Mitotic Index Determined by Phosphohistone H3 Immunohistochemistry for Precise Grading in Follicular Lymphoma

Abstract

The World Health Organization classification recommends follicular lymphoma (FL) grading (G1-3) by considering centroblast number, while also suggesting its influence on disease outcome. As centroblast counting and other proliferation markers have limitations, we looked for more specific measures of cellular activity in FL. Phosphorylated histone H3 (pHH3) was widely applied for the objective detection of mitotic activity in different tumors. The aim was to evaluate the utility of pHH3 protein in FL grading and compare its value with the classical features of cell proliferation. Representative samples from 48 FL patients and 9 samples with follicular hyperplasia were examined. Hematoxylin-eosin-based mitosis index (HE-MI), number of mitotic figures based on anti-pHH3 immunohistochemical staining (pHH3-MI), and percentage of Ki-67-positive cells [proliferation index (PI)] were determined and compared with centroblast-based histologic grade. PHH3-MI showed significant correlation with HE-MI (r=0.85, P<0.0001) and PI (r=0.84, P<0.0001). All 3 cell proliferation parameters showed significant correlation with histologic grade: HE-MI versus grade, r=0.85 (P<0.0001); PI versus grade, r=0.74 (P<0.0001); pHH3-MI versus grade, r=0.80 (P<0.0001). PHH3-MI showed continuous increase with the histologic grade. The pHH3-MI value was distinctive between the G2 and the G1 FL groups (P<0.0001) and was increased in G3 FL compared with that in the G2 FL group (P=0.0020). In conclusion, easy-to-perform mitotic counting following phosphohistone H3 immunohistochemistry (pHH3-MI) correlates well with centroblast-based grading. PHH3 immunohistochemistry offers a reliable quantification tool supporting lymphoma grading and can be recommended as an additional parameter for the precise subcategorization of FL cases.

Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling

“…The intense staining was reported to be beneficial to demonstrate and count mitotic figures in histological sections in earlier studies. The specific presentation enabled the precise grading in different cancer types, such as breast carcinoma, astrocytoma, meningioma [19,[29][30][31] and follicular lymphoma by our group [20] . Beside intense labeling of the condensed chromatin during mitosis, premitotic cells can be frequently identified in histological sections with increasing amounts of intranuclear pH3S10 labeling.…”

“…In cases with follicular hyperplasia individual germinal centers were selected and evaluated at ×400 magnification in the microscope. Immunopositive events representing premitotic (dot-type) and mitotic pH3S10 staining pattern were counted for each field of view (Leica N Plan 40/0.65) for all changes; altogether at least 10 high power fields (HPF) were evaluated as described [20] . Premitotic G 2 cells were defined as cells with nuclei presenting at least 1 sharply occurring immunopositive dot.…”

“…The following B-cell lymphomas were analyzed: 24 follicular lymphomas, 10 small lymphocytic lymphomas, 44 diffuse large B-cell lymphomas (DLBCL), 6 Burkitt lymphomas, 5 lymphoblastic lymphomas. Follicular lymphomas were categorized as grade 1-3 by counting intrafollicular centroblasts as described [20] . Germinal center and postgerminal center subtypes of DLBCL were determined following CD10, bcl-6, and MUM-1 immunostainings using the algorithm proposed by Hans et al [25] .…”

“…Mitotic counts determined following phospho-H3 serine 10 (S10; pH3S10) immunohistochemistry (IHC) has been experimentally and clinically applied to measure cell proliferation in different types of cancer [13][14][15][16][17][18][19] . In a recent paper we could establish an excellent relation between H3S10-based mitotic counts and histological grade in follicular lymphoma [20] .…”

Will we all be replaced by machines?

“…Immunostains, including antiphosphohistone H3, have been shown useful in detecting mitotic figures in melanoma and other tumours, but the current American Joint Committee on Cancer (AJCC) staging guidelines are based on mitotic counts performed without immunostains. Immunostaining has been shown to correlate well with manual counts and improve interobserver variability in a variety of tumours, and image analysis has the potential to increase accuracy and reduce variability beyond what can be achieved with immunostaining.…”