A case of mucoepidermoid carcinoma arising in mature cystic teratoma

卵巣粘表皮癌は卵巣悪性腫瘍の中で極めてまれな組織型に分類される。今回、我々は成熟嚢胞性奇形種より発生した卵巣粘表皮癌の症例を経験したので報告する。症例は、69歳、女性、両側の成熟嚢胞性奇形腫を認めたが、SCC 高値とCT、MRI にて左側の腫瘍内に造影される充実性部分を認めたこと、小腸に浸潤を疑う所見を認めたこと、から悪性転化を疑い、手術を施行した。開腹時、両側卵巣腫瘍を認め、左卵巣腫瘍はS状結腸と強固に癒着していた。卵巣腫瘍充実性部分の迅速病理にて低分化癌と診断し、単純子宮全摘出術、両側付属器摘出術、S状結腸合併切除、骨盤リンパ節郭清術、大網切除術を施行した。病理組織学的には、左卵巣腫瘍の嚢胞壁肥厚部に皮膚付属器、脂肪織、軟骨組織、リンパ球集簇、卵巣間質を認め、充実成分に低分化な浸潤性扁平上皮癌を認めた。充実成分には、粘表皮癌に特徴的な、豊富な胞体粘液（PASおよびAlcian blue 染色陽性）を有する異型細胞が胞巣状~不完全な腺管状を呈する領域があり、成熟嚢胞性奇形腫より発生した卵巣粘表皮癌IIb期(pT2bN0M0)と診断した。術後補助化学療法としてDC（ドセタキセル、カルボプラチン）療法を施行し、術後1年8ヶ月現在、再発を認めない。雑誌掲載論

Urinary Polyamine Biomarker Panels with Machine-Learning Differentiated Colorectal Cancers, Benign Disease, and Healthy Controls

Colorectal cancer (CRC) is one of the most daunting diseases due to its increasing worldwide prevalence, which requires imperative development of minimally or non-invasive screening tests. Urinary polyamines have been reported as potential markers to detect CRC, and an accurate pattern recognition to differentiate CRC with early stage cases from healthy controls are needed. Here, we utilized liquid chromatography triple quadrupole mass spectrometry to profile seven kinds of polyamines, such as spermine and spermidine with their acetylated forms. Urinary samples from 201 CRCs and 31 non-CRCs revealed the N1,N12-diacetylspermine showing the highest area under the receiver operating characteristic curve (AUC), 0.794 (the 95% confidence interval (CI): 0.704–0.885, p < 0.0001), to differentiate CRC from the benign and healthy controls. Overall, 59 samples were analyzed to evaluate the reproducibility of quantified concentrations, acquired by collecting three times on three days each from each healthy control. We confirmed the stability of the observed quantified values. A machine learning method using combinations of polyamines showed a higher AUC value of 0.961 (95% CI: 0.937–0.984, p < 0.0001). Computational validations confirmed the generalization ability of the models. Taken together, polyamines and a machine-learning method showed potential as a screening tool of CRC

Mechanism of Fcγ Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

<div><p>The whole blood erythrocyte lysis method is the most common protocol of sample preparation for flow cytometry (FCM). Although this method has many virtues, our recent study has demonstrated false-positive results when surface markers of monocytes were examined by this method due to the phenomenon called Fcγ receptor (FcγR)-mediated trogocytosis. In the present study, similar FcγR-mediated trogocytosis-based false-positive results have been demonstrated when granulocytes were focused on instead of monocytes. These findings indicated that not only monocytes but also granulocytes, the largest population with FcγR expression in peripheral blood, could perform FcγR-mediated trogocytosis. Since the capacity of FcγR-mediated trogocytosis was different among blood samples, identification of factors that could regulate the occurrence of FcγR-mediated trogocytosis should be important for the quality control of FCM. Our studies have suggested that such factors are present in the serum. In order to identify the serum factors, we employed the <em>in vitro</em> model of FcγR-mediated trogocytosis using granulocytes. Investigation with this model determined the serum factors as heat-labile molecules with molecular weight of more than 100 kDa. Complements in the classical pathway were initially assumed as candidates; however, the C1 inhibitor did not yield an obvious influence on FcγR-mediated trogocytosis. On the other hand, although immunoglobulin ought to be resistant to heat inactivation, the inhibitor of human anti-mouse antibodies (HAMA) effectively blocked FcγR-mediated trogocytosis. Moreover, the inhibition rates were significantly higher in HAMA^high serum than HAMA^low serum. The collective findings suggested the involvement of heterophilic antibodies such as HAMA in the mechanism of false-positive results in FCM due to FcγR-mediated trogocytosis.</p> </div

Involvement of HAMA in FcγR-mediated trogocytosis.

<p>(<b>A</b>) Effects of HAMA blockade on FcγR-mediated trogocytosis (n = 10). Heparinized whole blood samples were added with or without 1 µl of HAMA inhibitor, TRU block, and then made to react with the PE-labeled anti-CD8α (HIT8a) and FITC-labeled anti-CD15 (H198) Abs. After depletion of erythrocytes, these cells were subjected to FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. Student <i>t</i>-test for paired samples was applied for statistical analysis. (<b>B</b>) Difference in the inhibition rates of FcγR-mediated trogocytosis by the HAMA inhibitor between HAMA^high sera and HAMA^low sera. The serum samples were divided into 2 groups, including HAMA^high serum (more than 10 ng/ml, n = 5) and HAMA^low serum (less than 10 ng/ml, n = 5). The decrease rates of CD8⁺ granulocytes by treatment with TRU block were calculated from the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052918#pone-0052918-g006" target="_blank">Figure 6A</a>. Student <i>t</i>-test for unpaired samples was applied for statistical analysis.</p

Characterization of serum factors that contribute to FcγR-mediated trogocytosis.

<p>(<b>A</b>) Heat instability of serum factors that contribute to FcγR-mediated trogocytosis (n = 5). PMNs and PBMCs were mixed in sera, which had been heated at 56°C for 30 min, or sera without heat inactivation. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. Student <i>t</i>-test for paired samples was applied for statistical analysis. (<b>B</b>) Molecular weight range of serum factors that contribute to FcγR-mediated trogocytosis. PMNs and PBMCs were mixed in sera, which had been fractionated into those with molecular weight of more than 100 kDa or less than 100 kDa. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. This experiment was repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.</p

Requirement of FcγRs, dynamism of plasma membrane, and actin recruitment in detection of CD8⁺ granulocytes.

<p>(<b>A</b>) Involvement of FcγRII (CD32) and FcγRIII (CD16) in the detection of CD8⁺ granulocytes. Heparinized blood samples were pre-incubated with the anti- FcγRII (CD32) Ab (AT10) or with the anti-FcγRIII (CD16) Ab (3G8). After the pre-incubation, the samples were made to react with the PE or PECy5-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS followed by reaction with the FITC or PE-labeled anti-CD15 Ab (H198), and then served for FCM. PE or PECy5-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. (<b>B</b>) Effects of plasma membrane fixation and inhibition of actin recruitment in the detection of CD8⁺ granulocytes. PMNs and PBMCs were mixed together. For fixation of the plasma membrane, the cells were exposed to 4% PFA for 10 min at room temperature. After washing 3 times with PBS, the cells were re-suspended in the autologous serum. The samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. For inhibition of actin recruitment, the mixture of PMNs and PBMCs was made to react with CyD (2 µg/ml) for 30 min at 37°C. After the pre-incubation, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 (H198), and then served for FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.</p

Detection of CD8⁺ granulocytes and correlation to CD8⁺ monocytes.

<p>(<b>A</b>) Detection of CD8⁺ granulocytes in human peripheral blood samples. Heparinized whole blood samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS, and then allowed to react with the FITC-labeled anti-CD15 Ab (H198). PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. The cells gated in R1 were regarded as PMNs. Among the PMNs, granulocytes were characterized by the high level of expression of CD15 (gated in R2). The cells in both R1 and R2 gates were examined for the expression of CD8α. (<b>B</b>) Correlation of CD8⁺ granulocytes and CD8⁺ monocytes (n = 32, r = 0.92, p = 9.46×10⁻¹² in Pearson correlation test).</p

Requirement of serum and cell-to-cell interaction with T cells in detection of CD8⁺ granulocytes.

<p>(<b>A</b>) mRNA expressions of CD3ε, CD11b, CD8α, and CD8β in granulocytes (CD15⁺ PMNs) and T cells (CD3⁺ cells) determined by RT-PCR. These cells were separated from blood samples as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052918#s4" target="_blank">Materials and Methods</a>. The quality of RNA samples was verified by the expression of GAPDH. (<b>B</b>) Requirement of serum for detection of CD8⁺ granulocytes. PMNs and PBMCs separated from heparinized peripheral blood were mixed in PBS or autologous serum. These cells were incubated with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. (<b>C</b>) Requirement of cell-to-cell contact with T cells for detection of CD8⁺ granulocytes. PMNs and T cells were separated from heparinized blood samples. PMNs were cultured with or without T cells in the autologous serum. In the co-culture wells, PMNs were cultured separately from T cells using the transwell chambers or mixed together with T cells. Subsequently, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. These experiments were repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.</p