Exploring B-Cell Migration and Adhesion Molecules CXCR4, CXCR5 and CD49d in Waldenström Macroglobulinemia/Lymphoplasmacytic Lymphoma and Correlation with MYD88L265P and CXCR4 Mutations
Authors: Urbano Mafalda Pereira , Baptista Mariana , Silva Isabel , Santos Susana , Coucelo Margarida , Simões Ana , Martins Madalena , Rebelo Ana , Luís Dino , Ribeiro Patrícia , Silva Fernando , Santos Catarina Geraldes , Roque Adriana Isabel , Paiva Artur , Duarte Sara 
Published: December 2024

Introduction: The majority of Waldenstrom macroglobulinemia (WM) patients (pts) harbor the activating mutation MYD88L265P and about ⅓ nonsense or frameshift mutations in the CXCR4 gene. Both mutations have prognostic as well as therapeutic implications. B-cell malignancies present differences in the migratory and invasive patterns of malignant cells. Little is known about the mechanisms governing migratory heterogeneity, despite growing evidence concerning the role of homeostatic chemokines and their receptors, mainly CXCR4 and CXCR5, and adhesion molecules such as CD49d, in the dissemination pattern of lymphoid malignancies. Also, there are no data about its correlation with genetic characteristics of WM.

Aims: To evaluate the relationship between CXCR5, CXCR4 and CD49d protein expression levels in normal (nr) and abnormal (ab) lymphocytes and their role as potential surrogate markers for MYD88L265P and CXCR4 gene mutational status in WM.

Methods: We prospectively studied bone marrow aspirate samples of 45 pts newly diagnosed with WM from 3 Portuguese centers. MYD88L265P mutation testing performed in 39/45 (87%) cases by Real Time ASO-PCR and CXCR4 gene mutations by Sanger sequencing in 30/45 (67%) cases, preferably after sorting of CD19+ cells. Flow cytometric (FCM) expression of CXCR5, CXCR4 and CD49d evaluated in nr (normal k/l ratio) and ab B cells (monoclonal). FCM performed using a BD FACSCanto II cytometer. Data acquisition carried out using FACSDiva software and ≥10^3 events/sample were collected. Median fluorescence intensities (MFI) of the 3 molecules evaluated and a log2 transformation applied to all MFI values, to allow a more interpretable scale and to reduce skewness. MFI and ratios nr/ab (normalized variables) statistically compared across populations by Mann-Whitney U test.

Univariate logistic regression conducted to explore the association between the phenotypic markers and the genetic findings. Factors with p-values &lt; 0.05 considered significant.

Results: We included 45 pts, median age 76 years, 57.8% male. Seventy-seven percent of pts were positive for MYD88L265P mutation and had no mutation in CXCR4 gene, 10% were positive for both and 13% were double negative. The median (min-max) of MFI was 11.83 (8.75-14.15) for CXCR5nr, 11.43 (8.71-14.24) for CXCR5ab, 10.50 (6.82-13.10) for CXCR4nr, 10.43 (7.48-13.63) for CXCR4ab, 9.95 (5.49-12.73) for CD49dnr and 9.83 (7.46-11.57) for CD49dab. There were no MFI differences between nr and ab lymphocytes in CXCR5 (p=0.233), CXCR4 (p=0.988) or CD49d (p=0.713). There was no correlation between CXCR5nr/CXCR4nr (r=-0.085; p=0.662), CXCR5ab/CXCR4ab (r=0.116; p=0.506), CXCR5nr/CD49dnr (r=0.001; p=0.996), CXCR5ab/CD49dab (r=0.211; p=0.186), CXCR4nr/CD49dnr (r=0.028; p=0.887) and CXCR4ab/CD49dab (r=0.280; p=0.115). When considering normalized variables, there was no correlation between CXCR5/CXCR4 (r=0.315; p=0.096) and CXCR4/CD49d (r=-0.006; p=0.978), but there was a positive correlation between CXCR5/CD49d (r=0.353; p=0.038). CXCR5 (p=0.707 and 0.343), CXCR4 (p=0.915 and 0.599) and CD49d (p= 0.227 and 0.062) were not significantly associated with MYD88 mutational status, when used the MFI in ab lymphocytes or the value normalized, respectively. There was also no significant association between CXCR5 (p=0.109 and 0.330), CXCR4 (p=0.701 and 0.109) and CD49d (p=0.414 and 0.109) with CXCR4 mutational status, for MFI in ab lymphocytes or the value normalized, respectively.

Conclusion: In our cohort, there was no differences in CXCR5, CXCR4 and CD49d MFI between nr and ab lymphocytes of WM pts. There was a positive correlation between normalized levels of CXCR5 and CD49d, which indicates that an increase in CXCR5 expression is associated with an increase in CD49d expression and suggests a functional relationship between these two markers. There was no significant association between CXCR5, CXCR4 and CD49d with MYD88 and CXCR4 mutational status. The findings propose that behavior of tumor cells may be influenced by adhesion and chemotaxis factors and that malignant cells may use these mechanisms to promote their survival and proliferation, possibly facilitating homing to specific sites in the tumor microenvironment. More studies are needed to validate our findings and to better understand its clinical impact, namely in expansion to secondary lymphoid organs.