Concomitant peripheral blood smears from the 2nd and 3rd bone marrow biopsies (the slide from the first peripheral blood smear was lost) revealed 53-90% of total cells as atypical LGLs with oval to occasional irregular nuclear contours, some of which eccentrically located, coarse chromatin, absent nucleoli, and abundant pale basophilic cytoplasm and sparse azurophilic granules (). While not present in the peripheral blood smears from the 2nd and 3rd bone marrow specimens, the 1st peripheral blood smear was reported to have approximately 20% medium-sized atypical lymphoid cells with abundant cytoplasm exhibiting circumferential cytoplasmic projections, consistent with hairy cells (data not shown). In addition, there was an agranulocytosis and monocy-topenia seen.
Figure 1 Morphologic features of the T-LGL leukemia, HCL, and PCM in the peripheral blood and bone marrow. A. The predominant cells seen in the concurrent peripheral blood smear of the 2nd bone marrow biopsy were numerous medium-sized lymphoid cells with oval (more ...)
Due to lack of spicules, bone marrow aspirate smear from the 1st bone marrow specimen was not made. Bone marrow aspirate from the 2nd specimen showed occasional (less than 5%) atypical LGLs with similar morphologic features to those from the peripheral blood. However, the prominent feature of the bone marrow aspirate from the 2nd specimen was presence of numerous plasma cells (). These plasma cells accounted for approximately 80% of total cellularity based on 200-cell differential count.
Bone marrow core biopsy from the 1st specimen was approximately 80-90% cellular (), and approximately 80% of which were composed of small to medium sized lymphoid cells with irregular nuclear contours and abundant clear to pale cytoplasm with the so-called “fried egg” appearance (). However, no plasma cells were noted. Other normal hema-tolymphoid cells were present but in decreased numbers.
Bone marrow core biopsy from the 2nd specimen showed an uneven and patchy cellular distribution from less than 5% to focal 60% with an average cellularity of 30%. The cardinal feature was the interstitial plasma cell infiltrate forming sheets and small clusters. These plasma cells constitute -70% of total cellularity (). Of note, atypical lymphoid cells were not easily noted. Bone marrow biopsy from the 3rd specimen showed a marrow of approximately 20% cellularity with scattered plasma cells.
Immunophenotypic analysis of the 1st bone marrow aspirate by flow cytometry revealed two distinct populations. The first population (yellow) represented 44% medium-sized cells which were positive for CD2 (), CD3 (), CD5 (), CD7 (), CD8 (), CD16/CD56 (), CD20 (dim) (), CD57 (variable) (data not shown), TCRa/p (not shown), but negative for CD1a (data not shown), CD4 (), CD19 (), CD23 (), and TCRg/5 (data not shown), consistent with T-LGLs. The second population (red) represented -20% medium-sized cells which were positive for CD19 (), CD20 (bright) (), CD25 (dim) (), CD103 (dim) (), and lambda immunoglobulin light chain (), but were negative for CD5 (), CD10 (), and kappa light chain (data not shown). This population (red) was consistent with hairy cell leukemia. There were no plasma cells seen by flow cytometry.
Figrue 2 Flow cytometric analysis of the T-LGL leukemia, HCL, and PCM on bone marrow aspirates. Compared to the small mature normal T-cells (population in green. A, D-E), the neoplastic T-lineage cells (population in yellow) were slightly larger in size (A), positive (more ...)
Flow cytometric analysis of the bone marrow aspirate from the 2nd bone marrow specimen revealed three separate populations, two of which had similar immunophenotype to those described above from the 1st specimen, consistent with residual T-LGL leukemia and HCL leukemia. The 3rd population was bright positive for CD38 (data not shown), CD138 (), and cytoplasmic lambda (), consistent with monotypic plasma cells.
To confirm our suspicion that the 40% of aberrant LGL cells as revealed by flow cytometry on the 1st bone marrow aspirate was due to peripheral blood contamination, we resorted to immunohistochemical stains on the bone marrow core biopsy. As shown in , there were less than 1% of CD3 (+) (), CD8 (+) () but CD4 (-) () T-cells in the marrow, indicating that the bone marrow was minimally, if any, involved by the patient's T -LGL leukemia.
Figure 3 Immunohistochemical features of the T-LGL leukemia and PCM in the bone marrow. A-C: Scattered CD3(+) (A), CD8(+) (B) and CD4(-) (C) T-cells were seen in the clot section (A-C: Original magnification 200x); D-F: The CD138 (+) (D) plasma cells were positive (more ...)
In order to corroborate the percentage of plasma cells obtained by differential count based on the bone marrow aspirate, a panel of immunohistochemistry was performed on the clot section from the 2nd bone marrow biopsy. CD138 staining () showed that plasma cells accounted for -70% of total cellularity. These plasma cells were positive for lambda () but negative for kappa () mmunoglobulin light chain expression.
Conventional cytogenetics and FISH
Conventional cytogenetic and FISH analysis on cells from the aspirate of the 1st bone marrow specimen revealed no abnormalities. However, conventional karyotyping on cells from the aspirate of the 2nd bone marrow specimen () showed a complex abnormal karyotype as follows: 40-43,XY,-1,add(3)(p26), add(4)(p14),-6, psu dic(6;19)(q25;p13.3),der(8)t(8;17) (p21;q21),-10,add(11)(q23),-14,-17,-20,+mar [cp10] / 46,XY 
Figure 4 Cytogenetic aberrancy by conventional karyotype. Complex cytogenetic aberrancies including loss of chromosomes 1, 6, 14, 17, 20, and additional uncharacterized materials at 3p26 (first arrow), 4p14 (second arrow), 11q23 (fourth arrow) were shown here. (more ...)
Conventional cytogenetic analysis of the bone marrow aspirate on the 3rd specimen showed a complex aberrant karyotype as follows: 4041,XY,der(1)t(1;1)(?;?),?2q,-3, add(4)(p11.2),6,del(7)(q31),der(8)t(8;17) (p11.2;q11.2),-10, add(11)(q23),add(14)(q32),del(17)(p12), add (19)(p13.3),-20,-20,+1-2mar[cp15]/46,XY . nuc ish (FGFR3,IGH)x2,(EGR1x2), (MYBx2), (D7S486x2), (MYCx2) ,(CCND1,IGH)x2,(CCND1,IGH) ×2, (D13S319x2), (IGHx1)[MAFx2)[25/100] (IGH,BCL2) ,(p53x1)[73/100], (D17Z1x3) [78/100].
Since breakpoint at 8p11.2 was seen on 2 consecutive bone marrow aspirates, a FGFR1 breakapart FISH analysis was performed on both CD138 sorted and unsorted cells. No 8p rearrangement involving the FGFR1 locus was observed among 200 cells analyzed.