CD69 Expression Predicts Favorable Outcome in Multiple Myeloma Patients Treated with VTD

Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (PC). Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification but the clinical and prognostic value of immunophenotyping in MM remains questionable. We have analyzed the prognostic impact of a relatively new marker as CD69. CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous laboratoristic data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. In a previous abstract, we described for the first time in a clinical report, the CD69 expression on pathological PCs of MM patients. Our preliminary data also suggested an intriguing role of CD69 in patients treated with chemotherapy in different stages of disease. In this study, we report a larger setting of 97 patients where we confirmed the expression of CD 69 in 48 of them (49%) (see table I). Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was > 20%.

After an induction regimen of treatment with four cycles of VDT (bortezomib, dexametasone, thalidomide), 69 patients were evaluable. 40/69 (65%) of patients obtained at least of a very good partial response or better (Responding pts). In this subgroup of patients 30/45 (66.6%) showed the expression of CD 69. On the contrary only in a little part of partial or less responding patients (NR pts) 9/24 (37.5%) CD69 was detected (see table II) (p=0.02 using a chi squared test and p=0.019 using a Fisher’s exact test). Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in >90% of patients. Clinical data were available in all patients and CD69 maintained its association with different response, independently of other prognostic variables.

In conclusion CD69 is often expressed in PCM cases, and the expression of this marker is useful to reveal poor prognostic categories and delineate a risk stratification. This molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs.

Table I Pts Characteristics	Total	CD69+
	97	48/97
Sex
	Male	51(52%)
	Female	46(48%)
Clinical status
	MM non evaluable MM after VTD	28/97 69/97
	in VGPR/CR 45/69      in PR/SD/PD 24/69

Table II Pts treated with VTD		Responding pts	NR pts
		45	24
CD69+ 39/69		30/45	9/24
CD69-  30/69		15/45	15/24