GIST is the most common mesenchymal tumor of the gastrointestinal tract. Most GISTs are activated due to mutations of c-kit proto-oncogene or platelet-derived growth factor receptor alpha gene. Its immunophenotype is positive for CD117, CD34, and DOG-1. The biological behavior of GIST has malignant potential and it is difficult to assess its prognosis accurately. The 2008 NIH modified assessment system is usually used to evaluate the postoperative recurrence risk of primary and resectable GISTs. The test parameters include primary location, tumor size, MI, and tumor rupture. Among these parameters, primary location, tumor size, and tumor rupture are easily established, while MI quantification is performed more subjectively and its repeatability is poor. However, MI determination in GIST or other soft tissue tumors is less repeatable among observers due to the diverse morphological manifestation of mitosis and maldistribution in tumors [4, 5]. In addition, accurate quantification of mitosis on H&E-stained sections depends on the ability to locate "hot spot" regions with the highest mitosis rate and to identify true mitosis from apoptotic cells and nuclei fragments. Furthermore, this may underestimate MI in some cases and result in false assessment of biological behavior of GIST if the definition of mitosis is too strict [6]. Therefore, it is necessary to identify a biomarker to represent the true status of the MI.
A histone is an important component of the nucleosome, which is a basic unit of chromatin in eukaryotes. Researchers have identified five types of histones, including H1, H2A, H2B, H3, and H4 [7]. Histones H2A, H2B, H3, and H4 can be phosphorylated under the effect of protein kinases. The modified sites of histone H3 protein are positions 3 and 11 of threonine and positions 10 and 28 of serine. In the process of mitosis, histone H3 begins to phosphorylate in the late G2 phase and diffuses to the entire chromosome as mitosis progresses. The phosphorylation reaches a peak level in the M phase, while dephosphorylation occurs at the end of the M phase. The phosphorylation degree of histone H3 is very low in the interphase, which means that phosphorylated histone H3 (PHH3) is mainly expressed in G2 and M phases [2, 3]. At present, many studies consider that PHH3 is a specific mitotic marker, which can help to distinguish mitosis, nuclear fragments, and apoptotic cells in tissue sections [8]. It can be easier and quicker to perform mitosis quantification objectively in meningiomas by detecting the expression status of PHH3. This test has important value for assessing meningioma prognosis [9–12]. Similarly, the histological grading of breast cancer has been changed by quantifying mitosis with PHH3 labeling. The PHH3 expression status is closely related to the prognosis of breast cancer [13–16]. In addition, MI quantification using MI-H&E has a strong correlation with PHH3-labeled MI in breast fibrous epithelial tumors, which can make MI quantification more convenient and is an important tool for diagnosis. In addition, researchers have discovered that PHH3-positive index has a significant correlation with tumor grading and prognosis in uterine smooth muscle tumor [17], astrocytoma [18–19], malignant melanoma [20–21], esophageal squamous cell carcinoma [22], ovarian serous adenocarcinoma [23], and other tumors.
Ki67 is a nuclear proliferation antigen that is expressed in all cell cycle phases, except for G0 phase, and is widely used to evaluate the proliferative activity of tumor cells. The Ki67-positive cells are not necessarily occurring in the M phase because Ki67 is expressed in all cell cycle phases, except for the G0 phase [24]. Although studies have shown that Ki67 is related to biological behavior of GIST, there is no system that uses it as an evaluation parameter for GIST grading [25–28]. Moreover, using it will result in overestimation of tumor proliferation activity because infiltrating lymphocytes in the tumor may also have an immunoreaction to Ki67.
The present study evaluated the relationship between MI-H&E and PHH3-positive index and clinicopathological characteristics of 125 cases of GIST. There were no statistically significant differences among MI-H&E, PHH3-positive index, and groups of different gender, age, primary location, and histological subtype. Correlation analysis showed that the PHH3-positive index has a good correlation with MI-H&E, which is consistent with previous research results [29–30]. Similarly, Ki67 index is correlated with MI-H&E. In the 125 cases of GIST, both PHH3 and Ki67 had a high expression in 13 cases and low expression in 57 cases. Correlation analysis results showed that the Ki67 index was positively correlated with the PHH3-positive index, but the correlation was lower than that between the PHH3-positive index and MI-H&E. This is due to the fact that Ki67 is expressed in all cell cycles except the G0 phase and Ki67 is also expressed by inflammatory cells in tumors. Comparatively, PHH3 is mainly expressed in the G2 and M phases and can represent cell mitosis status more accurately.
For a metastatic GIST in the present study, MI-H&E (31/5 mm2) revealed a higher value than the PHH3-positive index (1/5 mm2). The tumor risk stratification of this case has not been changed because the GIST was large enough in diameter (16 cm) to be graded as high-risk and therefore did not depend on the MI value for evaluation. However, correlation between MI-H&E and prognosis is better than that with the PHH3-positive index in prognosis analysis of this special case. There are two possible reasons for this. First, false negative results during immunohistochemical staining may be due to various factors, such as insufficient tissue fixation or improper operating procedure. Second, apoptotic cells or nuclear fragments affect the mitosis interpretation on H&E-stained sections. Our data show that the Ki67 index is related to GIST prognosis as well, which is consistent with previous research [25–28]. This suggests that both PHH3 and Ki67 are prognostic markers of GIST.
In the present study, tumor risk stratification of five cases was changed due to PHH3-IHC results. Compared to MI quantification using H&E-stained sections, two cases received a higher risk classification and three cases received a lower risk classification based on PHH3-IHC results. It is more difficult to accurately determine the "hot spot" regions and evaluate mitosis on H&E-stained sections due to interference from apoptotic cells and nuclear fragments [31]. This may lead to MI-H&E producing values that are higher than those for the PHH3-positive index, resulting in higher tumor risk stratification using H&E-stained sections. By contrast, it is easy to identify the "hot spot" mitosis regions and distinguish real mitosis in a lower-powered field using PHH3 IHC staining. Therefore, it is more convenient and accurate to evaluate mitosis using PHH3-stained IHC sections. However, the false negative PHH3 IHC staining results may also affect mitosis quantification due to various factors [32–35]. Previous studies on PHH3 expression in malignant tumors have shown that PHH3-IHC is highly correlated with MI-H&E, although it is slightly higher than MI-H&E. However, only H&E-stained sections can be used to perform mitosis quantification during risk stratification of GIST at this time [36–38]. For this reason, using PHH3-stained IHC sections to perform mitosis evaluation may lead to grading changes in tumor risk stratification of GIST and affect further therapy. Accordingly, MI quantification using PHH3-stained IHC sections can be used as an assistive tool, rather than a replacement, for tumor risk stratification of GIST when a pathological diagnosis is made.
In summary, PHH3-positive IHC index is positively correlated with MI-H&E, which is a more pronounced relationship than that between Ki67 in GIST. PHH3 can play an assistive role in evaluation of risk stratification and prognosis in GIST. MI determined using PHH3-stained IHC sections can be used as an assistive tool, but not a replacement, for tumor risk stratification of GIST using H&E-stained sections.