H2AFZ: A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment

Abstract

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.

Diagnosis and histopathologic prognostication of canine melanocytic neoplasms: A consensus of the Oncology‐Pathology Working Group

“…In one study, nuclear survivin expression was significantly greater in malignant cutaneous melanomas compared to melanocytomas, and increased expression (≥8% of neoplastic cells) was related to the presence of metastasis and death of the animal due to melanoma 3 . In a recent study, survivin gene expression from formalin fixed‐paraffin embedded (FFPE) tumour tissue was assessed in both cutaneous and oral melanocytic tumours, and confirmed a correlation of this marker with tumour related death and Ki67 index 37 . In the same study, gene expression from FFPE tissue of another marker, H2AFZ, showed promising results as a new prognostic marker, being correlated with MC, Ki67 index, presence of metastasis, and death due to melanoma 37 …”

“…Several molecular markers have been investigated for their potential as prognostic markers for canine melanocytic neoplasms (Table 2). Currently, immunohistochemical evaluation of Ki67, as the Ki67 index or Ki67 count, is the only established prognostic molecular marker, and it has been shown to be highly predictive of behaviour for both oral and cutaneous melanocytic tumours in dogs in multiple studies, including four that were published after 2011 1,4,12,20,27,31,37 . Whereas MC only accounts for cells in the M phase of the cell cycle, Ki67 is a nuclear protein that is expressed in all phases of the cell cycle, except the resting phase; therefore, it is a measure of growth fraction.…”

“…3 In a recent study, survivin gene expression from formalin fixed-paraffin embedded (FFPE) tumour tissue was assessed in both cutaneous and oral melanocytic tumours, and confirmed a correlation of this marker with tumour related death and Ki67 index. 37 In the same study, gene expression from FFPE tissue of another marker, H2AFZ, showed promising results as a new prognostic marker, being correlated with MC, Ki67 index, presence of metastasis, and death due to melanoma. 37 RACK1 is another marker that has been investigated as a potential diagnostic, as well as a potential prognostic, marker.…”

“…37 In the same study, gene expression from FFPE tissue of another marker, H2AFZ, showed promising results as a new prognostic marker, being correlated with MC, Ki67 index, presence of metastasis, and death due to melanoma. 37 RACK1 is another marker that has been investigated as a potential diagnostic, as well as a potential prognostic, marker. 4 In one study, RACK1 distribution differed between benign and malignant canine melanocytic neoplasms and a RACK1 homogeneous labelling pattern was highly correlated with other criteria such as classic histologic features of malignancy, Ki-67 index and MC.…”

“…The consensus was made available to OPWG membership at large, which approved the report by popular vote, and then it was made available online on the OPWG webpage ( http://vetcancersociety.org/vcs-members/vcs-groups/oncology-pathology-working-group/ ) in August 2020. While preparing the current manuscript, 14 additional articles 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 were identified that were published from November 2019 to August 2021. Again, to expedite review of these 15 additional articles, the co‐chair (RCS) completed the reviews and distributed them to the subgroup for comments and discussion.…”