Phosphohistone H3: new standard IHC marker for grading tumors via mitotic indexing

M

icroscopic evaluation of mitotic activity is a routine procedure in assessing the grade of tumors,¹ such as soft tissue sarcomas, meningiomas, or breast carcinomas. Recent evidence has shown that the entry into the M phase of the cell cycle sensitizes tumor cells to new anti-cancer strategies. For pathologic grading of tumors, a value of proliferating activity is established by observing and scoring the number of mitoses per 10 high-power field (MF/10 HPF). Nevertheless, the method of counting is subjective, time-consuming, and associated with problems of reproducibility in differentiating mitotic figures from apoptotic cells, necrotic cells, or karyorrhexis, requiring experience of trained pathologists.^2-3 In addition, the number of cells per unit area varies wildly in tumors. Because mitotic count is based on tumor area and not on cell number, determining mitoses/10HPF is not a true index of proliferation but, rather, an estimate that is inherently confounded by tumor cellularity. Moreover, for several reasons, including hypoxia, fixation delay, and suboptimal fixation, mitotic figures can become more condensed and, thereby, less easily identifiable.

Clearly, an accurate immunohistochemical (IHC) proliferation index marker would be valuable. Unfortunately, most available IHC markers are much weaker predictors than the mitotic activity index.⁴ This may be due to the fact that mitotic figures reflect a very small time window in the cell cycle (the M phase), whereas other markers such as MIB1/Ki-67 stain cells in nearly all phases of the cell cycle.

Immunostaining of proliferating cell nuclear antigen, or PCNA, or MIB-1/Ki-67 has been proposed to be an additional or alternative method for evaluating the proportion of proliferation activity. Unlike mitoses/10HPF, the MIB1/Ki-67 labeling index is normalized for cellularity and can be considered a true index. Ki-67, however, is expressed by cells throughout the cell cycle from late G1 phase, except G0,⁵ and many Ki-67+ nuclei may not survive the cell cycle and are driven into apoptosis, thereby blurring the prognostic value of the Ki-67 index. Also the count of Ki-67-positive nuclei is higher than the hematoxylin and eosin-based mitotic count. Because of technical variability in staining protocols and counting methods, the MIB1/Ki-67 index is also subject to interlaboratory variability⁶ and is difficult to standardize. Mitoses are very late cell cycle markers and, once formed, are reliable proliferative markers. Therefore, ideally, any substitute IHC proliferation markers should be expressed only in late G2 and M phases.

Applications

Recently, studies have demonstrated histone H3 is a core histone protein which, together with other histones, forms the major protein constituents of the chromatin in eukaryotic cells. In mammalian cells, phosphorylation of the Serine 10 residue of histone H3 is negligible during interphase but reaches a maximum for chromatin condensation during mitosis.^7-8 An antibody directed against phosphohistone H3 (PHH3) should have the potential to be a reliable and technically robust method for identifying mitotic activity.⁹

Immunohistochemical studies performed with anti-PHH3 antibody have shown that the antibody detects specifically the core protein histone H3 only when phosphorylated at serine 10 or serine 28. The phosphorylation of histone H3 is a rare event in interphase cells but is a process almost exclusively occurring during mitosis.^7,9 Studies have also revealed no phosphorylation on the histone H3 during apoptosis.¹⁰ Therefore, PHH3 can serve as an effective mitotic marker.

Kim, et al, found the PHH3 counting method in the detection of mitotic figures was more sensitive compared with the traditional H&E-stained mitotic figure counts; and if the mitotic thresholds suggested by the WHO were applied to the PHH3 MI results in 28 cases, there would be a change in grade. Twenty-seven cases of WHO grade I meningiomas would have increased in grade to atypical meningiomas (WHO grade II), and one atypical meningioma would have increased to anaplastic meningioma (WHO grade III). Of these, three of the WHO grade I meningiomas and the atypical meningioma recurred. This study also demonstrated that six or more PHH3 mitoses per 10 HPF indicate meningiomas with significantly higher risk of recurrence or death within five years after primary surgery, independent of patient age or extent of tumor resection. Based on the traditional H&E MI, 36% of patients at risk for recurrence could be identified; the PHH3 MI indicated recurrent cases with a sensitivity of 56%. Thus, the PHH3 mitosis counting method also increases the sensitivity in the identification of cases at risk of recurrence.¹¹ 

According to the current WHO grading system, the distinction between infiltrating astrocytomas of malignancy grade II versus grade III is largely based on the assessment of the presence and degree of proliferative activity of neoplastic cells, and also is important both for prognosis and for treatment decision making. This is routinely accomplished by determining the total number of mitoses on the examination of H&E sections. PHH3 stains only cells in mitosis, offering the possibility of obtaining true mitotic index.

Colman, et al, studied PHH3 MI (per 1,000 cells) in 103 cases of grade II and grade III astrocytomas from patients with clinical follow-up and compared with MIB/Ki-67 MI and H&E mitoses/10 HPF. The PHH3 MI value was generally similar to MIB/Ki-67 results and better than mitoses/10HPF for dividing patients in distinct groups. Among the 19 cases carrying a grade II diagnosis, three cases had a PHH3 MI above 3/1,000 cells, and in retrospect, would have been upgraded to grade III. Among the 84 cases carrying a grade III diagnosis, 53 had an MI of three or less and, in retrospect, would have been downgraded.¹² 

PHH3 also finds application in rapidly assessing mitotic figures in melanoma diagnosis. Nasr and El-Zammar studied 66 melanocytic lesions (18 malignant melanomas, eight Spitz nevi, 20 dysplastic nevi, and 20 compound nevi) using anti-PHH3 and Ki-67 antibodies. They revealed that PHH3 expression was higher in malignant melanomas, averaging 25/10HPF than in Spitz nevus, averaging 0.5/10HPF. They concluded that PHH3 and Ki-67 can be useful adjuncts to histopathology to separate malignant melanoma from benign nevus.¹³

George G. Yang, MD
who obtained his medical education in China, completed his anatomic and clinical pathology residency at University of Southern California with subspecialty fellowship trainings in surgical pathology, IHC, and hematopathology at University of California-San Francisco, Stanford University, and University of Southern California. A U.S. board-certified pathologist with a focus in IHC and hematopathology, Yang is a Fellow of the American Society of Clinical Pathology and the College of American pathologists. He is currently staff hematopathologist at Cell Marque Corp. in Rocklin, CA.

References

1. Rosai J. Gastrointestinal stromal tumor and its mimics. Int J Surg Pathol. 2005;12:155.
2. Van Diest PJ, et al. Reproducibility of mitosis counting in 2,369 breast cancer specimens: results from the Multicenter Morphometric Mammary Carcinoma Project. Hum Pathol. 1992;23:603-607.
3. Molenaar WM, et al. Observer reliability in assessment of mitotic activity and MIB-1-determined proliferation rate in pediatric sarcomas. Ann Diagn Pathol. 2000;4:228-235.
4. Van Diest PJ, et al. Prognostic value of proliferation in invasive breast cancer: a review. J Clin Pathol. 2004;57:675-681.
5. Endl E, Gerdes J. The Ki-67 protein: fascinating forms and an unknown function. Exp Cell Res. 2000;257:231-237.
6. Hsu CY, et al. Interobserver reproducibility of MIB-1-labeling index in astrocytic tumors using different counting methods. Mod Pathol. 2003;16:951.
7. Gurley LR, et al. Histone phosphorylation and chromatin structure during mitosis in Chinese hamster cells. Eur J Biochem. 1978;84:1-15.
8. Juan G, et al. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry. 1998;32:71-77.
9. Shibata K, et al. Cell cycle-dependent suppressive effect of histone H1 on mitosis-specific H3 phosphorylation. J Biol Chem. 1993;268:18431-18434.
10. Hendzel, MJ, et al. Chromatin condensation is not associated with apoptosis. J Biol Chem. 1998;273:24470-24478.
11. Kim YJ, et al. Prognostic significance of the mitotic index using the mitosis marker anti-phosphohistone H3 in meningiomas. Am J Clin Pathol. 2007;128:118-125.
12. Colman H, et al. Assessment and prognostic significance of mitotic index using the mitosis marker phospho-histone H3 in low and intermediate-grade infiltrating astrocytomas. Am J Surg Pathol. 2006;30:657-664.
13. Nasr MR, El-Zammar O. Comparison of pHH3, Ki-67, and survivin immunoreactivity in benign and malignant melanocytic lesions. Am J Dermatopathol. 2008;30:117-122.