Hematopathology

B-cell Lymphoma, Unclassifiable, with Features Intermediate Between Diffuse Large B-cell Lymphoma and Burkitt Lymphoma

Steven H. Swerdlow


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Clinical History:
88 year old woman who hit her head on the sink when she bent to pick up a brush and developed a lump on her forehead. Initial CT was interpreted as a small left frontal scalp hematoma. She had persistent shoulder/neck pain, went to the ER and her shoulder was injected with steroids and she was given oral steroids as well. After some pain relief, the patient presented again 4 weeks following her injury to the hospital with diplopia, a persistent "hematoma" and severe neck pain. Following a repeat CT and an MR, an excisional biopsy was performed and is submitted for your review. She developed pericardial tamponade and left sided cardiac failure unresponsive to medical therapy, was found to have involvement of the liver and probably other sites and expired about one week following her biopsy.


Slide 1
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Figure 1
Note the diffuse proliferation associated with a "starry sky" related to the scattered tingible body macrophages.
Figure 2
This high magnification field looks very much like a Burkitt lymphoma with rather uniform intermediate sized transformed lymphocytes and scattered tingible body macrophages.
Figure 3
Note how some of the neoplastic cells have very clefted nuclei.

Figure 4
Here the neoplastic cells are even more pleomorphic and include a very large multinucleate cell.
Figure 5
The Wright-Giemsa stained cytospin shows intermediate to large sized transformed cells with sometimes vacuolated basophilic cytoplasm.
Figure 6 - CD3
There are relatively few scattered CD3+ cells.

Figure 7 - CD20
There are numerous CD20+ cells.
Figure 8 - CD10
CD10 staining is equivocal.
Figure 9 - BCL6
Many cells are bcl-6+.

Figure 10 - BCL2
The cells are strongly bcl-2+.
Figure 11 - MUM-1
Many cells are MUM-1+.
Figure 12 - Ki-67
Virtually all nuclei are Ki-67+.


Histopathology:
The histologic sections demonstrated a diffuse proliferation of intermediate to larger transformed lymphoid cells associated with a starry sky pattern due to scattered tingible body macrophages. There were individually apoptotic cells as well as larger areas of apoptosis. The transformed cells varied in size, some had very clefted nuclei and there were even some very large multinucleate forms. The cells had variably vacuolated basophilic cytoplasm as seen on a cytospin of the cell suspension used for the flow cytometric studies.

Flow cytometric immunophenotypic studies:
CD5-, CD10-, CD20+, kappa monoclonal population with admixed heterogeneous T-cells.

Paraffin section immunohistochemical & in situ hybridization stains:
CD20+, CD10 some possible weak positivity, bcl-6+, bcl-2+, Ki-67 -- almost 100% positive, MUM-1+, ALK1-, EBER ISH for EBV-, CD3 -- scattered positive small cells.

Classical cytogenetics:
No metaphases.

Cytogenetic FISH studies:
nuc ish 3q27(BCL6sp)[80]/3q27(BCL6st[29], nuc ish 8q24(MYCsp)[86]/8q24(MYCst)[14], nuc ish 14q32(IGHx2), 18q21(BCLx2)[69]. In other words, BCL6 and MYC rearranged but not BCL2.

Diagnosis
B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (MYC and BCL6 rearranged) (aka, Diffuse large B-cell lymphoma with "high grade" features)

Discussion

Differential diagnosis
  • Burkitt lymphoma (atypical BL)

  • Diffuse large B-cell lymphoma

  • B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma

  • Primary cutaneous diffuse large B-cell lymphoma, leg type

  • Blastoid (pleomorphic) mantle cell lymphoma

  • Unusual lymphoblastic lymphoma
We will concentrate on the major differential diagnoses here which include the first three on the list. Primary cutaneous diffuse large B-cell lymphoma, leg type must be considered because clinically the mass would have been consistent with a deeply invasive cutaneous lymphoma. The pure population of transformed B-cells and the phenotype (CD10-, bcl6+, MUM-1+, bcl-2+) are typical for this entity (although not specific), MYC or BCL6 translocations have been reported to be present in a majority of cases, these lymphomas occur most frequently in older females and they are not restricted to the leg. [1] However, the diagnosis of a primary cutaneous lymphoma requires no evidence of extracutaneous disease at diagnosis (although they can then disseminate afterward -- I didn't write the rules!). Blastoid (pleomorphic) mantle cell lymphomas can resemble a lymphoblastic lymphoma or diffuse large B-cell lymphoma. The upcoming WHO Bluebook will use the term blastoid for the mantle cell lymphomas that resemble lymphoblastic lymphomas and pleomorphic for the more pleomorphic cases with larger cells and often prominent nucleoli that have diffuse large B-cell in the differential. None of the features of the case being presented specifically suggested such a diagnosis with the absence of CD5 expression, the strong expression of MUM-1 and presence of both MYC and BCL6 translocations (no individual feature is specific, however). If there was more concern for this possibility, documenting a lack of cyclin D1 and/or absence of a CCND1 translocation would also help to exclude such a possibility, even though they are also not absolute indicators since rare cases lack cyclin D1 abnormalities. The possibility of an unusual-appearing B-lineage lymphoblastic lymphoma could also be considered far down on one's list; however, in this case both the cytologic appearance and presence of surface immunoglobulin would be very unlikely. When dealing with only paraffin embedded tissue sections, use of a TdT stain is helpful since the vast majority of lymphoblastic neoplasms are positive.

Diffuse large B-cell lymphoma (DLBCL)
DLBCL is "a diffuse proliferation of large neoplastic B lymphoid cells with nuclear size equal to or exceeding normal macrophage nuclei or more than twice the size of a normal lymphocyte." [2] One should, however, recognize that there are other types of lymphoma that would also fulfill this definition and some DLBCL that do not. DLBCL is a heterogeneous entity that includes a number of different variants and subtypes. A complete discussion of DLBCL is far beyond the realm of this handout. Only a skeletal discussion is included here. The reader is referred to the references including our recent review of aggressive nodal B-cell lymphomas. [2, 3] One should also look for the 2008 WHO Bluebook when the stork deposits it for an expanded discussion of DLBCL and its many subtypes/variants.

Histopathology
There are four major morphologic variants of DLBCL that are well described in the WHO monograph: centroblastic, immunoblastic, T-cell/histiocyte rich (T/HRBCL) and anaplastic. [2] Although not recognized in the WHO monograph, another important morphologic variant of DLBCL is the type composed of often intermediate sized very blastoid-appearing cells. [4] Some of the blastoid type cases may also correspond to lymphomas described as being composed of "small centroblasts."

Other variants/subtypes of DLBCL, some of which will shortly become "distinct entities" include plasmablastic lymphomas, DLBCL with expression of anaplastic lymphoma kinase (ALK), mediastinal large B-Cell lymphoma, intravascular large B-cell lymphomas, primary effusion lymphoma and DLBCL associated with chronic inflammation . Each of these lymphomas has its own distinctive, although not necessarily specific morphologic appearance. Similarly they have distinctive immunophenotypic and cytogenetic/molecular features but are not further discussed here.

Immunophenotypic studies
Most but not all DLBCL express the B-cell-associated, but not B-cell-specific, CD20 antigen and/or other pan-B-cell antigens. [5, 6, 7] Surface immunoglobulin, if present, is monoclonal but it may be non-detectable. They are cyclin D1 and TdT negative. The detailed phenotype of DLBCL is variable. [2, 8, 9, 10, 11] Some cases mark like cells in a follicular lymphoma (CD10+, bcl-6+) and others more like later B-cells with loss of follicular center cell markers (CD10, bcl-6) and acquisition of transitional (MUM-1) and overt plasma cell-associated markers (CD138). Bcl-2 is variably expressed. Some cases express the CD5 T-cell associated antigen. [12]

Cytogenetic abnormalities
DLBCL demonstrate BCL-2 rearrangements in 20-30% of the cases and BCL-6 rearrangements in up to 30%. [13, 14] A small proportion have MYC translocations. [15, 16, 17] In our experience, classical cytogenetic studies are successful in about 70% of DLBCL with >90% of those studies demonstrating an abnormal karyotype. [18]

Burkitt/Atypical Burkitt Lymphoma (BL)
BL are diffuse, and much less often in part follicular, proliferations of relatively small transformed cells with amphophilic cytoplasm. [19] They have an extremely high proliferation fraction, prominent apoptosis and a "starry sky" appearance. Some cases, usually found in immunodeficient patients, have plasmacytoid features. Current convention is that Burkitt and atypical Burkitt (Burkitt-like) lymphomas represent the same entity but with the latter showing more cytologic variation. The terms atypical Burkitt or Burkitt-like lymphoma are not to be used simply because the cells of a DLBCL are on the smaller side.

Immunophenotypic studies
Most BL are surface immunoglobulin positive, CD5-, CD10+, and usually bcl-2- with virtually 100% of their nuclei Ki-67+, a phenotype shared with some DLBCL.

Cytogenetic studies
Cytogenetic studies demonstrate a translocation involving MYC at chromosome 8q24 and usually either the IgH locus at 14q32 or less commonly either the kappa or lambda light chain loci at 2q11 or 22q11, respectively. There are usually few other abnormalities. [20] It is critical to realize that a MYC translocation is not specific for BL, so that if one is going to use FISH to help document the diagnosis of BL one should also look for BCL-2 and BCL-6 translocations that at least usually ought to be absent.

Burkitt lymphoma versus diffuse large B-cell lymphoma
Although it is considered very important to distinguish BL from DLBCL to prevent overtreatment of DLBCL and undertreatment of BL, it can be very difficult, especially in adults, and gold standard criteria are lacking. [20, 21, 22, 23, 24, 25] As recently reviewed in the editorial written about the two BL gene profiling papers, "BL is not…cured by the treatment for diffuse large-B-cell lymphoma,…", and, at the same time, adults "often do not tolerate very well the side effects" of BL-type therapy. [25] Hence, "Diagnostic accuracy is therefore essential to prevent undertreatment or overtreatment of patients".

The problematic cases usually have many Burkitt-like features but either the phenotype/genotype/karyotype is too "atypical", the cytologic appearance is beyond that described for "atypical" BL, or both. Depressingly, Stein and Hummel wrote in 2006 that "at present, there are no reliable criteria that can be applied to distinguish BL from DLBCL." [26] Studies that have addressed this problem either have found only a minority of cases that fulfill all of the typical BL criteria described above and one study found no adult cases. [21, 23, 27]

Recent gene profiling studies have demonstrated the existence of a molecularly homogeneous group of BL that are morphologically, phenotypically and even cytogenetically heterogeneous. [20, 24, 25] While this suggests that the criteria we use to diagnose BL cannot always be relied upon, gene profiling studies are not standardized and not currently a clinical laboratory test. Furthermore, consistent with our daily experience, one of the major gene profiling studies also demonstrated the presence of cases that were "intermediate" between BL and DLBCL, substantiating the existence of a true gray zone. [20] The 2008 WHO Bluebook will have a chapter specifically about these cases that seem to have features intermediate between a BL and a DLBCL.

B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (diffuse large B-cell lymphoma with "high grade" features)

B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma is characterized as having:
  • Some morphologic features of BL including apoptosis and frequent starry sky although usually with other features unlike BL (larger cells, more prominent single nucleoli, non-amphophilic cytoplasm)

  • A phenotype usually with some features of BL (CD10+, bcl6+, sometimes bcl2-) but with other atypical but inconstant features (eg, bcl2+ (~50%), MUM1+, often Ki-67 not >95% although usually >80%).

  • MYC rearrangements present in ~35-50% of cases, sometimes with other translocations as well. BCL2 translocations in ~15% sometimes with MYC.

  • MYC translocations often with non-IG genes and associated with a complex karyotype. This is unlike most BL.

A pragmatic approach to cases with a major differential diagnosis between Burkitt lymphoma and diffuse large B-cell lymphomas

Our current imperfect modus operandi is to diagnose BL when the morphologic appearance is acceptable (but not necessarily typical), the phenotype is typical and a MYC but not a BCL-2 or BCL-6 translocation is present. Most other cases require a more descriptive diagnosis such as the one used here or a term like "high grade B-cell lymphoma, not otherwise specified" or DLBCL with "high grade" features with the cases having the fewest discrepancies from the typical constellation of BL findings being considered closer to a "real" BL. [23] In addition, although not a quantitative science and beyond what I can attempt to impart today, the reality is that some discrepancies also carry less weight than others, especially if the finding is not entirely convincing, such as possible weak bcl-2 protein expression in an otherwise typical BL. Once one is dealing with a case with BL at least in the differential, reliance solely on "biologic markers" (Ki-67>90%, CD10+, bcl-6+, bcl-2-, MYC translocated but not BCL-2 or BCL-6) seemed to identify a more homogeneous population of patients, than use of a consensus histologic/immunophenotypic diagnosis. [21] However, many of the lymphomas in the BL/DLBCL gray zone region that were not felt to fulfill the criteria for a BL still had "a phenotypic and/or genetic shift to BL." This study also highlighted hematopathologists' problems with reproducibly categorizing these types of cases. We would specifically designate cases with coexistent MYC and BCL-2 and/or BCL-6 translocations as, at least those with dual MYC and BCL-2 translocations, are known to be extremely aggressive. [15, 28, 29] Although BCL-2 translocations are rarely found in molecularly-defined BL, Hummel, et al, found them more commonly in their intermediate or non-BL groups and BCL-6 translocations were not found in molecularly defined BL. [20]

Therapeutic implications of B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (diffuse large B-cell lymphoma with "high grade" features)

The best therapy for the DLBCL with some BL-like features remains to be defined, although some report that cases with some of the BL features do not do well when they receive more typical DLBCL therapy. [24, 30] Whether finding MYC translocations independent of other parameters is a prognostic indicator in DLBCL is controversial.

Acknowledgement:
Some of this handout is adapted and/or exerpted from a recent review article that I co-authored. [3]

Take Home Messages
  • Not everything is what it seems to be in diagnostic hematopathology.

  • Reliance on any single ancillary study to help categorize a lymphoma can be very misleading.

  • Be proud and not ashamed of recognizing that a lymphoma has features that do not allow for it to fit neatly into one of the standard categories.

  • The new WHO Bluebook monograph is on its way.

References
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  2. Gatter KC, Warnke RA. Diffuse large B-cell lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World Health Organization Classification of Tumours Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001:171-174.

  3. Prakash S, Swerdlow SH. Nodal aggressive B-cell lymphomas: A diagnostic approach. J Clin Pathol. 2007;60:1076-1085.

  4. Natkunam Y, Warnke RA, Zehnder JL, Jones CD, Milatovich-Cherry A, Cornbleet PJ. Blastic/blastoid transformation of follicular lymphoma: immunohistologic and molecular analyses of five cases. Am J Surg Pathol. 2000;24:525-534.

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  17. Au WY, Horsman DE, Gascoyne RD, Viswanatha DS, Klasa RJ, Connors JM. The spectrum of lymphoma with 8q24 aberrations: a clinical, pathological and cytogenetic study of 87 consecutive cases. Leuk Lymphoma. 2004;45:519-528.

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  22. Anonymous. A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin's lymphoma. The Non-Hodgkin's Lymphoma Classification Project. Blood. 1997;89:3909-3918.

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  28. Kanungo A, Medeiros LJ, Abruzzo LV, Lin P. Lymphoid neoplasms associated with concurrent t(14;18) and 8q24/c-MYC translocation generally have a poor prognosis. Mod Pathol. 2006;19:25-33.

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  30. Braziel RM, Arber DA, Slovak ML, et al. The Burkitt-like lymphomas: a Southwest Oncology Group study delineating phenotypic, genotypic, and clinical features. Blood. 2001;97:3713-3720.