The Utility of Phosphohistone H3 (PHH3) in Follicular Lymphoma Grading

A Comparative Study With Ki-67 and H&E Mitotic Count

Michelle L. Khieu, MD; Devin R. Broadwater, MD; James K. Aden, PhD; Jean M. Coviello, DO; David T. Lynch, MD; Jordan M. Hall, MD


Am J Clin Pathol. 2019;151(6):542-550. 

In This Article

Abstract and Introduction


Objectives: To determine the utility of phosphohistone H3 (PHH3) mitotic count (MC) in grading follicular lymphoma (FL).

Methods: FL cases were identified (2005-2017). Three hematopathologists recorded their average Ki-67 proliferation index, MC/high-power field (hpf) using PHH3 and H&E stains, and number of centroblasts/hpf. Results were assessed for correlations and interobserver variability.

Results: Forty-three cases of FL were studied. PHH3 MC resulted in the strongest correlation to grade (r = 0.701, P < .0001) compared with Ki-67 proliferation index (PI) (r = 0.681, P < .0001) and H&E MC (r = 0.536, P = .0002) and the strongest linear relationship to centroblast count (R2 = 0.453). Agreement among pathologists was strongest for PHH3 (intraclass correlation coefficient [ICC] = 0.86) followed by Ki-67 PI (ICC = 0.85) and H&E MC (ICC = 0.78).

Conclusions: PHH3 correlates best to histologic grade and has less interobserver variability compared with Ki-67 PI and H&E MC. These results support using PHH3 as an adjunct in FL grading.


Follicular lymphoma (FL) is a common B-cell lymphoma of germinal center B-cell origin. Approximately 20% of all lymphoma cases are diagnosed as FL.[1] Current recommended histologic grading according to the 2017 World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues continues to use criteria adapted from the original Mann-Berard criteria,[2] which are based on the average number of large transformed cells or centroblasts in 10 high-power fields (hpf) from 10 random and representative neoplastic follicles. Those with five or fewer centroblasts/hpf are designated as grade 1, with six to 15 centroblasts/hpf as grade 2, and with more than 15 centroblasts/hpf as grade 3. Grade 3 FL is further subdivided into grades 3A and 3B, based on the presence (3A) or absence (3B) of centrocytes. FLs designated as grades 1 and 2 are considered low grade, while grade 3 is high grade.[3]

There is a significant difference in treatment between low-grade and high-grade FLs, as established by the National Comprehensive Cancer Network (NCCN) guidelines; therefore, the ability to accurately distinguish between the two grades is vital. On the other hand, there is still controversy regarding the clinical significance of subdividing high-grade FL. Some more recent investigations do suggest a difference both biologically and clinically between grade 3B FL and the other FL grades. In a study by Horn et al,[4] 3B FL was shown to have cytogenetic and immunohistochemical features more in line with diffuse large B-cell lymphoma (DLBCL) than other FLs. Another study found the clinical course of 3B FL to resemble DLBCL, with the possibility of cure after therapy,[5] while yet another study found no difference in outcome between grades 3A and 3B.[6]

Although several studies suggest that the current grading system predicts which FLs may behave more aggressively, the clinical significance and best method of grading are still controversial. However, the WHO deems there to be insufficient evidence to recommend an alternative method, and therefore, although certainly not free of problems, centroblast count in 10 random (representative) follicles has remained the recommended system of grading. One obvious issue with using the cutoff values determined by Mann and Berard as described by the 2017 WHO is that there is no mention of field of view. The cutoffs set by Mann and Berard apply to an hpf of 0.159 mm2, which correlates to a ×40 objective with an 18-mm field number ocular. However, microscopes now often use oculars with 20- or 22-mm field numbers. To accurately correlate with Mann and Berard's cutoffs, when using an ocular with a field number other than 18 mm, one would need to convert the count to centroblasts/0.159 mm2. For example, if using a 22-mm field number ×10 ocular, you would need to count only 7 hpf and then divide by 10 or count 10 hpf and then divide by 15. Yet this is not specifically addressed and therefore is not often done. Another overarching concern associated with centroblast count is the high interobserver variability and therefore lack of reproducibility.[7–10] To help ameliorate this problem, several studies have assessed the value of proliferation rate in FL, mainly focusing on H&E mitotic count (MC) and/or Ki-67 proliferation index (PI). These studies all reached a similar conclusion, in that there is significant prognostic value in assessing proliferative rate in FL.[11,12] In the study by Wang et al,[13] 18% of their low-grade FL (LGFL) cases actually had high Ki-67 PIs (LG-HPI) with a higher early death rate than in LGFL cases with low PI, leading them to propose classifying them separately. These data led the WHO to recommend Ki-67 PI assessment as a useful adjunct in evaluating cases of FL in the 2008 fourth edition[14] and to continue this recommendation in the 2017 revised fourth edition.[3] However, both H&E MC and Ki-67 PI also suffer from high interobserver variability.[11,12] Besides the inherent differences among individual pathologists, these methods are also affected by technical differences. Darker H&E staining can make searching for mitotic figures difficult. The presence of apoptotic bodies can be confused with mitotic figures on H&E. Nonspecific background staining and variable staining intensity can lead to problems with Ki-67 interpretation. Therefore, although prognostically significant, the issue of interobserver variability still remains.

Phosphohistone H3 (PHH3) is a commercially available antibody targeted against phosphorylated histone H3, a core histone protein that becomes increasingly phosphorylated during chromatin condensation as a cell moves from the late G2 phase to the M phase. Differences in staining pattern allow the observer to differentiate cells in the G2 phase (speckled nuclear staining) from the M phase (diffuse, clumpy staining). Due to these properties, PHH3 is considered a mitotic-specific immunohistochemical (IHC) stain.[15,16] PHH3 has been studied and found useful in detecting mitotic figures in the analysis of many tumors, such as those of neurologic,[17,18] breast,[19,20] melanocytic,[21,22] pancreatic,[23] and uterine[24] origins. As a mitotic-specific IHC marker, PHH3 allows for easy and more precise recognition of cells in mitosis. It does not stain apoptotic bodies. Our study assesses the utility of performing a mitotic count with the aid of PHH3 immunohistochemistry in cases of FL. We assess its correlation with histologic grade based on centroblast count as well as the degree of interobserver variability compared with that seen with H&E MC and Ki-67 PI. To our knowledge, this is only the second study to date investigating the utility of PHH3 MC in grading FL.[25]