0.075 for Ve generated the best combination of sensitivity (80%) and specificity (75%) in tumor gradation. Leakage was the only permeability parameter related to OS (P=0.006) and PFS (0.012); with prolonged survival for leakage values lower than 1.2. IDH-mutated high-grade tumors showed lower leakage and Ktrans values. High-grade tumors with loss of ATRX presented lower leakage and Vp values. CONCLUSIONS: Both DSC and DCE permeability parameters serve as non-invasive method for glioma grading. Leakage was the unique permeability parameter related to survival and the best discriminating high-grade gliomas classified according to IDH and ATRX status.

ATRX loss is an independent predictor of poor survival in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms accounting for 1% to 2% of all pancreatic tumors. The biological behavior of PanNETs is heterogeneous and unpredictable, adding to the difficulties of clinical management. The DAXX (death domain associated protein) and ATRX (α-thalassemia/mental retardation syndrome X-linked) genes encode proteins involved in SWI/SNF-like chromatin remodeling. Somatic inactivating mutations in DAXX and ATRX are frequent in PanNETs, mutually exclusive, and associated with telomere dysfunction, resulting in genomic instability and alternate lengthening of telomeres. We sought to assess the clinical significance of the loss of the ATRX and DAXX proteins as determined by immunohistochemistry (IHC) in patients with PanNET. From an unselected cohort of 105 patients, we found ATRX loss in 10 tumors (9.5%) and DAXX loss in 16 (15.2%). DAXX and ATRX losses were confirmed mutually exclusive and associated with other adverse clinicopathological variables and poor survival in univariate analysis. In addition, ATRX loss was also associated with higher AJCC stage and infiltrative tumor borders. However, only ATRX loss, lymphovascular invasion, and perineural spread were independent predictors of poor overall survival in multivariate analysis. In conclusion, loss of expression of ATRX as determined by IHC is a useful independent predictor of poor overall survival in PanNETs. Given its relative availability, ATRX loss as determined by IHC may have a role in routine clinical practice to refine prognostication in patients with PanNET.

Phosphorylated Hsp27 is mutually exclusive with ATRX loss and the IDH1R132H mutation and may predict better prognosis among glioblastomas without the IDH1 mutation and ATRX loss.

AIM: To identify biomarkers for accurate classification of glioma. PATIENTS AND METHODS: We evaluated the heat shock protein 27 (Hsp27), phosphorylated Hsp27 (p-Hsp27), ATRX and IDH1R132Hproteins using immunohistochemistry in 421 glioma tissues. The χ2 test was used to assess the relationship between molecular alterations and clinico-pathological parameters. Kaplan-Meier survival curves were constructed, and differences were detected by the log-rank test. RESULTS: We found that Hsp27 and p-Hsp27 were mainly expressed in aggressive astrocytic gliomas. However, neither Hsp27 nor p-Hsp27 expression was related to survival time for any grade of glioma. Interestingly, p-Hsp27 was mutually exclusive with ATRX loss (ATRX-) and the IDH1R132H mutation, except for one case of anaplastic astrocytoma. We classified glioblastomas (GBMs) into three subtypes: ATRX-/IDH1R132H, high p-Hsp27 expression (p-Hsp27+) and none of these three markers. ATRX-/IDH1R132Hshowed the longest median survival (19.6 months). The prognostic difference between p-Hsp27+ and none of these three markers was significant (15.0 vs 13.1 months, P=0.045). Moreover, p-Hsp27+ predicted better sensitivity for standard therapy among GBMs without the IDH1 mutation and ATRX loss (26.3 vs 15.5 months, P=0.008). CONCLUSION: p-Hsp27 is a novel biomarker of glioma and might have important clinical value for further classification of patients with wild-type IDH1 and normal ATRX expression, for evaluating prognosis and for guidance for adjuvant therapy.

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.

Radiation-induced gliomas: a report of four cases and analysis of molecular biomarkers.

Radiation-induced glioma (RIG) is a rare secondary glioma. The tumors morphologically resemble their sporadically arising counterparts. Recently, the WHO classification of tumors of the central nervous system was revised to incorporate molecular biomarkers together with classic histological features. The status of molecular biomarkers in RIG, however, remains unclear. The objective of this study was to investigate if commonly accepted glioma-specific biomarkers are relevant in RIGs. Among 269 gliomas diagnosed as WHO grade 2, 3 and 4 in our institution, four were diagnosed as RIGs. Immunohistochemical (IHC) staining for isocitrate dehydrogenase 1 (IDH1), p53, alpha thalassemia/mental retardation syndrome X-linked (ATRX), and H3K27M, and direct DNA sequencing of IDH1/2, telomerase reverse transcriptase (TERT) promoter, Histone H3.3 (H3F3A) and B-Raf (BRAF) genes was performed. All tumor specimens were IDH1-, p53- and H3K27M-negative. The nuclei of tumor cells in all cases exhibited positive staining for ATRX. In direct DNA sequencing analysis, no IDH1, IDH2, TERT promoter, H3F3A or BRAF mutations were found in any of the cases. Our findings suggest that these characteristic glioma-associated molecular mutations may be rare events in RIGs. More RIGs need to be tested for analysis of molecular biomarkers to clarify the clinical and histopathological spectra of this tumor.

Modified rapid immunohistochemical staining for intraoperative diagnosis of malignant brain tumors.

Rapid immunohistochemistry (R-IHC) has been developing mainly as a support technique in the rapid diagnosis of central nervous system tumors; however, there have been problems regarding instability in specimen preparation and immunostaining. To overcome the weakness of this technology, the instability of immunostaining, we developed a modified R-IHC. This was achieved by switching to 4% paraformaldehyde as the fixative solution and utilizing a commercially available Polymer Refine Detection Kit, as a high-sensitivity kit, in place of the secondary antibodies. In this study, we tested the modified R-IHC by evaluating rapid immunostaining on new staining items in 94 brain tumor removal cases, which took place at Tokyo Women's Medical University from 2014 to 2015. The results showed that, based on GFAP and p53 markers, the modified method obtained a higher stability in specimens than the standard rapid immunostaining method. It also achieved stainability on the same level as that of a permanent specimen. The modified method tested 86.6% (46/53) and 82.8% (24/29) in pHH3 and ATRX, respectively, in the percentage of correct classification (PCC) against the permanent specimens, and 100% (7/7) in the PCC against malignant lymphomas and gliomas that used CD20/CD3 for discrimination. We concluded that the modified R-IHC method indicated a higher stainability and PCC against the permanent specimens in comparison to the standard method in GFAP, p53, CD20/CD3, pHH3, and ATRX.

Molecular classification of adult diffuse gliomas: conflicting IDH1/IDH2, ATRX, and 1p/19q results.

Until recently, the diagnosis of brain tumors was primarily based on microscopic examination of hematoxylin and eosin-stained tissue sections. The updated World Health Organization (WHO) Classification of Tumours of the Central Nervous System incorporates genetic alterations into the classification system, with the goal of creating more homogenous disease categories with greater prognostic value. Hence, under the new classification system, the diagnosis of diffuse gliomas incorporates the evaluation of mutations in the IDH1 and IDH2 genes and simultaneous deletion of chromosomes 1p and 19q. For example, although under the 2007 WHO classification system, oligodendrogliomas could be diagnosed based solely on the presence of characteristic histologic features, the newly molecularly defined entity of "oligodendroglioma, IDH-mutant and 1p/19q codeleted" requires the presence of both an IDH1 or IDH2 mutation and 1p/19q codeletion. Given that diagnosis requires evaluation of critical genetic alterations, molecular diagnostics is becoming an increasingly important aspect of clinical oncologic neuropathology practice. As molecular testing is applied more frequently to the diagnosis of brain tumors, inconsistent or conflicting molecular information will create diagnostic challenges. Here we present 6 cases of diffuse glioma that presented a diagnostic challenge due to conflicting molecular testing results. These cases exemplify some of the potential complications that arise when introducing the new 2016 central nervous system WHO classification system diagnostic criteria into routine clinical practice. We aim to alert the general practice pathology community to these potential conflicts to help mitigate the risk of potential misdiagnosis.