Supplementary Material for: <b><i>DAXX</i></b>, <b><i>ATRX</i></b>, and MSI in PanNET and Their Metastases: Correlation with Histopathological Data and Prognosis

<b><i>Introduction:</i></b> Studies on pancreatic neuroendocrine tumors (PanNETs) regarding loss of <i>ATRX</i>, <i>DAXX</i>, or frequency of microsatellite instability (MSI) show inconclusive results. So far, data on corresponding metastaseshave not been published. <b><i>Methods:</i></b> We performed immunohistochemistry (IHC) of <i>ATRX</i>, <i>DAXX</i>, <i>MSH2</i>, <i>MSH6</i>, <i>MLH1</i>, and <i>PMS2</i> on 74 PanNETs and 19 metastases. <i>ATRX-</i> and <i>DAXX</i>-negative PanNETs were further sequenced for mutations. We used polymerase chain reaction for MSI on cases with IHC loss of <i>MSH2</i>, <i>MSH6</i>, <i>MLH1</i>, and <i>PMS2</i>. <b><i>Results:</i></b> Immunohistochemical loss of <i>DAXX</i> and <i>ATRX</i> was observed in 8/74 (11%) and 6/74 (8%) PanNETs. Loss of <i>DAXX</i> immunoreactivity was statistically associated with higher tumor grade and showed a tendency toward a decreased overall survival. Sequencing of <i>DAXX-</i> (7/11 [64%]) and <i>ATRX-</i>negative (5/11 [45%]) PanNETs revealed a mutation in 6/7 (86%) and 2/5 (40%). The specificity of immunohistochemical loss of <i>DAXX</i> and <i>ATRX</i> for mutation was 80% and 67%, respectively. The expression status of <i>DAXX</i> compared to primary tumor differs in 2/12 (17%) lymph node metastases. We further identified 3/74 (4%) tumors as MSI, associated with a poor prognosis. <b><i>Discussion/Conclusion:</i></b> Our study supports the hypothesis that a loss of <i>DAXX</i> immunoreactivity can identify a more aggressive subtype of PanNET with high confidence, while <i>ATRX</i> loss is a weaker indicator. Our results also strengthen the role of <i>DAXX</i> immunolabeling as a prognostic marker. We could show that <i>ATRX</i> might be less suitable as a surrogate for sequencing. Our results indicate that IHC of <i>DAXX</i> and <i>ATRX</i> may identify PanNET subtypes as targets for more aggressive therapy.<b><i>引言：</i></b>目前针对胰腺神经内分泌肿瘤（pancreatic neuroendocrine tumors, PanNETs）中ATRX、DAXX缺失或微卫星不稳定（microsatellite instability, MSI）发生频率的相关研究结论尚不明确，截至目前，尚未有关于其对应转移灶的相关数据发表。<b><i>方法：</i></b>本研究对74例胰腺神经内分泌肿瘤组织及19例转移灶组织进行了ATRX、DAXX、MSH2、MSH6、MLH1及PMS2的免疫组织化学（immunohistochemistry, IHC）检测。对ATRX阴性及DAXX阴性的胰腺神经内分泌肿瘤样本进一步开展基因突变测序。针对MSH2、MSH6、MLH1及PMS2免疫组化结果缺失的病例，采用聚合酶链反应（polymerase chain reaction, PCR）检测其微卫星不稳定情况。<b><i>结果：</i></b>74例胰腺神经内分泌肿瘤中，分别有8例（11%）、6例（8%）出现DAXX与ATRX的免疫组化缺失。DAXX免疫反应性缺失与更高的肿瘤分级存在统计学关联，且呈现出总生存期缩短的趋势。对11例DAXX阴性胰腺神经内分泌肿瘤中的7例（占比64%）以及11例ATRX阴性胰腺神经内分泌肿瘤中的5例（占比45%）进行测序后发现，分别有6例（86%）与2例（40%）存在基因突变。DAXX与ATRX免疫组化缺失对基因突变检测的特异性分别为80%与67%。在12例淋巴结转移灶中，有2例（17%）的DAXX表达状态与原发肿瘤存在差异。本研究还发现74例肿瘤中有3例（4%）为微卫星不稳定型，这类肿瘤预后较差。<b><i>讨论与结论：</i></b>本研究支持以下假说：DAXX免疫反应性缺失可高置信度地识别出恶性程度更高的胰腺神经内分泌肿瘤亚型，而ATRX缺失则是相对较弱的预后指标。本研究结果进一步强化了DAXX免疫标记作为预后标志物的价值。我们证实ATRX或许并不适合作为基因突变测序检测的替代指标。本研究结果提示，对DAXX与ATRX进行免疫组化检测，可用于区分胰腺神经内分泌肿瘤亚型，从而为更具针对性的强化治疗提供潜在靶点。