Marrow Plasma Cell Immunohistochemical Expression Profiles of Cyclins (A, D1, D2, D3), Cyclin Dependent Kinase Inhibitors (p16, p21) and Ki67: Predictors of Survival in Patients with Multiple Myeloma.
Authors: Beksac Meral , Soydan Ender , Toprak Selami Kocak , Kizil Merih , Kaygusuz Gulsah , Kuzu Isinsu 
Published: April 2020

Abstract
Background: Recently Bergsagel et al have published a hierarchical model of myeloma evolution based on cytogenetics and Cyclin (Cy) profiles. Previously we had reported flow cytometric expression profiles of these Cy and CDKI’s in unsorted marrow samples (Blood98 (11): 4230,2001) but had not been able to detect the parallelism between Cy-CDKI’s which we observed in the normals in the myeloma patients.
Aim: Here in this study we aimed to analyze the same Cy and CDKI’s in marrow plasma cells by multiparameter immunocytochemistry and to evaluate the association with clinical parameters and impact on survival.
Patients and methods: Fourty five myeloma patients diagnosed in our department between 1998–2004, aged 57(33–79), M/F:29/16 whose bone marrow biopsy specimens could be retrieved were included in the analysis. All patients were treated with VAD or MP as first line treatment. Staining was performed using monoclonal antibodies: Cyclins A, D1, p16, p21 (LabVision), Cy D3(DAKO) and Cy D2 (Santa Cruz) with Zymed ABC Px Kit manualy or Ventana Benchmark automated immunostainer for secondary visualization. Results were reported as positive (equal or more than 20% of plasma cells) or negative (less than 20%).
Results: expression results are: p16: 8/45, Cy D1: 10/45, Cy D2: 16/44, Cy D3: 3/45, Cy D(D1 or D2 or D3): 27/45, CyA:7/45, p21:7/43. Cyclins and their inhibitors were found to be expressed in different combinations by plasma cells. A decreased expression of Cy A or D and a relevant CDKI’s increased expression was evaluated as a low proliferative situation (P min). P min was observed 5/45 of the patients, resulted with 80% overall response and 100% survival at 5 years. On the contrary P max patients (high Cy and low CDKI expressing) were observed more frequently(19/45). 10/19 of these patients responded and performed 57% survival. P max and P intermediate patients(any combinations other than P min and P max) responded (7/17) and survived similarly.
Figure Figure
Prognostic factors such as age, B2MG and CRP were similar between P min, P max or P intermediate groups. Cy-CDKI patterns were associated with bone disease: P max patients had bone lesions more frequently (11/14) whereas P min patients had none( p=0.008). When we analyzed the role of Cy D1 as a prognostic factor on survival (OS), OS at 5 years was 80 vs 49% (p=0.85). Similar analysis for Cy D2 was:72 vs 35%(p= 0.04). Among the Cy D2 positive patients, presence of Ki67 was an adverse factor on 5 year OS: 50 vs 75% (p=0.01) but p16 did not show an impact. Multivariate analysis results will be presented later.
Conclusion: Our results show that combining Cy and CDKI expression profiles compared to analysis with only Cy or CDKI, gives better information about the proliferative status and survival. The role of bone disease as a prognostic factor but not it’s association with cell cycle regulators have been shown before.