Adult T-cell leukemia/lymphoma

A cytopathologic, immunocytochemical, and flow cytometric study

Authors


Abstract

BACKGROUND

Adult T-cell leukemia/lymphoma (ATLL) is a postthymic lymphoproliferative neoplasm of T cells caused by human T-cell lymphotropic virus (HTLV-1). Most cases are found in Japan, the Caribbean basin, and West Africa.

DESIGN

To identify diagnostic parameters for cytology in this neoplasm, the authors undertook a retrospective review of all ATLL samples from 1990 to 2000.

RESULTS

One hundred fourteen samples from 34 patients with the diagnosis of ATLL were reviewed: 80 cerebrospinal fluids, 7 pleural effusions, 4 bronchoalveolar lavages, 2 peritoneal effusions as well as fine-needle aspirations of 15 lymph nodes, 4 subcutaneous lesions, and 2 breast nodules. Twenty-one patients were women and 13 were men, with an age range of 30 to 71 years. Morphologically, all specimens were characterized by the presence of a polymorphous population of lymphocytes ranging from small bland-appearing lymphocytes to large atypical ones with bizarre, multilobulated nuclei (flower-like or clover leaf cells) with coarse chromatin and prominent nucleoli. The cytoplasm was deeply basophilic with occasional vacuoles. Immunocytochemistry was performed on 17 specimens from 14 patients. In all cases tested, tumor cells were immunoreactive for CD3, CD4, CD5, and CD25 and were nonimmunoreactive for CD7 and CD8. Flow cytometry was performed on 12 specimens from 9 patients. The tumor cells in all cases tested were positive for CD2, CD3, CD4, CD5, and CD25 and were negative for CD7.

CONCLUSIONS

Despite the polymorphous nature of ATLL, diagnosis can be established by close attention to nuclear cytologic features in conjunction with ancillary studies such as immunocytochemistry and/or flow cytometry. Cancer (Cancer Cytopathol) 2002. © 2002 American Cancer Society. Cancer (Cancer Cytopathol) 2002. © 2002 American Cancer Society.

Adult T-cell leukemia/lymphoma (ATLL) is a postthymic lymphoproliferative neoplasm of helper T lymphocytes caused by human T-cell lymphotropic virus, type 1 (HTLV-1). It is most frequent in areas where the virus is endemic: Japan, the Caribbean basin, and West Africa. It is essentially a disease of adults, characterized clinically by generalized lymphadenopathy, hepatosplenomegaly, skin lesions, and hypercalcemia. It is remarkable for the absence of thymic masses. The prognosis is poor. Morphologically, the leukemic cells have a peculiar polymorphic appearance with markedly lobulated nuclei, often assuming a flower-like shape.1–3 Owing to the rarity of this neoplasm in Western countries, it has rarely been addressed in the cytology literature.

At the National Institutes of Health/National Cancer Institute (NIH/NCI), an immunotherapy protocol for ATLL has been in place for more than a decade, during which the Cytology Section was closely involved in the follow-up of ATLL patients. This study was undertaken to report our experience with 114 ATLL specimens, from various anatomic sites, in an attempt to identify the cytologic features of this tumor. We also report the corresponding immunocytochemical and flow cytometric studies performed on some of these samples.

MATERIALS AND METHODS

We retrospectively reviewed all cases of ATLL received at the NIH/NCI between March 1990 and August 2000. This yielded 114 samples from 34 patients, consisting of 80 cerebrospinal fluids, 7 pleural effusions, 4 bronchoalveolar lavages, 2 peritoneal effusions, and 21 fine-needle aspirations (see Table 1). All patients had a history of ATLL; 21 patients were women and 13 were men, with an age range of 30 to 71 years. The cerebrospinal fluids, pleural and peritoneal effusions, and bronchoalveolar lavages were collected and prepared fresh and then processed as air-dried Diff-Quik –stained (Dade Diagnostics, Aguada, PR) cytospins. In samples showing numerous red blood cells, peripheral blood smears obtained on the same day were reviewed, whenever possible, to rule out peripheral blood contamination. In 10 cases, additional air-dried cytospins were used for immunocytochemistry. Fluid from 4 cases was submitted for flow cytometry.

All fine-needle aspirates (FNAs) were of superficial sites and were performed by a cytopathologist using a 23- or 25-gauge needle, with on-site evaluation of Diff-Quik–stained smears. The remainder of the sample was rinsed with RPMI-1640 (Gibco BRL, Grand Island, NY) and processed as Diff-Quik–stained cytospins. In seven cases, additional air-dried cytospins were used for immunocytochemistry. Eight cases were submitted for flow cytometry.

Immunocytochemistry

Cell suspensions from 17 cases were concentrated in 6-mm-diameter circles on charged (Fisher Scientific, Pittsburgh, PA) or salinated (Digene Diagnostics, Beltsville, MD) glass slides via centrifugation. The slides were air-dried and stored, desiccated at 2–8 °C for up to 48 hours. Slides were fixed in acetone for 10 minutes immediately before the immunoperoxidase assay. All stains were performed according to a modified avidin-biotin-peroxidase procedure with 3,3′-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) as the chromogen. Monoclonal antibodies utilized were as follows: CD3 (titered at 1:100; Becton-Dickinson, San Jose, CA), CD4 (1:20; Becton-Dickinson), CD5 (1:20; Becton-Dickinson), CD7 (1:30; Becton-Dickinson), CD8 (1:20; Becton-Dickinson), and CD25 (1:80; courtesy of Dr. T. Waldemann, NIH). A negative control was performed on each case utilizing mouse myeloma protein immunoglobulin IgG1 Kappa (Organon Teknika, Durham, NC).

Flow Cytometry

Flow cytometry was performed, as previously described,4 in 12 cases. An antibody panel specific for ATLL was chosen. It included CD3 FITC (clone UCHT1; Dako, Carpinteria, CA), CD2 PE (T11; Coulter Cytometry, Miami, FL), CD4 FITC (Leu3a; Becton-Dickinson), CD5 PE (DK23; Dako), CD7 PE (3A1; Coulter Cytometry), and CD25 PE (IL-2R; Becton-Dickinson).

RESULTS

Morphology

Cytologic assessment revealed a polymorphous population of lymphocytes ranging from small to intermediate to large. Atypical lymphocytes were easily noticed. They were characterized by having irregular nuclear contours (flower-like cells), variably prominent nucleoli, and deeply basophilic, occasionally vacuolated cytoplasm. The irregular nuclear contours were not confined to the large lymphocytes, with some small lymphocytes showing markedly convoluted nuclear shapes.

Immunocytochemistry

Immunocytochemistry was performed in 17 cases (Table 1). The atypical lymphocytes were consistently immunoreactive with CD3, CD4, CD5, and CD25 and nonimmunoreactive with CD7 and CD8.

Table 1. Samples Diagnostic for Adult T-Cell Leukemia/Lymphoma
Site of sampleNo. of samplesNo. of patientsNo. of samples for ICCaNo. of samples for FCb
  • ICC: immunocytochemistry; FC: flow cytometry; BAL: bronchoalveolar lavage.

  • a

    Corresponding immunocytochemistry: immunoreactive with CD3, CD4, CD5, and CD25. Nonimmunoreactive with CD7 and CD8.

  • b

    Corresponding flow cytometry: immunoreactive with CD2, CD3, CD4, CD5, and CD25. Nonimmunoreactive with CD7.

  • c

    The study population comprised 34 patients; some had multiple specimens submitted from different anatomic sites.

CSF802140
Pleural effusion7533
BAL4321
Peritoneal effusion2210
FNA211278
Total11434c1712

Flow Cytometry

Twelve cases were submitted to flow cytometry (Table 1). The atypical lymphocytes were immunoreactive with CD2, CD3, CD4, CD5, and CD25 and nonimmunoreactive with CD7.

Cerebrospinal fluid

Eighty air-dried Diff-Quik–stained cytospins from 21 patients were examined. Cytologic examination showed rare to few atypical lymphocytes, ranging from small to intermediate to large, in a variably cellular background (Fig. 1).

Figure 1.

Cerebral spinal fluid samples showing an atypical lymphocyte with irregular “flower-like” nuclear contours, prominent nucleoli, and deeply basophilic cytoplasm (A) and an atypical lymphocyte with irregular nuclear contours and deeply basophilic cytoplasm (B) (note comparison of size with a small lymphocyte) (Diff-Quik stain, original magnification × 1000).

Pleural and peritoneal effusions

Seven air-dried Diff-Quik–stained cytospins of pleural effusions from five patients and two similarly processed peritoneal effusions from two patients were examined. Morphologic assessment revealed few mesothelial cells admixed with a polymorphous population of atypical lymphocytes (Fig. 2).

Figure 2.

Pleural effusion showing a polymorphous population of lymphocytes, some with markedly irregular nuclear contours (flower-like cells) surrounding a mesothelial cell (A) and adjacent to a mesothelial cell cluster (B) (Diff-Quik stain, original magnification ×600).

Bronchoalveolar lavages

Four air-dried Diff-Quik–stained cytospins of bronchoalveolar lavages from three patients were examined. Cytologic examination revealed few bronchial cells and a moderate number of alveolar macrophages admixed with a polymorphous population of atypical lymphocytes (Fig. 3).

Figure 3.

(A) Bronchoalveolar lavage showing a polymorphous population of lymphocytes admixed with ciliated bronchial columnar cells and alveolar macrophages (Diff-Quik stain, original magnification ×400). (B) Bronchoalveolar lavage showing large atypical lymphocytes with irregular nuclei and basophilic, vacuolated cytoplasm (Diff-Quik stain, original magnification ×600).

FNAs

Twenty-one air-dried Diff-Quik–stained smears and cytospins of FNAs of various sites were examined: 15 lymph nodes, 4 subcutaneous nodules, and 2 breast masses. Cytologic examination revealed a polymorphous population of lymphocytes, including easily identifiable small, intermediate, and large cells having markedly irregular multilobulated nuclei, prominent nucleoli, and deeply basophilic cytoplasm. Scattered mitoses were noted in the background, but there was no evidence of necrosis (Fig. 4). The morphologic findings are summarized in Table 2 (two FNAs were not available for review).

Figure 4.

(A) Fine-needle aspirate (FNA) of an axillary lymph node showing large atypical lymphocytes with markedly irregular nuclear contours and basophilic, vacuolated cytoplasm (Diff-Quik stain, original magnification ×600). (B) FNA of an axillary lymph node showing a polymorphous population of lymphocytes with marked nuclear convolutions noted in small lymphocytes (Diff-Quik stain, original magnification ×600).

Table 2. Sites and Cytomorphologic Features of 19 Fine-Needle Aspirates of ATLL
Case no.SiteSize of lymphocytesCytoplasmNucleiNucleoliLGBLT
  1. ATLL: adult T-cell leukemia/lymphoma; LGB, lymphoglandular bodies; LT, lymphoid tangles; I, intermediate; L, large; B, basophilic; V, vacuolated; R, round; O, ovoid; Ir, irregular; S, small.

1Axillary lymph nodesI to LB, fine VR, O, Ir1–4++
2Abdominal noduleI to LBR, O, Ir1–4++
3Inguinal lymph nodesI to LB, rare VR, O, Ir1–2++
4Cervical lymph nodesS to LB, rare VR, O, Ir1–3++
5Inguinal lymph nodesS to LB and VR, O1–3++
6Leg noduleS to LB and VR, O1–2++
7Cervical lymph nodesS to LB and VR, O1–2++
8Left breastS to LB and VR, O, Ir1–3++
9Femoral lymph nodesS to LBR, O, Ir1–2++
10Inguinal lymph nodesS to LBR, O, Ir1–3++
11Inguinal lymph nodesS to LBR, O, Ir1–3++
12Inguinal lymph nodesS to LBR, O, Ir1–4++
13Axillary lymph nodesS to LBR, O, Ir1–4++
14Inguinal lymph nodesS to LBR, O, Ir1–2++
15Leg noduleI to LBR, O, Ir1–4++
16Neck massI to LB, rare VR, O, Ir1–2++
17Right breastS to LB, rare VR, O, Ir1–4++
18Inguinal lymph nodesS to LBR, O, Ir1–2++
19Cervical lymph nodesS to LBR, O, Ir1–3++

DISCUSSION

The human T lymphotropic virus type 1 (HTLV-1) is a complex type C retrovirus, which was first isolated and identified in 1980, from a patient with cutaneous T-cell lymphoma.5 Subsequent seroepidemiologic, morphologic, and molecular studies confirmed that HTLV-1 is the causative agent of ATLL cases that had been described a few years earlier in Japan.1 The virus spreads by sexual contact, intravenous drug use, breast milk, and blood transfusions.1, 3

Adult T-cell leukemia/lymphoma is a distinct clinicopathologic syndrome characterized by the presence of leukemic cells with highly convoluted nuclei “flower-like cells,” lymphadenopathy that spares the mediastinum, hepatosplenomegaly, skin lesions, hypercalcemia associated with lytic bone lesions, and generalized bone resorption.1–3, 6 Several clinical subtypes have been described: acute, lymphomatous, and chronic types and a rare smoldering type. The prognosis is generally poor.1 The tumor cells have a characteristic immunophenotype: they express CD2, CD3, CD4, CD5, and CD25 and lack CD7; rare cases are CD8 positive.7, 8 This profile distinguishes ATLL from other peripheral T-cell lymphocytic proliferations, such as T-cell prolymphocytic leukemia (CD4+, CD7+, CD8+/−, and CD25+/−) and mycoses fungoides/Sezary syndrome (CD4+,CD8−, and CD25−).8

The role of cytology in the primary diagnosis, subclassification, and management of non-Hodgkin lymphomas has long been a controversial subject.9–14 However, after the introduction and subsequent widespread use of the revised European–American Lymphoma (REAL) classification of non-Hodgkin lymphomas,7 with its emphasis on immunophenotypic analysis along with clinical features and morphologic criteria, it has become clear that cytology specimens in conjunction with ancillary studies are well suited for the diagnosis and classification of lymphomas. This has been evidenced by studies showing that non-Hodgkin lymphomas can be accurately diagnosed in cytologic specimens utilizing ancillary techniques such as immunocytochemistry and flow cytometry.4, 15–32

In the REAL classification, ATLL belongs to the category of peripheral T-cell and natural killer cell neoplasms. Although several reports of peripheral T-cell lymphoma have been published in the cytology literature,33–37 we are aware of only a single cytologic study of T-cell lymphoma in HTLV-1 positive patients.38 In our study, we reviewed 114 samples from 34 patients already diagnosed with ATLL, from diverse anatomic sites. Cytologically, we noted a polymorphous population of lymphocytes that ranged in size from small to intermediate to large. The nuclei varied from round to oval to irregular with some showing highly convoluted, flower-like shapes. Of note, the irregular nuclear contours were observed in small lymphocytes and in large ones. Variably prominent nucleoli were present, and the cytoplasm was deeply basophilic with occasional vacuolizations. An immunophenotypic panel characteristic of ATLL (CD2+, CD3+, CD4+, CD5+, and CD25+ and CD7−) was utilized to confirm the diagnosis in selected cases (17 by immunocytochemistry and 12 by flow cytometry).

Because this disease has well known peripheral blood involvement, in our exfoliative cytology samples (cerebrospinal fluids, bronchoalveolar lavages, pleural and peritoneal fluids) when numerous red blood cells were observed, we attempted to review the peripheral blood smears of the patients from the same day to rule out the possibility of peripheral blood contamination.

Oshima et al.38 studied fine-needle aspiration smears of seven lymphomas from HTLV-1 positive patients. They reported a predominance of round cells with irregular nuclei and moderately basophilic cytoplasm, with some smears also showing a few giant cells with cerebriform nuclei; a feature that was not observed in our cases. All but one of their cases were composed of T-helper cells with rearrangements of the T-cell receptor.

Few studies describe the histopathologic features of lymph nodes involved by ATLL. All stress the pleomorphic or polymorphous nature of ATLL lymphomas,2, 6 thus concurring with our observations. Jaffe et al.6 studied 13 lymph nodes from patients with ATLL that showed diffuse and pleomorphic lymphomas including diverse histologic subtypes: mixed small and large cell, large cell, large cell immunoblastic, and unclassifiable. They noted that, unlike B-cell lymphomas, there was no correlation between histologic subtype and clinical outcome. Kikuchi et al.,2 in their study of 39 lymph nodes involved by ATLL, characterized the lymphomas as diffuse pleomorphic type, composed of variable-sized and shaped atypical lymphocytes, with irregular nuclear contours and the occasional presence of giant cells with highly convoluted nuclei.

Unlike other peripheral T-cell lymphomas in which the diagnosis depends on lack of expression of B-cell markers and detection of aberrant expression of pan T-cell markers,36, 37 ATLL has a distinctive immunophenotypic profile. In conclusion, we believe that despite the polymorphous appearance of lymphocytes in ATLL, the diagnosis can be suspected morphologically by paying close attention to nuclear details and the spectrum of cell sizes and confirmed by utilizing a specific antibody panel for immunocytochemistry and/or flow cytometry.

Ancillary