Tumor budding is an independent prognostic marker in early stage oral squamous cell carcinoma: With special reference to the mode of invasion and worst pattern of invasion

<div><p>Pathologically proven regional lymph node metastasis affects the prognosis in early stage oral cancer. Therefore we investigated invasive tumor patterns predicting nodal involvement and survival in patients with clinically node-negative T1 and T2 oral squamous cell carcinoma (cT1,2N0M0 OSCC). Ninety-one cases of cT1,2N0M0 OSCC treated with transoral resection of the primary tumor were assessed based on 3 types of invasive tumor patterns on histopathologic and pancytokeratin-stained immunohistological sections: the mode of invasion, worst pattern of invasion (WPOI), and tumor budding. The correlations among invasive tumor patterns, regional metastasis, and disease-free survival were analyzed. Of the 91 cases, 22 (24%) had pathologically proven regional metastasis. The mode of invasion (p<0.01) and tumor budding (p<0.01) were associated with regional metastasis as well as lymphovascular invasion (p = 0.04) in univariate analysis. In logistic regression analysis, however, tumor budding was the only independent predictor of regional metastasis (hazard ratio (HR) = 3.05, 95% confidence interval (CI) = 0.29–5.30, p<0.01). All three invasive patterns, the mode of invasion, WPOI, and tumor budding, were found to be significant predictors of 5-year disease-free survival (p<0.01, p = 0.03, and p<0.01, respectively) as well as lymphovascular invasion (p = 0.02) and perineural invasion (p = 0.02). A final model for Cox multivariate analysis identified the prognostic advantage of the intensity of tumor budding (HR = 2.19, 95% CI = 1.51–3.18, p<0.01) compared with the mode of invasion and WPOI in disease-free survival. Our results indicate that the intensity of tumor budding may be a novel diagnostic biomarker, as well as a therapeutic tool, for regional metastasis in patients with cT1,2N0M0 OSCC. If the pancytokeratin-based immunohistochemical features of more than five buds, and a grade 4C or 4D mode of invasion are identified, careful wait-and-see follow-up in a short period with the use of imaging modalities is desirable. If there are more than ten buds, a grade 4D mode of invasion, or WPOI-5 in the same section, wide resection of the primary tumor with elective neck dissection should be recommended.</p></div

Representative photographs of intensity of tumor budding (immunohistochemical pancytokeratin staining, x200 magnification).

<p>(3A) Low intensity of tumor budding (<5 buds), (3B) intermediate intensity of tumor budding (≥5~<10 buds), and (3C) high intensity of tumor budding (≥10 buds).</p

Representative photographs of classification of mode of invasion (H&E staining, immunohistochemical pancytokeratin staining, x40 magnification).

<p>(1A,1B) grade 1, (1C,1D) grade 2, (1E,1F) grade 3, (1G,1H) grade 4C, and (1I,1J) grade 4D.</p

Kaplan-Meier curves of disease-free survival (DFS) in patients in combined groups with the intensity of tumor budding and mode of invasion (A) or worst pattern of invasion (WPOI) (B).

<p>In group A, the 5-year DFSs were 70.4% with low tumor budding and grade 1+2+3 mode of invasion ①, 66.7% with low tumor budding and grade 4C mode of invasion ②, 50.0% with intermediate tumor budding and grade 1+2+3 mode of invasion ③, 0% with intermediate tumor budding and grade 4C mode of invasion ④, 20.0% with high tumor budding and grade 1+2+3 mode of invasion ⑤, 33.3% with high tumor budding and grade 4C mode of invasion ⑥, and 0% with high tumor budding and grade 4D mode of invasion ⑦. In group B, the 5-year DFSs were 72.8% with low tumor budding and WPOI-1+2+3+4 ①, 44.4% with low tumor budding and WPOI-5 ②, 36.4% with intermediate tumor budding and WPOI-1+2+3+4 ③, 60.0% with intermediate tumor budding and WPOI-5 ④, 33.3% with high tumor budding and WPOI-1+2+3+4 ⑤, and 0% for high tumor budding and WPOI-5 ⑥.</p

Representative photographs of classification of worst pattern of invasion (H&E staining, immunohistochemical pancytokeratin staining, x200 magnification).

<p>(2A,2B) Worst pattern of invasion (WPOI)-1, (2C,2D) WPOI-2, (2E,2F) WPOI-3, (2G,2H) WPOI-4, and (2I,2J, x40 magnification) WPOI-5.</p

Photographs of immunohistochemical pancytokeratin staining show representative examples of grade 4D mode of invasion and worst pattern of invasion (WPOI)-4 at x40 magnification.

<p>The region in the rectangle is shown at x200 magnification in the lower panel, which shows 12 buds.</p

Kaplan-Meier survival curves by mode of invasion (A), worst pattern of invasion (WPOI) (B), tumor budding (C), lymphovascular invasion (D), and perineural invasion (E).

<p>5-year disease-free survivals were 58.0% for grade 1+2+3 mode of invasion, 36.4% for grade 4C mode of invasion, 0.0% for grade 4D mode of invasion (A), 58.4% for WPOI-1+2+3+4, 33.3% for WPOI-5, 65.0% for low-intensity tumor budding (<5 buds), 37.5% for intermediate-intensity tumor budding (≥5 buds-<10 buds), 23.1% for high-intensity tumor budding (≥10 buds), 58.1% for absence of lymphovascular invasion, 33.3% for presence of lymphovascular invasion, 56.1% for absence of perineural invasion, and 20.0% for presence of perineural invsion in patients with clinically node-negative T1 and T2 oral squamous cell carcinoma.</p

Photographs of immunohistochemical pancytokeratin staining show representative examples of grade 3 mode of invasion and WPOI-5 at x12.5 magnification.

<p>The yellow line measures a distance >1 mm between the main tumor and the next focus of dispersed islands. The region in the rectangle is shown at x200 magnification in the lower panel, which shows 1 bud.</p

Anatomical Transcriptome of G Protein-Coupled Receptors Leads to the Identification of a Novel Therapeutic Candidate GPR52 for Psychiatric Disorders

<div><p>Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a <i>Cre-lox</i> system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.</p></div

Gene expression distribution and protein localization of GPR52.

<p><b>A,</b> GPR52 mRNA is abundantly expressed in human brain. GPR52 mRNAs were quantified by qPCR throughout human tissues. Data represent the ratios of GPR52 to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA. <b>B–C,</b> ISH investigation of GPR52 mRNA was performed in rat striatum and medial prefrontal cortex. Double-ISH analysis of GPR52 with DRD1/DRD2 in adult male rats. Localizations of GPR52 and DRD1/DRD2 mRNAs were shown as brown and blue signals, respectively, in striatum (<b>B</b>) and medial prefrontal cortex (<b>C</b>). Allow indicates the double-stained neurons. Bar: 25 µm. <b>D,</b> Distribution of LacZ signals in GPR52-LacZ Tg mouse brain. Serial frontal brain sections (rostral → caudal) were stained with X-Gal in GPR52-LacZ Tg mouse. Black characters on the left side of the pictures show LacZ-positive cell bodies and fibers while purple characters on the right side show the fibers. Results and abbreviations were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090134#pone-0090134-t002" target="_blank">Table 2</a>. Bar: 1 mm. <b>E,</b> Double detections of GFP signals with DRD2 in frontal brain sections of hGPR52-GFP Tg mouse. Green and red colors show GFP and DRD2 immunopositive signals, respectively. Bar: 200 µm.</p