—  SPECIALTY CONFERENCE  —

Hematopathology

Case 2 - T-Cell Large Granular Lymphocytic Leukemia

William G. Morice
Mayo Clinic
Rochester, MN


Click on each slide thumbnail image for an enlarged view
Peripheral Blood
Peripheral blood smear evaluation is a fundamental element in the evaluation of unexplained cytopenias. In this case, the most noteworthy finding is an absolute lymphocytosis. The lymphocytes have small, bland nuclei and relatively abundant pale-staining cytoplasm containing variable numbers of azurophilic granules. Although the red blood cells and granulocytes are reduced in number they are cytologically unremarkable. In this case it is pertinent that no hypochromic-microcytic red blood cells, hypogranular neutrophils, or other features to suggest a diagnosis of myelodysplasia are seen.


Case 2 - Figure 1 - Review of a the peripheral blood smear reveals the majority of the lymphocytes to have small nuclei and relatively abundant cytoplasm containing variably prominent granules.
Case 2 - Figure 2 - The bone marrow biopsy is normocellular to slightly hypercellular with decreased erythropoesis, increased granulopoesis, and unremarkable megakaryopoesis. The most evident lymphoid element on morphologic review are the bland appearing interstitial lymphoid aggregates.
Case 2 - Figure 3 - Immunoperoxidase staining of the bone marrow biopsy reveals the interstitial lymphoid aggregates to predominantly contain CD3 positive T-cells (left) with lesser numbers of CD20 positive B-cells (right).


Case 2 - Figure 4 - Additional immunoperoxidase stains demonstrate that the CD3 positive T-cells (left) in the lymphoid aggregates do not express the cytotoxic granule protein granzyme B (right). Note the presence of small granzyme B positive cells at the periphery of the lymphoid aggregate.
Case 2 - Figure 5 - Increased numbers of CD3 positive T-cells were present in the marrow interstitium (stain not shown). These interstitial T-cells are CD8 positive, with clusters of CD8 positive cells present.
Case 2 - Figure 6 - On closer examination a linear of distribution of CD8 positive cells in the bone marrow biopsy is evident. This is attributable to the presence of these cells in small vessels.


Case 2 - Figure 7 - The intravascular T-cells are also positive for the cytotoxic granule proteins TIA-1 (top) and granzyme B (bottom).
Case 2 - Figure 8 - Flow cytometric immunophenotyping analysis performed on the bone marrow aspirate reveals the presence of an immunophenotypically aberrant T-cell population with diminished expression of CD3 and CD7 (right, indicated by arrow). Subsets of these cells show both diminished expression and complete loss of CD5 (left, arrows).
Case 2 - Figure 9 - Flow cytometry demonstrates the T-cells to be CD8 positive (left, arrow). These cells aberrantly coexpress CD16 (right, arrow).


Case 2 - Figure 10 - 3-color flow cytometry allows for selective gating on the CD3 and CD16 positive T-cells (left, cells are in gate R2 and highlighted green). This technique is used to confirm that these cells coexpress CD57 (right).
Case 2 - Figure 11 - The same gating strategy used in Figure 10 is used to demonstrate that the CD3 and CD16 positive cells show uniform expression of the KIR antigen CD158b. These cells do not express CD158a or CD158e (also known as p70).


Bone Marrow Aspirate and Biopsy
The bone marrow aspirate is cellular. Decreased numbers of erythroid precursors are present, those seen are left shifted in their maturation. No dyserthropoietic forms are noted. Maturing granulocyte precursors and megakaryocytes are both quantitatively and cytologically unremarkable. Increased numbers of lymphocytes are present in the aspirate smear. Similar to the lymphocytes in the peripheral blood these have small, bland nuclei and many appear to have cytoplasmic granules; although these cytoplasmic features are more difficult to appreciate than in the peripheral blood smear.

The bone marrow biopsy is normocellular with an estimated cellularity of 30% to 40%. The changes in hematopoiesis mirror those seen in the bone marrow aspirate with markedly diminished erythropoesis, and unremarkable granulopoeisis and megakaryopoeisis. Scattered benign-appearing interstitial lymphoid aggregates are present. These are well-circumscribed and are populated by lymphoid cells with small, minimally irregular nuclei. No discrete lymphoid infiltrates are seen. Detailed examination does, however reveal the presence of a very subtle interstitial lymphocytosis.

The lymphocytes in the interstitium have small, elongate nuclei with minimally irregular nuclear contours.

Immunophenotype
Ancillary immunophenotyping techniques including both immunohistochemistry and flow cytometry are tools required for arriving at the diagnosis in this case. Flow cytometric immunophenotyping analyses performed on the bone marrow aspirate reveal the presence of an immunophenotypically distinct CD8-positive T-cell population with aberrantly diminished expression of the T-cell associated antigens CD5 and CD7. These cells also coexpress the NK-cell associated antigen CD16.

Immunoperoxidase stains were performed on the bone marrow biopsy using antibodies to CD3, CD8, CD20, and the cytotoxic granule proteins TIA-1 and granzyme B. These stains reveal a prominent CD8-positive interstitial T-cell infiltrate which exceeds that expected based on the marrow histology in the H&E stained slide. The CD8 positive T-cells also stain with antibodies to TIA-1 and many, but not all, are positive for granzyme B. These stains not only demonstrate an increase in interstitial CD8-positive cytotoxic T-cells, they also illustrate distinctive patterns of marrow infiltration. The most striking feature are linear configurations of antigen positive cells which can be appreciated at low magnification, at higher magnification it becomes evident that this pattern is due to the presence of antigen positive cells in microvascular structures. Interstitial clusters of 8 or more antigen positive cells are also present. In contrast to the interstitial T-cells, those which are associated with the CD20 positive B-cells in the lymphoid aggregates do not stain with antibodies to cytotoxic lymphocyte associated antigens.

Molecular Genetic and Cytogenetic Findings
Clonal TCR gene rearrangements were detected in the bone marrow aspirate by polymerase chain reaction (PCR) analysis using primers specific for the T-cell receptor gamma chain gene.

Cytogenetic studies performed on the bone marrow aspirate revealed a normal male karyotype ( 46,XY) in 20 analyzed metaphases.

The morphologic, immunophenotypic, and molecular genetic findings in this case support a diagnosis of T-cell large granular lymphocytic leukemia.

Discussion
T-cell large granular lymphocytic leukemia (T-LGL) is an indolent lymphoproliferative disorder of cytotoxic T-cells typified by the presence of an immunophenotypically distinct, clonal population of these cells in the peripheral blood [1]. The clinical features of T-LGL are largely attributable to the disease associated cytopenias which are almost invariably present and include recurrent infections due to neutropenia and fatigue due to anemia [2]. An association between T-LGL and clinical and/or laboratory features of autoimmune disease has also been well documented.

In Wrights-Giemsa stained peripheral blood smears T-LGL cells typically have small nuclei, minimally irregular nuclear contours, and relatively abundant amphophilic cytoplasm containing azurophilic granules. The size and number of cytoplasmic granules vary between cases and even among cells in individual cases. In rare instances the granules may be very difficult to identify. These bland cytologic features do not allow the abnormal cells of T-LGL to be reliably distinguished from their non-neoplastic counterparts. Given protean clinical manifestions of T-LGL and the difficulties in morphologic disease recognition early diagnostic criteria included a persistent absolute peripheral blood granular lymphocyte count of > 2,000 cells/ml. It was subsequently recognized that in some bona fide T-LGL cases there was a modest increase in granular lymphocytes which did not meet these numeric threshold [3]. For this reason, the detection of a distinctive cytotoxic T-cell population by ancillary immunophenotyping techniques has become a mainstay in T-LGL diagnosis.

Flow cytometric immunophenotyping analysis performed on peripheral blood or bone marrow aspirate specimens is extremely useful in identifying and characterizing the abnormal cells of T-LGL and thereby establishing a diagnosis. In the vast majority of T-LGL cases the abnormal cells are CD8-positive T-cells expressing ab T-cell receptor heterodimers. These CD8-positive T-cells often exhibit abnormalities in the expression of T-cell associated antigens such as CD2, CD3, CD5, and CD7. Aberrantly diminished or lost expression of CD5 and/or CD7 are the most commonly encountered in T-LGL and are present in the majority of cases. Although infrequent, abnormally diminished expression of CD2 or CD3 may also be seen [4].

A pathognomic feature of T-LGL is the coexpression of NK-cell associated antigens by the abnormal cells. Early studies emphasized the importance of CD57 expression in the diagnosis. While CD57 is expressed by the majority of cells in most T-LGL cases, this antigen is also normally expressed memory cytotoxic T-cells. Therefore a reactive expansion of CD8 and CD57 positive T-cells can be seen in a variety of conditions and does not represent a disease specific finding [4, 5, 6, 7, 8, 9] . CD16 is another NK-cell associated antigen that is expressed by most T-LGL. In contrast to CD57, this C16 expression is a distinguishing feature of T-LGL only a minor subset of normal T-cells is CD16-positive. CD56 expression in T-LGL is uncommon.

A novel group of NK-cell-associated receptors which recognize MHC I and related antigens on potential target cells has recently been described. These receptors are normally expressed on NK-cells and a subset of memory cytotoxic T-cells and include the CD94/NKG2 heterodimeric complexes and the killing inhibitory receptors, or KIRs [10, 11, 12] . The KIR antigens, which include CD158a, CD158b, and CD158e, are found on approximately one-half to two-thirds of T-LGL. In KIR positive T-LGL cases typically a single KIR antigen is expressed by all of the neoplastic cells. This restricted pattern of KIR expression not only aids in identifying the abnormal T-LGL cells, it also provides indirect evidence of clonality [4, 13] .

Bone marrow biopsy may reveal a variety changes in hematopoiesis in T-LGL ranging from essentially normal trilineage hematopoiesis to pure red blood cell aplasia, although a left-shifted granulocytic hyperplasia appears to be most commonly encountered [14, 15, 16] . As in peripheral blood specimens, T-LGL cells are not easily detected in bone marrow aspirates and biopsy specimens by morphologic assessment alone. In the aspirates, the cytoplasmic granules of the lymphocytes are difficult to visualize. Furthermore, as illustrated by the current case, T-LGL infiltrates the marrow interstitium in a subtle interstitial pattern which is not readily apparent in H&E stained bone marrow biopsy specimens. Given the lack of distinguishing morphologic features early descriptions of the bone marrow involvement by T-LGL focused on the presence of identifiable interstitial lymphoid aggregates [15]. However, the advent of antibody reagents which allow for the identification of cytotoxic lymphocytes in paraffin-embedded tissues have proved invaluable in elucidating disease-specific patterns of marrow infiltration by this disorder. Immunoperoxidase staining of bone marrow specimens from T-LGL patients with antibodies to CD3, CD8, and the cytotoxic granule proteins TIA-1 and granzyme B frequently reveals distinctive interstitial clusters of 8 or cells positive for one or more of these antigens. In addition, careful review also commonly reveals linear staining of antigen positive cells due to their accumulation in marrow sinusoids and microvascular structures. At least one of these immunoperoxidase features (interstitial clusters or intravascular staining) is present in the majority of T-LGL cases (>80%). These stains demonstrated that the neoplastic cells of T-LGL were present in subtle interstitial foci that were not readily apparent in H&E stained slides and that they were not associated with the discrete lymphoid aggregates which may be seen in some T-LGL marrows but do not represent a disease specific feature. Moreover, while increased numbers of CD3 positive cytotoxic T-cells may be seen by immunoperoxidase stains in reactive conditions associated with numbers of granular lymphocytes the distinctive interstitial clusters and intravascular staining are not present in these cases(16). Hence, bone marrow immunohistochemistry using these antibodies provides both a sensitive and specific tool for detecting involvement by T-LGL.

Demonstrating T-cell clonality remains a fundamental, requisite element in establishing a diagnosis of T-LGL. Traditionally this has been accomplished through PCR and/or Southern blot analysis of T-cell receptor gene rearrangements. Recently however, it has been demonstrated that flow cytometric immunophenotyping analyses using antibodies to specific T-cell receptor beta chain variable region families may also be used for this purpose [17, 18] . Advantages of the flow technique is that is rapid and multicolor gating strategies can be used to assess clonality specifically in the CD8-positive T-cell compartment. There are drawbacks to this flow method, however, including that the standard antibody reagents do not detect all of the known T-cell receptor V-beta families and that the method works best on fresh peripheral blood with other specimens types, including bone marrow aspirates, only yielding suboptimal (often un-interpretable) results.

Differential Diagnosis
NK-Cell Large Granular Lymphocytic Leukemia

Unlike B-cells and T-cells, natural killer-cells (NK-cells) do not contain uniquely rearranged antigen receptor genes and therefore establishing NK-cell clonality is problematic. For this reason, NK-cell lineage large granular lymphocytic leukemia (NK-LGL) remains a somewhat controversial entity [2, 19] . Given the difficulties in documenting NK-cell clonality some required for the diagnosis of NK-LGL more overt clinical features of malignancy than are typically seen in T-LGL. Based on early descriptions using these criteria, NK-LGL was considered a more aggressive entity [15]. It now appears however that these studies may have included under the heading of NK-LGL cases of peripheral blood involvement by a number of subsequently recognized rare, aggressive NK-cell lymphoproliferative disorders.

The identification of NK-cell associated receptors such as the KIRs and the development of antibody reagents to these receptors have provided tools for more comprehensive NK-cell evaluation and have allowed for the recognition of an indolent lymphoproliferative disorder of NK-cells similar to T-LGL. This disorder has been ascribed a number of monikers including chronic NK-cell lymphocytosis and NK-cell lymphoproliferative disease of granular lymphocytes (NK-LGDL} [4, 20] . Indolent NK-LGL is less common than T-LGL. The clinical features of the disorders are similar although NK-LGL may be associated with a slightly higher peripheral blood large granular lymphocyte count, slightly less severe cytopenias (particularly anemia], and less frequent splenomegaly [21]. The association with autoimmune phenomenon associated with T-LGL are not present in NK-LGL (neben).

As in T-LGL, the detection of an immunophenotypically distinct cell population is a central element in making a diagnosis of indolent NK-LGL. A primary difference between the two entities is that NK-cells by definition lack surface CD3 expression as this requires the presence of rearranged T-cell receptor gene products, which NK-cell lack [19]. NK-LGL is universally CD16 positive, and often shows bright expression of this antigen, CD56 expression is often diminished or lost. Complete delineation of the abnormal NK-cells requires assessment of additional NK-cell associated antigens such as CD94 and the KIRs. When a comprehensive panel is used an aberrant NK-cell immunophenotype can be detected in all indolent NK-LGL cases [4, 20] . Distinguishing features of NK-LGL detected by these studies include uniform bright expression of CD94 and either uniform expression of a single KIR isoform, as seen in T-LGL or complete absence of KIR expression. This latter finding (complete lack of KIR expression) is aberrant when compared to normal NK-cells.

Bone marrow biopsy immunohistochemistry is also useful in evaluating for possible NK-LGL. By immunoperoxidase staining the interstitial clusters and intravascular staining of cells positive for TIA-1 and granzyme B strongly associated with clonality in T-LGL disorders are frequently identified in NK-LGL [4]. One cannot not reliably distinguish between T-LGL and NK-LGL by paraffin immunohistochemistry however as NK-cells can show a somewhat paradoxic reactivity with CD3 antibodies this method due to the normal expression of some subunits of the CD3 complex [19]. Flow cytometric immunophenotyping analysis is required to accurately determine the lineage of the abnormal cells. As there are no readily applied methods to document the clonality in indolent NK-LGL other possible secondary causes for NK-cell expansion such as recent viral infection should be excluded and persistence of the process over a relatively prolonged period (greater than 6 months) should be documented before an unequivocal diagnosis is rendered.

Myelodsyplasia and Other Causes of Unexplained Cytopenias
Given that a number of disorders can present with unexplained cytopenias it is important that other possible causes be considered before the diagnosis of T-LGL is made. Perhaps chief among these differential diagnostic possibilities is myelodysplasia (MDS). As a complete description of the elements required to make a diagnosis of MDS is beyond the scope of this brief discussion, important distinguishing features will be emphasized. When reviewing the peripheral blood smear morphologic features typical of MDS such as red blood cell dimorphism with coincident hypochromic-microcytes and macrocytes, hypogranular neutrophils or neutrophils with hypolobulated nuclei, and hypogranular platelets should be carefully sought out. Examination of the bone marrow is also critical. While LGL may be associated with marrow hypercellularity and granulocyte maturation arrest, increased numbers of blasts or dysplastic maturational changes in hematopoiesis such as the presence ringed sideroblasts or multi-nucleated megakaryocytes should not be present. Cytogenetic studies should also be performed to exclude the presence of myelodysplasia associated genetic abnormalities.

There have been anecdotal reports of coincident MDS and T-LGL, although the precise nature of the T-cell expansions described is unclear. The function of normal cytotoxic T-cells and NK-cells is to recognize and destroy self cells altered by viral infection, neoplastic transformation, or other stressors. Furthermore, limited clonal diversity, or oligoclonality, is seen in cytotoxic T-cells responding to chronic antigenic stimulation [22]. Hence it is quite possible that the cytotoxic T-cell populations described in these reports represent a physiologic response to the clonal myeloid stem cell disorder and not an autonomous lymphoproliferative process. Regardless of whether these cytotoxic T-cell expansions in T-LGL are primary or secondary in nature they may indeed contribute to the suppression of productive hematopoiesis and thereby contribute to the severity of the cytopenias [23, 24] .

As an increase in normal cytotoxic T-cells can be seen in response to neoplasia it is important to consider that LGLs present in the peripheral blood of patients with unexplained cytopenias may indeed be secondary to the presence of another malignancy. As such, when evaluating bone marrow specimens for potential involvement by T-LGL one should continue to assiduously search for evidence of other neoplasms such as Hodgkin lymphoma and diffuse large B-cell lymphoma. As such, it is prudent to include antibodies to a B-cell associated antigen such as CD20 to exclude the presence of histologically occult large B-cell lymphoma when performing immunoperoxidase stains to pursue the possibility of marrow involvement by T-LGL [25].

Hepatosplenic T-cell Lymphoma
Hepatosplenic T-cell lymphoma (HSTCL) is a rare, aggressive malignancy of cytotoxic T-cells which, like T-LGL, can involve the marrow in a sinusoidal pattern and therefore bears consideration in the differential diagnosis. HSTCL typically effects young adult males and often has a fulminant clinical presentation with massive hepatosplenomegaly, thrombocytopenia and anemia, and B-symptoms (fever, night sweats, weight loss). HSTCL has a dire prognosis, even in those patients which initially respond to treatment, with a median survival of less than 1 year [26, 27] .

Unlike T-LGL there are typically few circulating malignant cells in HSTCL, however the peripheral blood disease burden may increase with disease progression. When present in Wright-Giemsa stained peripheral blood smears the neoplastic HSTCL cells are cytologically malignant and usually have intermediate to large-sized, variably irregular nuclei and moderate amounts of basophilic cytoplasm which may or may not contain granules. In contrast to T-LGL and most other T-cell malignancies, HSTCL frequently expresses gamma-delta T-cell receptor heterodimers although cases expressing alpha-beta T-cell receptor heterodimers have also been described [28]. Also dissimilar from T-LGL, which is most often a disorder of CD8 positive T-cells, HSTCLs typically are CD4 and CD8 negative ("double negative T-cells"). Despite these differences there are a number of immunophenotypic attributes of HSTCL which closely resemble those of T-LGL including loss of CD5 and CD7 expression and co-expression of NK-cell associated antigens such as CD16, and CD56.

Bone marrow involvement by HSTCL is present in most, if not all, cases [29, 30] . In H&E stained sections the neoplastic cells most often have medium-sized rounded or slightly irregular nuclei with slightly dispersed chromatin and small, distinct nucleoli. These cells infiltrate the marrow in an intrasinusoidal and subtle interstitial pattern, that, like T-LGL, can be very difficult to recognize in H&E stained biopsy sections. As in T-LGL, immunoperoxidase stains using antibodies to TIA-1, which is universally expressed by HSTCL, and T-cell associated antigens such as CD3 are of great utility in revealing the presence of neoplastic infiltrates which may be missed by routine morphologic evaluation [27, 29] . Interestingly, HSTCL usually is granzyme B negative; this TIA-1 positive and granzyme B negative phenotype cannot be used to reliably differentiate T-LGL from HSCTCL however as only about one-half of T-LGL cases stain with antibodies to granzyme B. With HSTCL disease progression increased numbers of large atypical cells become evident in the bone marrow aspirate and biopsy and interstitial marrow infiltration becomes a more prominent histologic feature, allowing for more ready recognition of the marrow involvement on morphologic review [29, 30] .

Despite these many feature common to T-LGL and HSTCL it usually not difficult to distinguish these entities due to the different age groups effected and the more overt cytologic and clinical features of malignancy in the latter as compared to the former. If uncertainty regarding classification in an individual case persists cytogenetic studies may be helpful as a clonal isochromosome 7q abnormality (either in isolation or associated with other clonal cytogenetic abnormalities including trisomy 8) can be identified by karyotypic analysis in many HSTCL cases [27-29].

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