Investigating status of tumorigenic barriers in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM)

Abstract

Multiple myeloma (MM), an incurable late stage B-cell malignancy characterized by the presence of monoclonal plasma cells in the bone marrow, is the second most common haematological malignancy after non-Hodgkin¿s lymphoma. It is mostly preceded by the pre-malignant tumor stage, monoclonal gammopathy of undetermined significance (MGUS). MGUS progresses sporadically to MM with a probability of about 0.6-3% per year. Until now, all the disease-initiating genetic abnormalities are found in MGUS at a similar frequency as in MM. Thus, the genetic abnormalities causing the transformation of MGUS to MM are still unknown. Recent studies have shown that two important tumorigenic barriers, DNA damage response (DDR) and oncogene-induced senescence (OIS), are activated in various premalignant tumors, and malignant transformation is accompanied by defects in these barriers. The aim of my thesis project is to study whether defects in one or both of these barriers might also mediate transformation from MGUS to MM. Double staining IHC method was optimized and applied to compare the differential status of DDR checkpoint and OIS between MM and MGUS. As activation of DDR and OIS give rise to senescence or apoptosis in cells, different markers of cell cycle checkpoint, proliferation, apoptosis, and senescence such as Cyclin D1, Ki67, p53, Bax, Bcl-2, CC3, and p16 were combined with CD138 as plasma cell markers and optimized in the Ventana automated stainer (Roche). The double staining IHC was important in determining the pattern of expression of these specific markers in the bone marrow plasma cells, as CD138 stained the membrane of the plasma cells whilst the other markers had either cytoplasmic or nuclear staining. Our results showed that CC3 expression as a phenotypic marker of apoptosis was significantly (p-value=0.03) increased in MM compared to MGUS samples. However, we did not see overexpression of the apoptosis and senescence markers in MGUS compared to MM. For example, p16expression as a senescence marker did not change in both groups (p-value=0.09). Due to failure to find primary evidence of DDR or OIS activation in MGUS, we are not able to suggest that defects in OIS or DDR causes transformation of MGUS to MM, based on our work thus far. Furthermore, Bax and Bcl-2 overexpression was observed in MM samples compared to MGUS (Bax p-value=0.001, Bcl-2 p-value<0.001). Whilst Bcl-2 overexpression was also associated with short overall survival (log-rank p-value=0.04) in our MM patients, Bax overexpression was not similarly associated (log-rank p-value=0.46). Expression of Cyclin D1, Ki67, p53 did not vary between MGUS and MM samples (p-values were 1, 0.18, and 0.10, respectively). Neither was overall survival of MM patients associated with expression of Cyclin D1, Ki67, p16, and CC3. In contrast, p53 expression showed significant association with poor prognosis (lon-rank p-value=0.003). In conclusion, increased levels of pro- and anti-apoptotic markers were found in MM compared to MGUS patients; however, the senescence and proliferation markers were not varied between these two groups of patients.