Chronic Lymphocytic Leukemia FISH Panel: Impact on Diagnosis

Beverly P. Nelson, MD; Rohit Gupta, MD; Gordon W. Dewald, PhD; Sarah F. Paternoster; Steven T. Rosen, MD; LoAnn C. Peterson, MD

Disclosures

Am J Clin Pathol. 2007;128(2):323-332. 

In This Article

Results

Summary of Cases

A total of 136 cases of presumed CLL were referred for the FISH panel. Three cases were excluded from analysis because neither immunophenotypic nor morphologic data were available to correlate with the FISH results, and 22 were not CLL based on review of the morphologic features and immunophenotype. The remaining 111 cases included 71 men and 40 women ranging in age from 25 to 84 years (median, 61 years). Specimens used for FISH analysis were peripheral blood (61), bone marrow (49), and lymph node (1).

FISH Results

Cases With CCND1/IGH Fusion Classified as Mantle Cell Lymphoma. Two cases initially regarded as CLL based on clinical manifestations demonstrated CCND1/IGH fusion, indicating bcl-1 gene rearrangement. One patient, a 41-year-old man, had marked lymphocytosis in the peripheral blood (WBC count, 155,400/µL [155.4 × 109/L]; hemoglobin, 10.6 g/dL [106 g/L]; mean corpuscular volume [MCV], 90 µm3 [90 fL]; platelet count, 343 × 103/µL [343 × 109/L]; lymphocytes, 96.5% [0.97]; and neutrophils, 3.5% [0.04]). The lymphocytes varied from small cells with condensed chromatin and round nuclei to medium-sized cells with slightly irregular nuclei Figure 1. Flow cytometric immunophenotyping showed monotypic B cells that were CD19+, CD20+, CD5+, bright CD23+, bright FMC7+, and bright CD79b+. In addition to CCND1/IGH fusion, FISH also showed deletion of chromosomes 11q and 13q.

Figure 1.

Mantle cell lymphoma in peripheral blood. The lymphocytes show condensed chromatin, with round nuclei, but cells with slightly irregular nuclei and visible nucleoli that are unusual for chronic lymphocytic leukemia are also present (Wright-Giemsa, ×1,000).

The second patient, a 76-year-old man, had absolute lymphocytosis (WBC count, 11,500/µL [11.5 × 109/L]; hemoglobin, 10.4 g/dL [104 g/L]; MCV, 76 µm3 [76 fL]; platelet count, 155 × 103/µL [155 × 109/L]; lymphocytes, 50.2% [0.50]; neutrophils 35.4% [0.35]; monocytes, 10.3% [0.10]; eosinophils, 3.1% [0.03]; and basophils, 1.0% [0.01]). Lymphocyte morphologic features were unusual for CLL; the cells were primarily small with condensed chromatin and scant cytoplasm but included occasional larger cells with visible nucleoli. Flow cytometric immunophenotyping of the blood showed monotypic B cells that were CD5+, CD10-, CD20+, dim CD23+, bright FMC7+, and bright CD79b+. In addition to CCND1/IHG fusion, 13q and 17p deletions were also identified.

Although both cases were clinically regarded as CLL, their morphologic features and immunophenotype were atypical for CLL. Even though both cases were CD20+, CD5+, and CD23+, the bright FMC7 and CD79b positivity are unusual for CLL. FISH demonstrated CCND1/IGH fusion, and the cases were classified as mantle cell lymphoma (MCL).

CLL Cases. Of 109 CLL cases, 79 (72.5%) had 1 or more genetic abnormalities; the remaining 30 cases (27.5%) had normal FISH results. The majority of the patients with genetic alterations, 67% (53/79), had a single abnormality. Two alterations were present in 25% (20/79), and 8% (6/79) had 3 or more genetic alterations.

Chromosome 13q deletion (13q-) was the most common aberration Table 1 . Deletion of chromosome 13q was the only abnormality detected in more than half (33/53 [62%]) of the cases with 13q-. Trisomy 12 was the second most frequent abnormality and was the sole abnormality in 13 cases. Deletion of chromosome 11q was the third most common finding. Abnormalities of chromosomes 17p and 14q32 occurred with similar frequencies. The least common abnormality was deletion of chromosome 6q.

An abnormal signal pattern for the IGH gene was present in 11 CLL cases. Translocations were identified in 4 of these cases Table 2 . One or more extra IGH signals without an identifiable fusion partner were present in 6 cases, and the 3' end of the IGH variable region (IGHv) signal was absent in 1 case Table 3 .

FISH Results Correlated With Phenotypic, Morphologic, and Other Pathologic Findings

Immunophenotypic and Morphologic Findings. A total of 109 cases displayed typical phenotypes, and 2 were atypical with bright CD20+ results. The 2 CLL cases with bright CD20 staining had isolated trisomy 12. Nine CLL cases displayed atypical morphologic features for CLL; they included 2 with translocations involving chromosome 14q32 (bcl-3 and bcl-11a; discussed in the next section), 3 with isolated trisomy 12, 2 with isolated 13q deletion, and 2 with normal FISH results.

CLL Cases With Chromosome 14q32 Translocations. Eleven CLL cases demonstrated abnormalities involving the IGH gene located at chromosome 14q32. Of these, 4 had had translocations ( Table 2 ). These cases are discussed in more detail because the FISH results were informative for the diagnoses.

The first case, a 46-year-old man, had absolute lymphocytosis in the blood (WBC count, 32,100/µL [32.1 × 109/L]; hemoglobin, 14.3 g/dL [143 g/L]; hematocrit, 43.8% [0.44]; MCV, 84 µm3 [84 fL]; platelet count, 286 × 103/µL [286 × 109/L]; lymphocytes, 86% [0.86]; neutrophils, 10% [0.10]; bands, 2% [0.02]; monocytes, 1% [0.01]; and eosinophils, 1% [0.01]) associated with lymphadenopathy and splenomegaly. A bone marrow biopsy showed a lymphoid infiltrate with a diffuse growth pattern involving approximately 80% of the section. The lymphocytes were primarily small with condensed chromatin but included many cells with angulated or cleaved nuclei Figure 2A. The morphologic features of the lymphocytes were not typical for CLL and raised the possibility of another type of non-Hodgkin lymphoma.

Figure 2.

A, Chronic lymphocytic leukemia (CLL) with bcl-11a translocation involving the bone marrow aspirate. The lymphocytes are primarily small with condensed chromatin. However, many display angulated nuclei and do not resemble typical CLL cells (Wright-Giemsa, ×600). B, CLL with bcl-11a translocation involving the lymph node. The infiltrate is composed of small lymphocytes with a diffuse growth pattern and includes proliferation centers that are typical of CLL (H&E, ×100).

Flow cytometric immunophenotyping findings, however, were characteristic of CLL: k surface immunoglobulin (sIg) light chain-restricted B cells that were CD19+/CD20+/CD5+/CD10-/CD23+/CD79b-/FMC7-. Small lymphocytic lymphoma/CLL was confirmed with a lymph node biopsy that showed effacement of the normal architecture by a proliferation of small lymphocytes with a diffuse growth pattern that included proliferation centers Figure 2B. FISH analysis revealed fusion of IGH and bcl-11a, indicating t(2;14)(p13;q32), which rarely occurs in CLL.[10,11] Deletion of the long arm of chromosome 11 was also identified.

His illness was characterized by development of massive generalized lymphadenopathy that encased major blood vessels, including the inferior vena cava, aorta, common iliac arteries, portal vein, splenic vein, and superior mesenteric vein. Multiple bilateral pulmonary nodules, splenomegaly that required radiation therapy for symptom management, severe thrombocytopenia (platelet count, 4 × 103/µL [4 × 109/L]), and anemia (hemoglobin, 7.4 g/dL [74 g/L]) also developed. He was treated with several different chemotherapeutic regimens throughout the disease course. The therapies included fludarabine; fludarabine/cyclophosphamide; cyclophosphamide, doxorubicin, vincristine, and prednisone; and pentostatin/rituximab. He died of progressive disease 51 months after initial diagnosis.

The second case was a 52-year-old man who had absolute lymphocytosis in the blood (WBC count, 85,000/µL [85.0 × 109/L]; hemoglobin, 14.3 g/dL [143 g/L]; MCV, 90 µm3 [90 fL]; platelet count, 170 × 103/µL [170 × 109/L]; lymphocytes, 87% [0.87]; neutrophils, 11% [0.11]; and monocytes, 2% [0.02]) and lacked lymphadenopathy. The lymphocytes were morphologically atypical for CLL and included small cells with condensed chromatin and round nuclei Figure 3A and many cells with irregular nuclear contours and larger lymphocytes with more abundant cytoplasm and visible nucleoli. The bone marrow trephine biopsy section was hypercellular with an extensive lymphoid infiltrate that displayed interstitial Figure 3B and diffuse growth patterns. Flow cytometric immunophenotyping showed k sIg light chain-restricted B cells that were CD19+, CD20+, CD5+, CD10-, dim CD23+, and dim FMC7+.

Figure 3.

A, Chronic lymphocytic leukemia (CLL) with bcl-3 translocation involving the blood. The lymphocytes are morphologically heterogeneous and include small cells with condensed chromatin and round nuclei, many cells with irregularly shaped nuclei, and also larger cells with visible nucleoli (Wright-Giemsa, ×600). B, CLL with bcl-3 translocation involving the bone marrow core biopsy section. The infiltrate includes increased numbers of lymphocytes with visible nucleoli and more dispersed chromatin (H&E, ×200).

The phenotype was compatible with CLL, but the unusual morphologic features and dim CD23 staining raised the possibility of MCL. Although conventional chromosome banding of the bone marrow demonstrated a normal male karyotype, FISH showed trisomy 12 and fusion of the bcl-3 gene located at 19q13 to the IGH gene, resulting in t(14;19)(q32;q13), a translocation reported in rare CLL cases. Absence of CCND1/IGH fusion in this case excluded MCL. CLL with atypical morphologic features was diagnosed. After almost 2 years with a stable clinical course, he was treated with fludarabine, cyclophosphamide, and rituximab for progressive disease that responded well to therapy. He was alive with disease at 38 months.

The third patient with CLL with IGH fusion was a 47- year-old woman who also had a bcl-3 translocation. She was first given a diagnosis of Rai stage 0 CLL in May 1997 when a CBC count showed absolute lymphocytosis, anemia, and a normal platelet count (WBC count, 50,200/µL [50.2 × 109/L]; hemoglobin, 10.8 g/dL [108 g/L]; platelet count, 236 × 103/µL [236 × 109/L]; lymphocytes, 85% [0.85]; neutrophils, 12% [0.12]; and monocytes, 3% [0.03]). Flow cytometry performed on the blood in 2001 showed that the lymphocytes were dim k sIg light chain restricted, dim CD20+, CD5+, CD10-, CD23+, CD38+, CD52+, FMC7-, and dim CD79b+. A bone marrow biopsy specimen was hypercellular (80%) with a dense lymphoid infiltrate composed of small, mature-appearing cells.

Fludarabine was given for 6 months beginning in August 1997. She achieved and remained in remission until March 2000, when lymphocytosis recurred. Additional combination chemotherapy was given, resulting in a partial response. Autologous stem cell transplantation was performed in June 2002 because of progressive disease that was no longer responsive to chemotherapy; she achieved a complete morphologic and molecular remission with negative IGH gene rearrangement shown by polymerase chain reaction of blood and bone marrow specimens.

In June 2003, she was referred to NMH for recurrent disease. At this time, the blood smear showed many large lymphocytes with visible nucleoli in addition to small, mature-appearing lymphocytes consistent with prolymphocytoid transformation Figure 4. The immunophenotype was similar to that in the initial study except that CD23 was now negative. The initial history of CLL was not immediately available, and non-Hodgkin lymphoma, in particular MCL, was considered. However, FISH analysis of the blood (performed at the time of prolymphocytoid transformation) showed a hyperdiploid clone including 3 or 4 copies of chromosomes 6, 11, 12, 13, and 14 with 13q-, 17p-, and fusion of IgH and bcl-3, indicative of t(14;19)(q32;q13). Based on these data, the history of CLL, and review of the prior blood and bone marrow specimens, the patient was given a diagnosis of prolymphocytoid transformation of CLL and treated with more aggressive chemotherapy-cyclophosphamide, etoposide, methotrexate, bleomycin, and vincristine-without significant response, and she died 83 months after initial diagnosis.

Figure 4.

A, Chronic lymphocytic leukemia (CLL) with bcl-3 translocation involving the blood of a 47-year-old woman. The lymphocytes range from small cells with condensed chromatin and scant cytoplasm to medium-sized cells with more ample cytoplasm and visible nucleoli (Wright-Giemsa, ×600). B, CLL with bcl-3 translocation involving the bone marrow core biopsy section. The infiltrate includes increased numbers of lymphocytes with visible nucleoli and more dispersed chromatin (H&E, ×600).

The last CLL case with a translocation involving chromosome 14q32 was a 52-year-old man with absolute lymphocytosis in the blood (WBC count, 30,300/µL [30.3 × 109/L]; hemoglobin, 16.7 g/dL [167 g/L]; hematocrit, 47.9% [0.48]; MCV, 92 µm3 [92 fL]; platelet count, 300 × 103/µL [300 × 109/L]; lymphocytes, 84% [0.84]; neutrophils, 15% [0.15]; and monocytes, 1% [0.01]) that was first identified during an annual physical examination. Left axillary lymph nodes were also enlarged, but neither a bone marrow nor a lymph node biopsy was performed. The lymphocytes in the blood were small with condensed chromatin and scant cytoplasm Figure 5. Flow cytometric analysis of the blood showed CD19+, CD20+, CD5+, CD10-, CD23+, CD79b-, FMC7-, and k sIg light chain-restricted B cells. CLL was diagnosed.

Figure 5.

Chronic lymphocytic leukemia with bcl-2 translocation involving the blood. The lymphocytes are small with condensed chromatin, scant cytoplasm, and mostly round nuclei (Wright-Giemsa, ×600).

FISH analysis demonstrated fusion of the bcl-2 and IGH genes, resulting in t(14;18)(q32;q21), as well as deletion of the long arm of chromosome 13. Although an unusual presentation of follicular lymphoma with leukemic phase could not be completely excluded, the immunophenotype and morphologic features of the lymphocytes were considered most consistent with CLL. His disease was stable without treatment, and he was alive 44 months after initial diagnosis.

CLL Cases With Chromosome 14q32 Aberrations With No Translocation Partner Identified. Seven CLL cases without translocations had an abnormality involving the IGH gene. Six CLL cases had at least 1 extra signal for chromosome 14q32 without fusion of IGH with CCND1, bcl-2, bcl-3, bcl-11A, c-myc, or MALT1. They included 3 men and 3 women ranging in age from 53 to 74 years ( Table 3 ). All had typical lymphocyte morphologic features and immunophenotype for CLL. Four had Rai stage IV CLL, 1 had stage I, and 1 had stage 0. Deletion of chromosome 13q was detected in 3 cases, trisomy 12 in 2 cases, and 17p deletion in 1 case; no additional aberration was present in 2 cases.

The final CLL case with a chromosome 14q32 aberration and no identified translocation had loss of the 3' IGHv signal and trisomy 12. The results of chromosome banding analysis were similar; in 12 cells, they showed an interstitial deletion in chromosome 14 with breakpoints at 14q24 and 14q32, as well as trisomy 12. This patient, a 29-year-old man, had typical lymphocyte morphologic features and immunophenotype for CLL, Rai stage III disease at initial examination, and an aggressive clinical course that required therapy at the time of diagnosis because of bulky lymphadenopathy.

Correlation of FISH Prognostic Groups With CD38/ZAP-70 Status. The majority of the 109 CLL cases (63 [57.8%]) were in the favorable prognostic group with isolated 13q– or normal FISH results Table 4 . Thirteen cases (11.9%) were in the intermediate prognostic group with isolated trisomy 12; 25 (22.9%) were in the least favorable prognostic group, with 17p- or 11q-; this group included the 4 patients with 6q- because all 4 also had 17p-.

Of the 90 CLL cases analyzed for CD38, 81 were placed in prognostic groups. Nineteen (23%) of the 81 were CD38+. A similar percentage of CD38+ cases was present in cases with 17p (2/6 [33%]) and 11q deletion (4/11 [36%]) and cases with normal FISH results (9/27 [33%]) ( Table 4 ). CLL cases with trisomy 12 and isolated 13q- had the lowest percentage of CD38+ cases; 15% (2/13) and 8% (2/24), respectively. ZAP-70 was tested in 36 cases; 10 were positive. In this limited number of cases tested for ZAP-70, the ZAP-70 staining pattern did not correlate with different genetic prognostic groups and was negative in all 3 cases with IGH anomalies that were tested ( Table 3 ).

The majority of cases in the poor prognostic FISH groups of isolated 17p- (10/12 [83%]) and isolated 11q- (8/13 [62%]) had advanced disease with Rai stage III or IV. More patients in the intermediate and good prognostic groups had disease with a low Rai stage, 0 to II: trisomy 12 (7/11[64%]), normal FISH results (16/25 [64%]), and isolated 13q- (20/32 [62%]) Table 5 . Interestingly, the majority (5/7) of CLL cases with an IGH anomaly but without an identifiable translocation partner had Rai stage III or IV disease.

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