Fine-Needle Aspiration Evaluation of Lymphoproliferative Lesions in Human Immunodeficiency VirusPositive Patients

A Multiparameter Approach
Nina Shabb, MD, Ruth Katz, MD, Nelson Ordonez, MD, Angela Goodacre, BSc, Cheryl Hirsch-Ginsberg, MD, and Adel El-Naggar, PhD, MD
Forty-six fine-needle aspirates of lymphoproliferative lesions from 31 human immunodeficiency virus (H1V)-positivepatients were reviewed using cytomorphologic, immunocytochemical, flow cytometric (FCM), cytogenetic, and molecular studies. There were 29 lymphomas (15 small non-cleaved cell [SNCL], 11 large cell [LCL], one small lymphocytic, and two Hodgkins), 14 reactive hyperplasias, and three atypical lymphoid proliferations. The reactive hyperplasias were characteristically polymorphic and polyclonal lymphoid populations; six of seven were diploid on FCM, the seventh was hypodiploid. Higher proliferative indices (mean, 11.6%) and higher RNA indices (mean, 1.2) characterized this subgroup compared with published reactive lymphoid hyperplasias from patients without HIV positivity. Aspirates of SNCL showed monotonous populations of intermediate-sized cells except in one patient where a giant cell syncytial variant occurred. Nine of 13 SNCL aspirates showed light chain restriction. JH rearrangement revealed B-cell lineage in one aspirate in which immunocytochemical study was negative for Kappa, lambda, B1,and Leu-4. Nine of 12 SNCL were diploid; the mean proliferative index was 25.6% and the mean RNA index 2.3.Chromosomal translocations involving the c-myc locus were demonstrated in five of seven SNCL aspirates karyotyped. Five of eight LCL showed light chain restriction the remaining three showed null cell phenotype. Large cell lymphomas were diploid on tetraploid with the mean proliferative index of 22.0% and mean RNA index of 2.2. One of two LCL aspirates karyotyped demonstrated cmyc translocation. Despite the multiparameter approach, a definitive diagnosis could not be reached in three aspirates. Cancer 67:1008-1018,1991.

occurs in human immunodeficiency virus (HIV)-infected patients is most often due to a nonneoplastic lymphoid proliferation (HIV lymphadenitis),- malignant l y m p h ~ m a , ~or in- ~

HE LYMPHADENOPATHY that

From the Division of Pathology, Sections of Cytopathology and Surgical Pathology, and the Departments of Hematology and Laboratory Medicine, the University of Texas M. D. Anderson Cancer Center, Houston, Texas. The authors thank Dr. W. Velasquez for guidance and support of this study. Address for reprints: Nina Shabb, MD, Division of Pathology, Section of Cytopathology, Box 085, 15 15 Holcombe Blvd., Houston, TX 77030. Accepted for publication June 1 1, 1990.

fectious processes (usually mycobacterial). Rare causes include Kaposis sarcoma, metastatic nasopharyngeal carcinoma, and squamous cell carcinoma. 1 2 3 1 5 * 1 6 The nonneoplastic lymph nodes show a spectrum of morphologic changes, ranging from marked lymphoid hyperplasia (Stage I) to marked lymphocytic depletion (Stage III).1,2*5 The HIV-positive subjects are at almost 200-fold greater risk for developing lymphoma than their age-matched and sex-matched counterparts in the general population.* The increase is mainly in diffuse high-grade, B-cell non-Hodgkins ly rn p h ~ r n a s ,~although small but significant in-~ creases in the frequency of Hodgkins disease have also been demonstrated. 17-23 The Center for Disease Control

1008

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1009

has recently amended its definition of the acquired immune deficiency syndrome (AIDS) to include non-Hodgkins lymphoma of high-grade, B-cell type in the presence of a positive serologic or virologic test for H I V . ~ ~ The multiplicity of causes of lymphadenopathy in HIVpositive patients makes accurate tissue diagnosis important for selection oftherapy. Fine-needle aspiration (FNA), because of its minimal invasiveness, would seem an excellent technique in this setting; however, its use in the diagnosis of lymphoproliferative lesions has been controversial.16 At M. D. Anderson Cancer Center (Houston, TX), we have developed a multimodal strategy for using FNA in such cases examining cytologic analysis in conjunction with immunocytochemical, flow cytometric (FCM), cytogenetic, and molecular studies for a high degree of diagnostic accuracy.25Here we report results with this approach in 46 FNA specimens (40 nodal, six extranodal) from 3 1 HIV-positive patients.

Cancer Institutes working formulation for non-Hodgkins lymph~rna.~

Materials and Methods

Case Retrieval
Review of the July 1986 to July 1989 computerized records of the pathology division and hematooncology patient lists identified 46 aspirates from 3 1 HIV-positive patients. The patients charts were reviewed, HIV status confirmed, and pertinent information recorded.

Immunocytochemical Study Immunocytochemical studies were performed on airdried cytospin preparations fixed in absolute acetone at 4C for 10 minutes. Staining for Kappa (gK) and lambda (gl), light chains was done by a direct immunoperoxidase method using affinity-isolatedgoat anti-human kappa and lambda antisera (Tago, Burlingame, CA; 1:75 dilution). Specimens were also stained by the avidin-biotin-peroxidase complex (ABC) method2*for CD3 (Leu-4, Beckton-Dickinson, Mountainview, CA; 1: loo), CD20 (Bl, Coulter Electronics, Hialeah, FL; 1:25), and CD19 (Leu12, Coulter Electronics; 150). The immunocytochemical reaction was visualized by using 3-amino-9-ethylcarbaole as substrate. In each aspiration control cytospin preparation was stained with Wright-Giemsa for confirmation of the presence of lymphoid cells and for comparison with immunoperoxidase results. For each surface marker, the number of positive cells was visually estimated as a percentage of the total mononuclear cell population. Polyclonal immunostaining was defined by an admixture of Leu-4-staining, Kappa-staining, and lambda-staining cells in physiologic d i ~ t r i b u t i o nMonoclonality was defined .~~ by a Kappa:lambda or 1ambda:Kappa ratio of >6: 1 .25 Flow Cytometric Analysis Flow cytometric analysis was performed on an ICP-22 mercury arc-based cytometer (Phywe, Goettingen, Germany). DNA content and RNA content were evaluated after cell staining with acridine orange.30The DNA and RNA indices and the percentage of cells in the S G2M compartment were calculated as previously de~cribed.~

Cytologic Preparation
The aspirations were performed by a cytopathologist (37, palpable masses) or an interventional radiologist (nine, deep masses) using a syringe holder, a 20-ml plastic syringe, and 22-gauge or 23-gauge needles, with as many as five passes per lesion. Material was immediately smeared onto glass microscopic slides. Frosted-tip slides were air dried and stained with Diff-Quik (Harleco, Gibbstown, NJ), and totally frosted slides were immediately fixed in modified Carnoys (9: I 70% ethano1:glacial acetic acid) solution and then stained by the rapid Papanicolaou method. The syringe and needle were rinsed in 10 ml RPMI-I640 buffered with I % bovine serum albumin. One third of the specimen was set aside for molecular and cytogenetic studies. Mononuclear cells were isolated from the remaining RPMI- 1640 by Ficoll-Hypaque (Pharmacia Fine Chemical, Inc., Piscataway, NJ) density-gradient separatioqZ6and the enriched specimen was divided for surface marker studies and FCM. The average number of cells aspirated (before gradient separation) was approximately 2 X l 06/ml RPMI- 1640. Depending on cell count, three to seven cytospin preparations, using 2.5 X lo5cells/slide, were prepared for marker studies. Needle aspirates were classified on the basis of their cytologic appearance in accordance with the National

Cytogenetic Study
Pelleted tumor cells obtained from RPMI- 1640 medium were exposed to colcemid ( 1 pg/ml) and a hypotonic solution of 0.075 mol/l potassium chloride for 30 minutes. The cells were fixed in 3:1 methano1:glacial acetic acid for at least 20 minutes and air-dried slides made with cells suspended in 1:1 methano1:glacial acetic acid. Slides were Q-banded using standard methods32 and photographed, and karyotypes were designated according to the International System for Human Cytogenetic Nomenclature (ISCN) g ~ i d e l i n e s . ~ ~

Molecular Study DNA from the pelleted tumor cells was purified with standard phenol/chloroform extractions and ethanol precipitation after proteinase K digestion.34Ten-microgram samples of DNA were digested individually to completion with the restriction endonucleases, EcoRI, HindIII, and

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CANCER February 15 1991

TABLE Fine-Needle Aspiration of Reactive Lymphoid Hyperplasia: Clinicopathologic Data 1. Positive staining (%) FCM DI

Vol. 67

Patient no.

Age (yr) Sex

Risk factor Homosex. Bisex. Homosex. Homosex. Homosex. Denied Homosex. Denied Homosex. Homosex. IV drugs Denied Homosex.

Clinical data LCL PCP, Kaposis SNCL

FNA site Parotid LN Cervical LN Iliac LN Axillary LN Cervical LN Axillary LN Supraclavicular LN Cervical LN Left subdigatric LN Rt. subdigatric LN Cervical LN Cervical LN Cervical LN Inguinal LN

Size (em)

Leu-4

%S+GM
QI TNP

RI

36 M
41 M 43 M 44 M

2
1

30 20

20 20 35 25 30 15 30 20 30 30 30 30 30
30

50
60

33 M 68 M 30 M
46 M 41 M

HD, RCC LCL LCL SNCL Kaposis ARC ARC ARC

ARC ARC HD LCL

10 11 12 13

44 M

30 F 26 M 29 M

3 1 NA 2 5 2 2 3 2 NA 1.5 2

35 25
40

60
40 50 40 40 40

30 50 30 25 30 20 30 30 30
40

1.0

1.o

1.o I .o

30 10
40

1.o 0.9
1.o

60 30

18 TNP QI 12 QI 8 13 TNP 12 11 7 0 1

0.8

1.3
I .4 1.2

1.1
1.4

1.3

DI: DNA index; HD: Hodgkins disease; NA: not available; PCP: Pneumocystis curinii pneumonia; QI: quantity insufficient; RCC: renal cell carcinoma; RI: RNA index; T N P test not performed: FNA: fine-

needle aspiration; FCM: flow cytometry; LCL: large cell lymphoma; SNCL: small non-cleaved cell lymphoma; ARC: AIDS-related complex; LN: lymph node.

BamHI. The samples underwent electrophoresis on a Probe Tech I (Oncor) using a 0.7% agarose gel and transferred to a nylon filter (Oncor, Gaithersburg, MD) in accordance with the manufacturers instructions. Hybridization studies were performed with the oligolabeled DNA probes JH (immunoglobulin heavy-chain joining region; Oncor), JK (immunoglobulin kappa light chain region; Oncor); C (immunoglobulin lambda light chain constant X region; courtesy of Dr. P. Leder by licensing

arrangement35;and TPC (T cell-receptor chain gene constant regions CPl and Cp2; Oncor). After hybridization, the Southern blots were exposed to Kodak (Rochester, NY) X-OMAT AR radiograph film for 3 days at -70C. In samples lacking sufficient DNA to set up the required enzyme digestion for all the lineage probes simultaneously, previously probed membranes were stripped of the radioactive probe and rehybridized with additional probes in accordance with the manufacturers instructions.

TABLE . Fine-Needle Aspiration of Small Noncleaved Cell Lymphoma: CIinicopathologic Data 2

Positive staining (YO) Patient no. 6 14
15

FCM DI

Age (yr) Sex 68 M 44 M 32 M

Risk factor Denied Homosex. Homosex.

Clinical stage IVA 1VB IVA

FNA site Liver masses Axillary LN Cervical LN Paraspin. LN Pelvic LN Cervical LN Axillary LN Kidney mass Inguinal LN

Size (cm) 1 15
NA

Leu-4
0
10

Other

%S

+G M
TNP 32 27 II 12 TNP 26 27 30

RI TNP TNP

Cytogenetics
~

Molecular studies
~~

9 5

2 85
0 20 20 <I <I I 5

1.0

2.3

16 17

45 M 34 M

Denied Denied

IVB IVB

NA NA 20 4
10

0 20 20 100 98 95 9 0

20 20 15 <I <I 5 0

B,50

1.0 1.0 1.0

2.0 1.2 1.9

18
19

50 M

Bisex. Homosex. Bisex. Bisex.

IVA IVB
IVB IA

Suprapubic mass Cervical LN Abdominal mass Sternal mass Axillary LN Abdominal mass

8
II
10 5

TNP 70 95
15

15
<I <I

B1O

1.0 1.0 1.0 1.0 1.0

34 25 37 TNP
19

24M 34M 32 M

20 21

4 7

5 TNP 99 < I 0 0

B,O

27

4.6 46, XY, t(8; 22) 0.9 (q24;qll) 1.4 TNP 2.6 4.1 46, XY t(8; 14)(q24; 32) 1.7 47, XY (+13t(3; 11) (q21; ~ 1 3t(8:~14) ) (~124; q32) 1.3 4.0 46, XY, t(8; 14) ~ 4q32) ; 1.5 46, XY, t(2; 8)(p12; q24) TNP 1.8 TNP 1.8 47,XY, (+12del (6) (q21), del(17) ( ~ 1 3 )

TNP R:JH,G K , A T,C, c-myc TNP I

TNP

TNP TNP R:JH

G:,K,A,T&

DI: DNA index; G: germline; LN: lymph node; R rearranged; R1: RNA index; T N P test not performed; FCM: flow cytometry; FNA: fine-needle aspiration: NA: not availale; IVA: stage disseminated disease with no B symptoms (fever, night sweats, etc.); IVB: disseminated disease with B symptoms (fever, night sweats, etc.); IA: disease restricted to one lymph node group with no B symptoms.

* 74, XY. +3+3+5+7+8+9+11+12+13+14+15+16+18+18+22, del(1) (p31; p36), +21 inv (1) (q31, q43). ins (2, 3) (432: q12, q23); is0 (5 p). +4 del (6) (p 12), +iso. 8 (9), der (13) t (8; 13) (pl I , q l I), +del(l6) (q22), +del (17) (q12), +Ml+M2+2M3.

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TABLE Fine-Needle Aspiration of Large Cell Lymphoma: Clinicopathologic Data 3.

Positive staining
(%)

FCM Leu-4

Patient

no.
1

Age (yr) Sex 37M

Risk factor Homosex.

Cytologic diagnosis LIBL

Clinical stage Kaposis PCP, IllA ARC, IIIA IIA IIA IVB AFB sepsis IIA IA
IIlA

FNA site Am Epitochlear LN Cervical LN Cervical LN Inguinal LN Abdominal mass Scalp Skin (face) Gingival mass

Size (cm) NA NA 3 2 NA 6 2
4

DI

%S

+G M
TNP 30 23 TNP QI 20 TNP TNP 13

RI

Cytogenetics

Molecular studies TNP TNP TNP TNP TNP TNP TNP TNP QI

QI
75 85

10

5
0 0
1

4 22 23 24 25

44 M

32M 32 M 32 M 45 M

Homosex. Homosex. Denied Homosex. Homosex.

LCL LIBL LCL LCL LIBL

0 0
85

15 15 0

I 2.1

2.6 2.5

0
15

QI QI
10
15
15 4.1

26 21

61 M 25 M

Homosex. Homosex.

LCL LCL

Parotid LN Axillary LN

5
15

80
10

20
10

1.0
1.0

38
8

80

TNP TNP TNP TNP TNP 1.0 TNP TNP TNP 1.4 46,XY,der(12) (PABR), del ( 17) (pl1.2; q32) 3.6 46, XY, t(8; 14) (q24; q32) 1.9 TNP

TNP TNP

AFB: acid-fast bacillus; D1: DNA index; LN: lymph node: PCP: Pneurnocyslic currnii pneumonia: QI: quantity insufficient; RI: RNA index: TNP: test not per-

formed; FCM: flow cytometry; LIBL large cell immunoblastic lymphoma; LCL large cell lymphoma; A R C AIDS-related complex; NA: not available.

Results Thirty of the 31 patients were men. Mean patient age was 39.5 years (range 24 to 68 years). Human immunodeficiency virus was presumably contracted by homosexual or bisexual activity in 22 patients and by intravenous illicit drug use in one patient (the sole woman). The other eight patients denied any high-risk activity. Twenty-six patients had lymphoma; the other five had AIDS-related complex (ARC). Two patients also had Kaposis sarcoma and one had renal cell carcinoma. Forty aspirates were from enlarged lymph nodes; six were from extranodal sites (one kidney, one liver, and four soft tissue masses). Surgical biopsy material was available for 19 of the 46 aspirate lesions, in 12 instances obtained at an outside hospital before patient referral to M. D. Anderson. In these 19, aspiration was performed to confirm and subclassify lymphoma and to obtain tissue for special studies. . . Fine needle aspiration in conjunction with immunocytochemical study, FCM, cytogenetics, and molecular studies, was the only tissue diagnostic modality performed in 12 lesions diagnosed as reactive lymphoid hyperplasia and in 15 diagnosed as lymphoma by FNA. In five of the 26 patients with lymphoma (four with small non-cleaved
~

cell [SNCL] and one with large cell immunoblastic lymphoma [LIBL]) diagnosis and therapy were based on FNA results without any corresponding histologic study. Five of the surgical biopsy specimens showed reactive lymph nodes: four Stage I and one Stage I11 HIV lymphadenitis. Fourteen surgical biopsy specimens showed lymphoma with a diffuse pattern of involvement in 13 and a diffuse pattern with focal follicular areas in one (large cell lymphoma [LCL]). A definitive diagnosis was rendered in 43 of 46 aspirates. Fourteen demonstrated reactive lymphoid hyperplasia (Table 1); 29 showed lymphoma: 15 SNCL (Table 2), 11 LCL (Table 3), two Hodgkins (Table 4), and one small lymphocytic (Table 4). Findings were indefinite in the remaining three aspirates (Table 5).

Reactive Lymphoid Hyperplasia

The fourteen aspirates diagnosed as reactive lymphoid hyperplasia by FNA were from 13 patients. Eight patients had lymphoma, in seven before the present involvement and in one at a site separate from the aspirate. The aspirated nodes were fairly small (range, 1 to 5 cm; mean, 2.2 cm).

TABLE Fine-Needle Aspiration of Other Lymphomas: Clinicopathologic Data 4.

Positive staining Patient no. Age (yr), sex 43 M 49 M
(W)

FCM Leu-4 DI
1.o

Risk factor Homosex. Denied

Cytol. dx.

Histol. dx. NSHD

Clinical data/stage RCC, IlIA

FNA site Cervical LN Axillary LN CervicalLN

Size (cm)

%S + G M
2 TNP 4

RI 0.9
1.1

3 28

HD HD SLL

SLL

IVB

2 NA 4

TNP TNP 90

10

1.0

DI: DNA index; H D Hodgkins disease; L N lymph node; N A not available; NSHD nodular sclerosing Hodgkins disease; RCC: renal cell

carcinoma; RI: RNA index; SLL small lymphocytic lymphoma; TNP: tests not performed; FCM: flow cytometry.

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TABLE. Fine-Needle Aspiration of Atypical Lymphoid Proliferation: Clinicopathologic Data 5
Positive staining (%) FCM

Vol. 67

Patient Age (yr). no. sex

Risk factor

Cytol. dx.

Histol. dx.

Clinical data

F N A site

Size (cm) 5
3

Leu-4 DI %S
5
1
1

+G M
19

RI

Molecular studies

29
30

41 M

43 M

31

59 M

Homosex. ALP suggestive (Parotidectomy) Parotid mass Parotid LN (Stage I HIV lymphadenitis) of LCL Denied ALP (Nasopharyngeal) Mixed cell Nasopharyngeal Parotid LN lymphoma suggestive of and parotid PTCL (revised dx Stage mass I11 HIV lymphadenitis) Denied ALP sueaestive (Parotidectomv) Parotid mass Parotid LN ., of LCL (Stage I HIV lymphadenitis)
~~

15 10

1.5 TNP
1.1 TNP

30 20

50

Difficult to interpret

Too few cells

G:JH,K , X T&

ALP atypical lymphoid proliferation; DI: DNA index; LN: lymph node; RI: RNA index; T N P tests not performed; FCM: flow cytometry; HIV: human im-

munodeficiency virus; PTCL peripheral T-cell lymphoma; RNA: fine-needle aspirate.

The 14 aspirates were similar in cytologic pattern: a polymorphic population of lymphoid cells, most of them small round lymphocytes, with interspersed small and large cleaved cells, large non-cleaved cells, and immunoblasts. Plasma cells, tingible-body macrophages, and mitotic figures were present in the background (Fig. 1). In Case 7, there was also an increased number of epithelioid histiocytes, which occasionally formed small granulomas; culture in this case showed Mycobacteritim tuberculosis. Immunocytochemical studies revealed a consistent polyclonal pattern whereby the lymphoid cells marked positively for Kappa, lambda, and Leu-4, in approximately equal proportions. Flow cytometric study demonstrated a diploid cell population in six of seven aspirates. The seventh showed a low-degree hypodiploid peak. The proliferative indices were intermediate to high (%S GzM: range, 7 to 18; mean, 11.6), and RNA indices were elevated (range, 0.8 to 1.4; mean, 1.2). Molecular and cytogenetic studies were not performed in this group.

Small Non-Cleaved Cell Lymphoma

The 15 aspirates diagnosed as SNCL by FNA were from nine patients. Seven patients presented with disseminated disease (Stage IV). The aspirated masses were large (mean, 8 cm; range 2 to 20 cm). Twelve of the aspirates showed a monotonous population of intermediate-sized cells with scant to moderately abundant, occasionally vacuolated cytoplasm and coarsely clumped chromatin with multiple (two to five) prominent nucleoli (Fig. 2). Mitotic figures and tingible body macrophages were abundant. In three of four aspirates from one patient (Case 17) and in tumor cell lines established from two patients (Cases 17 and 18), a giant cell (syncytial) variant occurred that morphologically resembled SNCL in its cytoplasmic and nuclear characteristics.

Immunocytochemical analysis showed immunoglobulin light chain restriction in nine of 13 aspirates in which the tests were performed (eight kappa, one lambda). In four aspirates, neither light chain immunoglobulin nor pan-B-cell or pan-T-cell antigens could be demonstrated; hence, lineage could not be determined by this method. Flow cytometric study showed a diploid cell population in nine of 12 aspirates. The three aspirates with an aneuploid peak were from the syncytial-variant case (Case 17). The proliferative indices in the 12 aspirates were high (%S G2M: range, 1 1 to 37; mean, 25.6), and the RNA indices were high (range, 0.9 to 4.6; mean, 2.3). Five of seven samples karyotyped showed one of the three recognized translocations associated with SNCL. The classic t(8; 14)(q24;q32) was present in three; the variant t(8;22)(q24;q 1 1) and t(2;8)(p12;q24)were present in one case each. The two other karyotypes did not fall into any pattern associated with a histologic subtype of lymphoma. Karyotypes from three separate tumor sites in Case 17 showed different cytogenetic abnormalities. Cells from the axillary lymph node showed a complex but nonspecific abnormality; cells from both the kidney mass and the inguinal lymph node showed the t(8; 14)(q24;q32) translocation, with the inguinal lymph node showing in addition +13t(3;1 l)(q21;p13). The corresponding cytologic smears were also discordant. Cells from the axillary lymph node were monomorphic and of intermediate size, whereas those from the kidney and inguinal lymph node were pleomorphic, with many bizarre and giant forms present. Case 2 1 was diagnosed as a B-cell lymphoma (despite no immunocytochemical detection of either immunoglobulin light chain or pan-B-cell markers) by demonstrating gene rearrangements in the immunoglobulin heavy chain (JH), thus confirming the presence of a monoclonal population of B-cells (Fig. 3). In Case 14, immunocytochemical study demonstrated lambda light chain restriction. The corresponding molecular studies confirmed a monoclonal B-cell proliferation by demon-

No. 4

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1013

FIGS.IA-1D. FNA of a 2-cm subdigastric lymph node (Patient 9. reactive lymphoid hyperplasia). (A) The polymorphic lymphoid population consists of small and large lymphocytes. A mitotic figure is seen in the lower center (Diff-Quik, X665). (B-D) Polyclonality is demonstrated immunocytochemically by the following positive percentages: kappa, 30% (B): lambda, 30% (C); and Leu-4, 40% (D) (X425).

strating JH rearrangement; however, no rearrangements were detected by J kappa and C lambda probes. This discrepancy could be explained by possible comigration of the lambda and germline bands.

Large Cell Lymphoma

The 1 1 aspirates diagnosed as LCL by F N A were from eight patients. Four patients presented with advanced dis-

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Vol. 61

and showed either diploidy or tetraploidy (LIBL) high proliferative indices (%S G2M: range, 8 to 38; mean, 22.0), and high RNA indices (range, 1.0 to 3.6; mean, 2.2). The mean proliferative indices of the three LIBL and the three nonimmunoblastic LCL aspirates were the same (22.0%). Karyotyping was performed in two aspirates, from two patients. t(8; 14)(q24;q32) translocation was seen in one; the other karyotype was not one associated with any histologic subtype of lymphoma.

Other Lymphomas
There were two aspirates (from one patient, Case 3) diagnosed as Hodgkins disease and one aspirate (Case 28) diagnosed as small lymphocytic lymphoma. Both smears from the former case, showed atypical mononuclear and multinuclear Hodgkins cells in a polymorphic lymphoid background. The smears in Case 28 showed a monotonous population of small, mature plasmacytoid lymphoid cells demonstrating lambda light chain restriction and negative Leu-1 staining. Flow cytometric study demonstrated diploidy, a proliferative index of 4%, and an RNA index of 1.1 consistent with low-grade lymphoma.

Atypical Lymphoid Proliferation (Inconclusive)

FIG. 2. FNA of a 20-cm cervical lymph node (Patient 16, SNCL). The smear demonstrates a monotonous population of intermediate-sized lymphoid cells with prominent cytoplasmic vacuoles, coarse chromatin with multiple nucleoli (Diff-Quik. X665).

ease (Stages 111 and IV). The aspirated masses were fairly large (range, 2 to 15 cm; mean, 5 cm). The cytologic features were variable. In six aspirates, the smears showed a monotonous population of large cells with plasmacytoid basophilic cytoplasm; large, vesicular, eccentric nuclei; and prominent central nucleoli, all features of LIBL (Fig. 4). In the other five aspirates, cells were large without immunoblastic features. The cell composition varied from aspirate to aspirate with few small cells and many large cleaved or noncleaved cells with variable chromatin patterns, some of which contained multiple nucleoli (Fig. 5). Mitotic figures and tingible body macrophages were present, but were not as abundant as in the smears of SNCL. lmmunocytochemical study showed immunoglobulin light chain restriction in five of eight aspirates in which the tests were performed (four kappa, one lambda). In the remaining three aspirates so examined, lineage could not be determined by this method. The large atypical cells in these three aspirates were negative for immunoglobulin light chains and pan-B-cell and pan-T-cell markers. Flow cytometric study was completed in six aspirates

All three aspirates (from three patients) in which a definitive diagnosis could not be made were from the parotid region. In Cases 29 and 3 1, the smears showed a mixed population of small and large lymphoid cells, the large cells predominated (Fig. 6A). In Case 29, the large cells did not demonstrate positivity for kappa, lambda, Leu4, or Leu-I2 (a pan-B-cell marker); however, there was diffuse faint staining for B, (a pan-B-cell marker). Flow cytometric study showed diploidy, a proliferative index of 19%, and an RNA index of 1.5 (Fig. 6B). The findings in this case were considered to strongly suggest LCL. In

FIG. 3. Rearranged bands (arrows) of the immunoglobulin heavy chain (JH) in DNA from Patient 21 (SNCL) by EmRI and Hind111 enzyme digestion. confirming a monoclonal B-cell proliferation. This aspirate was immunocytochemically negative for immunoglobulin light chains and pan-9- and pan-T-cell markers.

No. 4

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1015

were performed to obtain tissue to confirm T-cell lymphoma by gene rearrangement studies; however, the aspirates were serous fluid consistent with parotid cysts. The patient was given one course of chemotherapy and remains without evidence of lymphoma 3 years after initial biopsy. Review of the original nasopharyngeal biopsy was reinterpreted as a depleted lymph node of Stage I11 HIV lymphadenitis. Discussion Lymphadenopathy is a common occurrence in HIVpositive patients; its differential diagnosis comprises reactive, neoplastic, and infectious processes. Cause is difficult to establish by history, physical examination, and laboratory tests and usually requires tissue diagnosis. Our results show that this FNA multiparameter approach not only can accurately classify lymphoid hyperplasia and neoplasia in HIV-positive patients, but also can provide additional information regarding pathobiology in this setting. The cytologic features reported here for HIV lymphadenitis are similar to those in lymphoid hyperplasia in patients without HIV infection. In florid hyperplasia of Stage I HIV lymphadenitis, large cells may predominate thus the cytologic features may overlap with those of

FIG.4.FNA of a 3-cm gingival mass (Patient 25, LIBL). The smear demonstrates a monotonous population of large cells with plasmacytoid basophilic cytoplasm, large vesicular eccentric nuclei, and prominent central nucleoli (Papanicolaou, X425).

Case 3 1 the immunocytochemical markers and FCM parameters were difficult to interpret because of sparse specimen. Cytomorphologically, however, LCL was favored. Molecular studies performed on the aspirated cells from Case 31 demonstrated germline configuration of JH, J Kappa, C lambda, and T beta C, supporting a benign process. In both Cases 29 and 3 1, excisional biopsy of the parotid mass revealed florid reactive lymph node hyperplasia (Stage I HIV lymphadenitis) involving the parotid lymph nodes. Both patients remain without evidence of lymphoma at follow-up visits of 3 (Case 29) and 2 (Case 3 1) years, respectively. The third aspirate (Case 30) consisted of a mixed population of predominantly small and many large cleaved and non-cleaved, lymphoid cells. kappa, lambda, and Leu4 stained 30%, 20%, and 50% of cells, respectively. Flow cytometric study demonstrated a diploid cell population with an intermediate %S G2M value (9) and an RNA index of 1.1. Nasopharyngeal surgical biopsy taken 1 month before aspiration was interpreted as diffuse mixed cell lymphoma consistent with peripheral T-cell lymphoma (PTCL). Although the cytologic features and immunocytochemical study of the FNA were consistent with a reactive lymphoid process, in light of the previous surgical diagnosis of PTCL the interpretation of the FNA together with the limited immunocytochemical battery could not verify or refute the surgical diagnosis. Two repeat aspirations in this patient of recurrent parotid masses

FIG.5. FNA of a 15-cm axillary lymph node (Patient 27, LCL). The smears show a monotonous population of large non-cleaved cells (Papanicolaou, X425).

1016

CANCER February I5 1991

Vol. 67

. .. . ... ...
-1

.. ..

DNA

FIGS. AND 68. (A) FNA of a 5-cm parotid mass (Patient 29, Stage 6A 1 HIV lymphadenitis). The smear shows a mixed population of small and large lymphoid cells. The large cells predominate (Diff-Quik, X425). (B) FCM shows a diploid cell population (arrow), a proliferative index of 19% and an RNA index of 1.5. This aspirate was misinterpreted to strongly suggest LCL.

mixed or large cell lymphoma. When the aspirate is polymorphic, other diagnostic considerations are Hodgkin's disease and PTCL. T-cell lymphomas, however, are extremely uncommon in these patients. Immunocytochemical study is particularly helpful in confirming the benign nature of a lymphoid process by demonstrating polyclonality. Proliferative indices (mean, 1 1.6%)demonstrated by FCM in our examples of reactive hyperplasia from HIV-positive patients are usually significantly higher than in lymph node hyperplasia in patients who are not HIV-positive (mean, 5.6%).36 and may

reach extremely high levels, overlapping those seen in intermediate-grade and high-grade lymphoma^.^^-^^ Aneuploid peaks, although uncommon, occur in HIV lymphadenopathy, as reported by Srigley et aL3' and as demonstrated by Case 1 1 in our series. Our FCM data support the proposed pathobiology of HIV lymphadenopathy where immunosuppression predisposes to massive Epstein-Barr virus infection (a B-cell mitogen), which leads to sustained polyclonal B-cell proliferati~n.~,'~'' Knowles et al. " described one or more oligoclonal B-cell expansions in approximately 20% of ARC lymph nodes; they postulated that these represent a premalignant condition. The significance of aneuploidy (as detected by FCM) remains unknown, although the finding may also reflect a premalignant condition. When cytomorphologic and immunocytochemical analyses do not firmly establish a benign process, gene rearrangement studies may be used to demonstrate the absence of a monoclonal proliferation (Case 3 1). The high sensitivity of FNA in detecting and subclassifying lymphoid malignancies in HIV-positive patients derives from the fact that the great majority of these lymphomas are diffuse, where subclassification depends on cell size and cytomorphologic features seen to an equal or greater advantage in FNA preparations as compared with histologic sections. The smears from our cases of lymphoma usually contained a monotonous population of intermediate (SNCL) or large (LIBL or LCL) cells with variable numbers of mitotic figures, and tingible-body macrophages. Immunocytochemical study generally demonstrated immunoglobulin light chain restriction; however, in some aspirates no immunoglobulin light chain or pan-B-cell or pan-Tcell markers were expressed. In such instances, gene rearrangement studies may establish monoclonality and Bcell lineage by showing heavy-chain or light chain immunoglobulin gene rearrangements (Case 2 1). Nucleic acid FCM profiles generally demonstrated diploid neoplasms with high proliferative indices (mean, 25.6% in SNCL and 22.0% in LCL), reflecting the biologically aggressive nature of these lymphomas. Previous studies6-I5 have shown about two thirds of non-Hodgkin's lymphomas in HIV-positive patients to be high grade (SNCL or LIBL) and about one third to be of intermediate grade (LCL or rarely diffuse small cleaved cell lymphoma); a few (< 10%)low-grade lymphomas may also occur. Our study is in agreement: 12 of 19 patients had SNCL or LIBL, five had LCL, and one had SLL. The patients tend to present with advanced disease and extranodal involvement, most often in the central nervous system, gastrointestinal tract, bone marrow, or skin. In the great majority of our cases, classification between SNCL and LCL was apparent; however, overlapping fea-

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tures made this distinction difficult in the other few cases. Garcia et aL4 attempted to use monoclonal antibody immunohistochemical study to help differentiate the two entities and found a predominance of lambda light chain restriction with a higher incidence of common acute lymphoblastic leukemia antigen (cALLa) and Io-67 positivity in SNCL and a predominance of Kappa light chain restriction with fewer cALLa-positive or Ki-67-positive cases in diffuse LCL. In contradistinction, our results concurred with Knowles et al. with Kappa light chain restriction predominant in both SNCL and LCL (in our series, 8: 1 and 4: 1 Kappa:lambda, respectively). By these data, the type of light chain restriction cannot be used as a differentiating feature. Cytogenetic translocations involving the locus of the c-myc proto-oncogene on chromosome 8 band q24, i.e., t(8;14)(q24;q32) or the variant t(8;22)(q24;qll) or t(2;8)(p12;q24), are associated with but not specific for SNCL.5,4-44 Five of seven SNCL and one of two LCL in our series demonstrated such translocations; Knowles et al. reported similar cytogenetic c - m y translocations in their series of LCL in HIV-positive patients. The variant translocations t(2;22)(q24;q 1 1) and t(2;8)(p12;q24) are observed in 6% and 9%, respectively, of non-HIV SNCL. Initial studies suggested that the frequency of variant translocations was increased in AIDS-associated lymphomas42but the sample size is as yet too small to reach a c o n c l ~ s i o nIn our series, two of six c-myc trans.~~ locations were of variant form. The demonstration of different karyotypes from separate tumor sites in the same patient (Case 17) supports the hypothesis of lymphomagenesis in HIV-positive patients where multiple oligoclonal proliferations occurring in the lymphadenopathy stage evolve into malignant clones once additional rearrangements occur in particular c-myc translocations. Not included in our study aspirates were two from the parotid region in Case 30 (the above-mentioned repeat procedures) and two from parotid masses in two other HIV-positive patients that yielded variable amounts of turbid fluid consistent with parotid cysts. Parotid cysts in HIV-positive patients have been described only recently45 and appear to be related to lymphoepitheliallesions arising in intraparotid lymph It is postulated that the proliferation of lymphoid tissue results in salivary gland duct obstruction and consequent dilatation, and thus the development of cysts. In summary, our study has demonstrated that FNA when the assessment includes cellular and molecular studies may be used in lieu of surgical biopsy in HIVpositive patients presenting with enlarged lymph nodes. This multiparameter approach allowed a definitive diagnosis in 43 of 46 aspirates. Information regarding the pathobiology of lymphoid hyperplasia and neoplasia can

also be derived. Fine needle aspiration may be used in HIV-positive patients to diagnose subclassify and grade lymphoma, enabling rapid institution of specific therapy; to stage lymphoma; to differentiate HIV lymphadenopathy from malignant lymphoma; and to monitor ARC for evolution into lymphoma. The procedure obviates extensive, complicated surgical procedures when the masses are in deep abdominal or thoracic sites and minimizes the risk of HIV infection for surgeons and the operating team.
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