Immunoglobulin Mutational Status Detected through Single-Round Amplification of Partial VH Region Represents a Good Prognostic Marker for Clinical Outcome.

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Immunoglobulin Mutational Status Detected through Single-Round Amplification of Partial VH Region Represents a Good Prognostic Marker for Clinical Outcome in Chronic Lymphocytic Leukemia Roberto Marasca, Rossana Maffei, Monica Morselli, Patrizia Zucchini, Ilaria Castelli, Silvia Martinelli, Marcella Fontana, Sara Ravanetti, Monica Curotti, Giovanna Leonardi, Katia Cagossi, Giovanni Partesotti, Giuseppe Torelli The Journal of Molecular Diagnostics Volume 7, Issue 5, Pages 566-574 (November 2005) DOI: 10.1016/S1525-1578(10)60589-2 Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Comparison between nucleotide sequences obtained with upstream FR2 and downstream JHE primers in a single case. Sequence alignment was performed using Clustal tool of Sequence Navigator 1.0.1 software. Sense and complementary reverted anti-sense (*) nucleotide sequences were aligned together (above); correspondent sense and anti-sense electropherograms relative to CDR3 region were shown below. The Journal of Molecular Diagnostics 2005 7, 566-574DOI: (10.1016/S1525-1578(10)60589-2) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Differential CDR3 length between three groups of B-CLL patients with different mutational rate in IgVH region expressed by leukemic population. The horizontal line inside boxes indicates the median value. LM-CLL patients have a percentage of mutations ranging from 2 to 5%, HM-CLL patients have a percentage of mutations greater than or equal to 5%, and UM-CLL patients have mutations in a percentage inferior to 2%. A statistically significant difference is shown between M-CLL (LM-CLL + HM-CLL) and UM-CLL groups using a nonparametric Mann-Whitney test (P < 0.0001). The Journal of Molecular Diagnostics 2005 7, 566-574DOI: (10.1016/S1525-1578(10)60589-2) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Kaplan-Meier survival curve comparing 146 CLL patients with mutated and unmutated VH gene (homology level cutoff = 98%). Median survival for 63 unmutated CLL, 127 months; median survival for 83 mutated CLL, 206 months. The difference is significant at the P = 0.0023 level (log-rank test). The Journal of Molecular Diagnostics 2005 7, 566-574DOI: (10.1016/S1525-1578(10)60589-2) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Log-rank statistics were performed for the VH homology rate to test for a possible cutoff value separating two groups with different survival distributions. VH germ line homology rate from 96 to 98.5% is shown on the x axes. Corresponding P values calculated by log-rank test for the distinction of two groups with different survival probability are shown on the y axes. Classical 98% homology rate is able to best separate prognostic groups between B-CLL patients. The Journal of Molecular Diagnostics 2005 7, 566-574DOI: (10.1016/S1525-1578(10)60589-2) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Scatter diagram of IgVH mutation percentage determined analyzing both complete IgVH sequence obtained by VH family-specific leader primers (x axes) and partial sequence obtained by FR2 degenerate primer (y axes). A: Comparisons were performed in 41 cases of our cohort; median gap in percentage of mutations was 0.3%, ranging from 0 to 3.8%. These two parameters were positively correlated with Spearman's rank order correlation coefficient of 0.96 (P < 0.0001). B: Comparisons were also performed on 80 cases selected from literature (set 1,18 n = 40, circle; set 2,34 n = 40, triangle) considering only the portion of VH sequence 3′ downstream to the annealing site of the FR2 primer. Cases with 100% homology to germ line of the entire VH sequence were obviously excluded from the analysis. Median gap in percentage of mutations was 0.85% (range, 0 to 3.7%) in set 1 and 0.4% (range, 0.1 to 3.4%) in set 2. These two parameters were positively correlated with Spearman's rank order correlation coefficient of 0.96 (P < 0.0001) and 0.95 (P < 0.0001) in sets 1 and 2, respectively. The vertical and horizontal lines indicate the mutation cutoff point of 2% commonly used to distinguish B-CLL patients into mutated and unmutated subsets. All cases were assigned to the same prognostic group (mutated and unmutated CLL) by both methods. The Journal of Molecular Diagnostics 2005 7, 566-574DOI: (10.1016/S1525-1578(10)60589-2) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions