Clinical, histological, phenotypical and molecular findings in the present series of FL are summarized in Table . The diagnosis of FL was confirmed in all 82 cases retrieved from the archives: 77 were nodal FL, of which 67 were only constituted by FL and 10 showed FL in combination with a LBCL; the last 5 cases were cutaneous FL which appeared negative for extra-cutaneous disease at the diagnosis and during the follow-up and were therefore considered primary CFL.
Clinical, histological, phenotypical and molecular findings in the series of 82 FL
No difference for age and gender was found among the 3 subgroups ("pure" FL: 28 M, 39F, median age 59 yrs; FL in combination with LBCL: 5 M, 5 F, median age 54 yrs; primary CFL: 3 M, 2 F, median age 53 yrs). Of the nodal FL cases, the most numerous group were those showing grade 2 with a predominantly nodular pattern. Marginal zone differentiation was seen only in this group (7 cases, 9.9%). Six cases showed an "in situ" pattern: in all, the lesion was incidentally discovered; they were 3 males and 3 females, with no difference in age compared to the other subgroups of FL (median age, 61.5, range 43-79). The lesion was diagnosed during the pathological staging of epithelial neoplasms in regional lymph nodes in 4 cases (three women with breast carcinomas and the lesion involving the axillary lymph nodes, and one man with gastric cancer, with the lesion involving the peri-gastric lymph nodes). In the other 2 cases the lesion was found in slightly enlarged inguinal lymph nodes excised during vascular surgery. In all cases, in lymphoid tissue devoid of metastases or any other destructive process, besides normal looking follicles, a variable number of germinal centers, ranging from few to the majority, showed partial or total substitution by small B-lymphoid cells with centrocyte appearance, immunoreactive for CD10 and BCL2 in all cases, for BCL6 in 3 cases, and with a mean Ki67-index of 5%; in all cases the cell infiltrate did not show tendency to invade the interfollicular areas (Figure ). Cytoplasmic HGAL staining was confined to the atypical GCs with a stronger intensity in small atypical cells with strong staining for BCL2 (Figure ). The immunostaining for BCL2 was intense and strong in all cases. The difference of Ki67 proliferation index was statistically significant compared to the other invasive FL (ANOVA test, p = 0.003). According to WHO recommendations, patients were staged, but they did not receive any treatment as no evidence of "overt" FL elsewhere was found. By PCR analysis, four cases showed either monoclonal IGH@ gene rearrangement (2) or MBR-BCL2/JH rearrangement (2).
"In situ" follicular neoplasm. The follicle depicted in serial sections shows partial polarization loss and prevalence of small cells (a), all staining for CD10 (b), strongly for BCL2 (c) and for HGAL (d).
The other 61 nodal "pure" FL (Figure ) stained more frequently for BCL6 (96.7%) than for CD10 (90.1%); staining for both GC-markers was detected in 53 cases (86.9%; Figure ). Cytoplasmic positivity for HGAL was found both in the nodular and in interfollicular components of FL (Figure ). In the remaining cases BCL6 was positive in 6 and CD10 in two. BCL2 positivity was found in 53 cases (86.9%): it ranged from faint and partial, limited to a thin perinuclear distribution and recognizable only at higher magnification (Figure ), to strong and diffuse, visible at glance at low power. Ki67-antigen proliferative index was highly correlated with grade (ANOVA test, p < 0.001; Figure ). Molecular tests were positive in 49 cases (80.3%): in 14 both IGH@ gene rearrangement and MBR-BCL2/JH rearrangement were positive, in the remaining 35 only the test of clonality was positive.
Figure 2 Invasive follicular lymphoma. FL is formed by large nodules with interfollicular invasion (a) showing positive staining for CD10 (b), BCL6 (c), HGAL (d); the positivity for GC markers are more evident in the nodular component than in the interfollicular (more ...)
In 10 cases of nodal FL, there was a variable admixture of LBCL, displayed by the presence of a diffuse interfollicular infiltrate of cohesive large blasts. For the purposes of the present study, evaluation of immunostainings in these cases was done only in the areas considered as FL which mainly coincided with follicular areas highlighted by networks of dendritic follicular cells staining either with CD21 or CD23; only in one case there was also a diffuse areas of FL without cohesive blast clusters, not realizing the pattern of LBCL. Therefore the pattern in these cases was mainly nodular. FL cells stained positively for BCL6 and BCL2 in 9 cases and for CD10 in 8. HGAL was positive in all cases. Mean Ki67-antigen proliferative index was significantly higher than in "pure" FL (68.0 vs 31.82; ANOVA test, p < 0.0001). Molecular tests were positive in 9 cases (90%): in one case both IGH@ gene rearrangement and MBR-BCL2/JH rearrangement were positive, in the remaining 8 only test of clonality was positive.
In CFL all cases showed staining for CD10, BCL6, and HGAL, and 4 for stained for BCL2. Ki67 proliferative index was lower than that of FL combined with LBCL (ANOVA test, p < 0.001). Only monoclonal IGH@ gene rearrangement was detected in 4 cases.
The percentage of HGAL-positive cells ranged from 5 to 100% (mean, 64.89 ± 21.10). The number of neoplastic cells stained for HGAL was similar between CFL, FL and FL combined with LBCL. Applying the cut-off of 20%, 80 cases were considered positive (97.6%) and 2 (2.4%) negative. Immunostaining for HGAL was more frequently positive than that for BCL6 and CD10; the cases negative for BCL6 (5) or for CD10 (7) or for both marker (1) were all positive for HGAL, whereas the two cases negative for HGAL were positive with BCL6. Combination of positivity of the three GC-marker is reported in Table .
Combination of GC-marker immunoreactivity in the series of FL