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Follicular center cell lymphomas, now known as Follicular lymphomas, describe a subset of Non-Hodgkin's lymphoma where the lymphocytes are derived from neoplastic lymphoid follicular germinal centers. Many lymphomas are derived from this subset. These lymphomas may have small or large nuclear size and the nuclei may be cleaved or folded or non-cleaved. The diffuse large cell lymphoma is the most common histologic subtype in the Western world.


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FISH Is Superior to PCR in Detecting t(14;18)(q32;q21)–IgH/bcl-2 in Follicular Lymphoma Using Paraffin-Embedded Tissue Samples

Richard R. Einerson, MT(ASCP), etal.
Am J Clin Pathol 2005;124:421-429 Abstract quote

Detection of t(14;18)(q32;q21)–IgH/bcl-2, which is present in 70% to 95% of follicular lymphomas (FLs), might aid in diagnosing FL. The efficacy of routine polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques in detecting t(14;18) in paraffin-embedded tissue samples was compared on 5 normal tonsils and 28 FLs demonstrated to be t(14;18)+ by previous karyotyping.

There was technical failure in 14 (50%) of the FLs by PCR, likely due to B-5 fixation, and 4 (14%) of FLs by FISH, likely due to advanced specimen age. In the remaining successful cases, 5 (36%) of 14 were positive by PCR and 24 (100%) of 24 were positive by FISH. All 5 normal tonsils were negative by both methods.

FISH is superior to PCR for detecting t(14;18) from paraffin-embedded tissue samples because it is more sensitive and equally specific.
Abnormal patterns of antigenic expression in follicular lymphoma : a flow cytometric study.

Ray S, Craig FE, Swerdlow SH.

Department of Pathology, Division of Hematopathology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Am J Clin Pathol. 2005 Oct;124(4):1-8. Abstract quote  

In order to distinguish follicular lymphoma (FL) from reactive hyperplasia (RH), flow cytometric (FC) immunophenotypic studies have been used primarily to look for monotypic CD5- CD10+ B cells with much more limited use of bcl-2 stains.

We studied what additional diagnostic information could be extracted from routine FC studies in a retrospective study of 90 FL and 91 RH cases. The following significant differences were identified: dimmer CD19 on CD10+ B cells in FL (P < .0001), brighter CD10 and more numerous CD10+ B cells in FL (P < .0001), and brighter CD20 on neoplastic B cells than on other B cells in FL (P = .002) or in RH (P = .05). In the FL cases, no correlations could be documented between any phenotypic findings. Grade 3 FL had significantly dimmer CD10 expression than lower grades (P = .05).

Visual analysis of CD10+ vs CD10- smaller B cells showed dimmer CD19 on the CD10+ cells in 28 (44%) of 64 evaluable FL cases and 0 of 87 evaluable RH cases.

These findings expand the ways in which FC studies can be used to help diagnose FL and suggest that the phenotypic aberrations identified do not represent normal developmental pathways.

bcl-2 expression by multicolor flow cytometric analysis assists in the diagnosis of follicular lymphoma in lymph node and bone marrow.

Cook JR, Craig FE, Swerdlow SH.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Am J Clin Pathol 2003 Jan;119(1):145-51 Abstract quote

The expression of bcl-2, CD10, and CD20 was examined by multicolor flow cytometry in 78 samples including lymph node or other tissue biopsy specimens containing follicular lymphoma (FL; n = 17), reactive hyperplasia (RH; n = 28), or other malignant lymphomas (n = 20), as well as bone marrow aspirates (n = 13).

The presence of CD10+ cells with high bcl-2 expression predicted the presence of FL rather than RH with a positive predictive value of 100% and negative predictive value of 96%. CD10+ cells with high bcl-2 expression also were found in a subset of diffuse large B-cell lymphomas and were otherwise rare in other types of malignant lymphoma. In contrast with immunohistochemical studies, a reduced but apparently measurable level of bcl-2 was present in benign follicular center cells. Hematogones showed lower bcl-2 levels than did FL cells in the bone marrow, and neutrophils were bcl-2-.

Measurement of bcl-2 expression levels by multiparameter flow cytometry offers a rapid, quantitative assessment that may assist in the diagnosis of FL in lymph nodes or bone marrow, even when other CD10+ cells or admixed normal B cells are present.

Clonal Heterogeneity Assessed by Flow Cytometry in B-Cell Lymphomas Arising From Germinal Centers

Mar Bellido, MD, Enriqueta Rubiol, Josep Ubeda, Camino Estivill, Granada Perea, MD, Joana Rego-Araujo, Anna Aventín, MD, Ramón Bordes, MD, Jorge Sierra, MD, and Josep F. Nomdedéu, MD

Am J Clin Pathol 2002;117:864-870 Abstract quote

Patients with mature follicular B-cell lymphomas develop aggressive non-Hodgkin lymphomas (NHLs) during disease progression. It is controversial whether most diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) emerge as de novo lymphomas or from an original follicular lymphoma.

To distinguish clonally related populations in aggressive NHL, we studied the immunophenotypic features of 18 consecutive samples from 16 patients. Three flow cytometric patterns were distinguished: (1) a homogeneous neoplastic population of large B cells with phenotypic features of follicular center cells; (2) 2 atypical populations of B cells, small monoclonal B cells, and large B cells with loss of some surface antigens; and (3) 2 clonal populations of small and large B cells sharing the same light-chain isotype.

The 3 flow cytometric patterns were observed, respectively, in de novo DLBCL and BL, transformation into BL, and transformation into DLBCL. Flow cytometric data can provide valuable information about the natural history of NHL.

Immunophenotyping Large B-Cell Lymphomas Flow Cytometric Pitfalls and Pathologic Correlation

Heidi C. Bertram, MD, Irene J. Check, PhD, and Michelangelo A. Milano, MD

Am J Clin Pathol 2001;116:191-203 Abstract quote

Large cell lymphomas often challenge the diagnostic flow cytometrist. The purposes of this study were to improve our protocols for diagnosing large cell lymphomas and to correlate flow cytometric (FC) data with demographic and histologic features.

We identified 63 cases of large B-cell lymphoma between January 1, 1995, and July 30, 1999, and reviewed the diagnostic slides and FC light scatter and staining patterns. The 51 lymphomas with adequate material for systemic review fell into 2 light scatter patterns: "clear cut," with large abnormal cells (high forward scatter relative to normal lymphocytes), 17 cases (33%); and "complex," 34 cases (67%). Clear-cut cases were more mitotically active (average of 42 vs 25 per 10 high-power fields), with higher cellularity. Apoptosis, geographic necrosis, and sclerosis were present histologically in many cases, regardless of FC findings.

We conclude that morphologic features of large cell lymphomas do not predict which cases will be difficult to diagnose by FC. Gating strategies can be critical to improve the diagnostic yield.


Real-Time RT-PCR Assay for Quantifying Cyclin D1 mRNA in B-Cell Non-Hodgkin's Lymphomas.

Medeiros LJ, Hai S, Thomazy VA, Estalilla OC, Romaguera J, Luthra R.

Departments of Hematopathology (LJM, SH, VAT, OCE, RL) and Lymphoma/Myeloma (JR), The University of Texas M.D. Anderson Cancer Center, Houston, Texas.

Mod Pathol 2002 May;15(5):556-64 Abstract quote

Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin's lymphoma (NHL) characterized by the t(11;14)(q13;q32), in which the ccnd1 gene is juxtaposed with the immunoglobulin heavy chain gene, resulting in up-regulation of cyclin D1. Cyclin D1 overexpression is a useful finding that supports the diagnosis of MCL.

In this study, we used a 5' --> 3' exonuclease-based real-time reverse-transcriptase polymerase chain reaction (RT-PCR) method to quantify cyclin D1 mRNA in 108 B-cell NHL and nonneoplastic specimens, including 25 cases of MCL. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also quantified to normalize cyclin D1 mRNA levels, and the data were expressed as a cyclin D1 to GAPDH ratio. At each anatomic site, MCL cases had higher cyclin D1 levels than other types of NHL or nonneoplastic specimens, without overlap. For example, in lymph node specimens, the median cyclin D1/GAPDH ratio was 147 (range, 94-160) in MCL, compared with 8.6 (range, 4-18) in chronic lymphocytic leukemia/small lymphocytic lymphoma; 5.8 (range, 1.8-24) in follicular lymphoma; 4.8 in one case of marginal zone lymphoma; and 20.2 (range, 5.8-44) in reactive specimens. Statistical analysis using one-way analysis of variance (ANOVA) showed that MCL cases had significantly higher cyclin D1 levels than other groups (P <.05). In peripheral blood specimens involved by MCL, cyclin D1 levels correlated with extent of involvement.

We conclude that this real-time RT-PCR method to quantify cyclin D1 expression is helpful in distinguishing MCL from other types of B-cell NHL and from nonneoplastic specimens. This method is rapid, can be applied to the analysis of fluid specimens, and obviates the need for time-consuming and laborious detection methods that are required by traditional semi-quantitative RT-PCR methods.

Detection of bcl-2/J(H) Translocation by Polymerase Chain Reaction.

Hsi ED, Tubbs RR, Lovell MA, Braziel RM, Gulley ML.

Cleveland Clinic Foundation, Cleveland, Ohio (Drs Hsi and Tubbs); The Children's Hospital of Denver, Denver, Colo (Dr Lovell); Oregon Health and Science University, Portland (Dr Braziel); and University of North Carolina, Chapel Hill (Dr Gulley).

Arch Pathol Lab Med 2002 Aug;126(8):902-8 Abstract quote

Context.-The t(14;18)(q32;;t6q21) translocation, found in about 85% of follicular lymphomas, brings the bcl-2 gene on 18q21 under control of the immunoglobulin heavy-chain gene transcriptional regulatory elements on 14q32. Detection of this translocation in a clinical sample suspected of containing lymphoma can assist the pathologist in diagnosis and classification of lymphoma. Polymerase chain reaction is a technology that is frequently used to detect the t(14;18)(q32;q21) translocation (bcl-2/J(H)). This article reviews the utility of polymerase chain reaction testing for bcl-2/J(H) detection and summarizes the experience of participants in the Molecular Oncology Proficiency Survey of the College of American Pathologists from 1997 through 2000.

Objective.-To describe current practice and encourage improvement of bcl-2/J(H) testing in clinical laboratories.

Design.-Retrospective analysis of Molecular Oncology Proficiency Survey data.

Participants.-Laboratory participants in the College of American Pathology Molecular Oncology Proficiency Survey.

Results.-Twenty-four well-characterized specimens were sent to participants, of which 6 contained bcl-2/J(H) major breakpoint region translocations. Eight hundred nineteen major breakpoint region and 323 minor cluster region determinations were performed, with an overall correct response rate of 91% and 94%, respectively. No significant difference in correct response could be found for frozen versus paraffin-embedded tissues. Many laboratories did not know their assay sensitivity.

Conclusions.-Overall performance was good; however, there was great variability in the methods reported and lack of knowledge of the limits of detection was common. Continued participation in external quality control programs, such as the Molecular Oncology Survey; dissemination of information that impacts on test performance; and technical recommendations from the molecular diagnostics community are critical for improved testing for bcl-2/J(H).


GENERAL Taken from Chan JKC. Practical Lymphoma Diagnosis: A Simplified Approach. Presented at the 111th Semi-Annual California Tumor Tissue Registry. December 2001.
MAJOR CRITERIA Back to back follicles disposed throughout the entire nodal parenchyma, with little interfollicular tissue (abnormal architecture). This pattern is diagnostic of follicular lymphoma and is observed in about ~85% of all cases
MINOR CRITERIA Must have >/=3 if major criteria is not fulfilled
  Consistent lack of tingible body macrophages
Cellular monotony with predominance of centrocytes in the follicles
Mantles are lacking or incomplete
Consistent absence of cellular polarization in the follicles
Presence of follicles in perinodal tissues
Dysplastic follicular center cells, eg. extremely elongated nuclei, bizarre cells, signet ring cells, many multilobated cells
Presence of atypical cells (cells larger than small lymphocytes, and possessing irregularly folded nuclei) in interfollicular regions, indicative of interfollicular invasion

DEFN: An area of lymphomatous infiltrate completely devoid of neoplastic follicles. A simple broadening of the interfollicular zone does not qualify

Impact of a diffuse component on survival is controversial. In general, it is signficant only if diffuse areas occupy >25-50% of grade 1 or grade 2 follicular lymphoma. On the other hand, any diffuse component apparently worsens the prognosis of grade 3 follicular lymphoma.

Follicular Pattern of Bone Marrow Involvement by Follicular Lymphoma

Emina Torlakovic, MD,1 Goran Torlakovic, MD,1 and Richard D. Brunning, MD

Am J Clin Pathol 2002;118:780-786 Abstract quote

Five patterns of bone marrow infiltration by non-Hodgkin lymphoma or Hodgkin lymphoma are currently recognized, but a true follicular pattern of bone marrow involvement by follicular lymphoma has not been described.

In 260 bone marrow trephine biopsy specimens involved by follicular lymphoma, we identified 12 cases with a follicular pattern of bone marrow involvement. The paratrabecular pattern was not present at all in 9, and it accounted for less than 10% of tumor burden in 3 cases. Malignant follicles in the bone marrow were similar to malignant follicles in the respective lymph nodes. Follicular dendritic cells were identified by immunohistochemical analysis.

The true follicular pattern of bone marrow involvement by follicular lymphoma seems to be more frequent in women than in men. It is important to recognize this pattern of follicular lymphoma in the bone marrow because it is possible to misinterpret interstitial lymphoid aggregates as benign in the absence of the more characteristic paratrabecular pattern.

Fine-Needle Aspiration in Non-Hodgkin Lymphoma
Evaluation of Cell Size by Cytomorphology and Flow Cytometry

Jerald Z. Gong, MD, David C. Williams, Jr, MD, PhD, Katharine Liu, MD, and Claudia Jones, MD

Am J Clin Pathol 2002;117:880-888 Abstract quote

We studied 48 non-Hodgkin lymphoma (NHL) fine-needle aspiration (FNA) specimens with initial cytomorphology (CM) and flow cytometry (FC) and subsequent surgical biopsy of the same lesion to determine whether a reliable diagnosis of large cell lymphoma or large cell transformation could be made. CM was evaluated by examining 200 lymphocytes in each specimen.

FC was performed by analyzing monoclonal or abnormal B-cell populations. Percentages of large cells were evaluated by CM and FC and results correlated with the histologic diagnosis. All small cell NHLs showed fewer than 40% large cells by CM and FC; 100% (9/9; FC) and 67% (6/9; CM) of diffuse large B-cell lymphomas demonstrated greater than 40% large cells. Variable numbers of large cells were detected in grade III follicular lymphoma, low-grade lymphoma with partial large cell transformation, and large B-cell lymphoma containing fewer than 10% neoplastic cells. By using combined CM and FC, large cell lymphoma and large cell transformation can be diagnosed reliably by FNA if greater than 40% large cells are present.

Surgical biopsy is necessary when there is necrosis, fewer than 10% neoplastic cells by FC, or fewer than 40% large cells with clinical signs of transformation.


Am J Clin Pathol 2000;114:912-921
This identified 11 cases of a floral variant which had 4 cases coexpression CD5

All had rearrangement of bcl-2 locus

CD5+ follicular lymphoma: a clinicopathologic study of three cases.

Barry TS, Jaffe ES, Kingma DW, Martin AW, Sorbara L, Raffeld M, Pittaluga S.

Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Am J Clin Pathol 2002 Oct;118(4):589-98 Abstract quote

Follicular lymphoma (FL) is a low-grade lymphoma that typically lacks CD5 antigen expression.

We report 3 cases of FL with unusual expression of CD5. All cases showed histologic features of FL, including effaced nodal architecture, follicular growth pattern, and a spectrum of grades from 1 to 3 using World Health Organization criteria. In flow cytometric studies, all 3 cases showed a light chain-restricted, CD19+, CD20+ B-cell population coexpressing CD10 and low-level CD5. Immunohistochemical studies demonstrated an identical B-cell immunophenotype with weak expression of CD5 and coexpression of bcl-2 protein and the germinal center-associated markers, CD10 and bcl-6 protein.

None of the cases showed expression of CD43, cyclin D1, or IgD. By molecular analysis, immunoglobulin heavy chain gene rearrangements were demonstrated in all 3 cases, and 2 of 3 cases had a t(14;18).

These cases highlight the difficulty classifying these lymphomas by flow cytometric studies alone and emphasize the importance of recognizing FL in the differential diagnosis of CD5+ B-cell lymphomas.


Blastic/Blastoid Transformation of Follicular Lymphoma Immunohistologic and Molecular Analyses of Five Cases

Yasodha Natkunam, M.D., Ph.D.; Roger A. Warnke, M.D.; James L. Zehnder, M.D.; Carol D. Jones; Athena Milatovich-Cherry, Ph.D.; P. Joanne Cornbleet, M.D., Ph.D.

From the Department of Pathology, Stanford University Medical Center, Stanford, California, U.S.A.

Am J Surg Pathol 2000;24:525-534 Abstract quote

Progression of follicular lymphoma to a higher-grade malignancy frequently heralds a poor prognosis. Clinical transformation is variably accompanied by a spectrum of histologic changes characterized by alteration in growth and cytology. Although several cytogenetic events and potential oncogenes have been documented in this progression, the underlying molecular mechanisms are largely unknown.

We present five patients with an unusual histologic transformation of follicular lymphoma manifested by blastic/blastoid morphology. This transformation is histologically distinct from other types of transformation of follicular lymphoma.

All five cases exhibited the t(14;18) translocation and expressed the BCL-2 protein. In addition, two of the five patients showed increased levels of the p53 protein within neoplastic cells implicating a possible role for this oncogene in blastic/blastoid transformation.

The lack of BCL-1 and myeloid antigens by immunohistochemistry and flow cytometry studies served to distinguish blastic/blastoid transformation of follicular lymphoma from its morphologic mimics. This distinction is clinically important because lymphoblastic and myeloid leukemias require significantly different therapeutic modalities and show better prognosis. Moreover, the lack of Epstein-Barr virus-specific mRNA suggests that this virus is unlikely to participate in blastic/blastoid transformation of follicular lymphoma.


Intracytoplasmic immunoglobulin crystals in follicular lymphoma.

Wada R, Ebina Y, Kurotaki H, Yagihashi S.

Department of Pathology, Hirosaki University School of Medicine, Hirosaki, Japan.


Hum Pathol 2002 Nov;33(11):1141-4 Abstract quote

We report a case of follicular lymphoma with crystal inclusions. Swollen lymph nodes taken from the left neck of a 53- year-old Japanese woman were replaced by follicular proliferation of atypical centroblastic and centrocytic cells with intracytoplasmic crystal inclusions.

The crystals were confined to lymphoma cells and were not found in histiocytes. Lymphoma cells were positively immunostained with lambda light chain and mu heavy chain, but the crystals were only weakly so. In situ hybridization of light chains disclosed a monoclonal expression of lambda light chain mRNA in lymphoma cells. The crystals had a periodic linear substructure with about 5-nm intervals.

The worldwide literature reports 8 cases, including the current case of non-Hodgkin's lymphoma with crystals confined to the neoplastic cells. The cases did not accompany paraproteinemia and crystal-storing histiocytosis and appear to follow a favorable clinical outcome.


Am J Clin Pathol 1087;88:264-269
Lymphomatous nodules surrounded and infiltrated by small lymphocytes of the follicular mantle, resulting in nodules with an unusual serrated configuration, mimicking a floral arrangement

Must distinguish from benign progressive transformation of germinal centers

Low Histologic Grade Follicular Lymphoma With High Proliferation Index: Morphologic and Clinical Features.

Wang SA, Wang L, Hochberg EP, Muzikansky A, Harris NL, Hasserjian RP.

From the Departments of *Pathology and daggerInternal Medicine, Division of Hematology/Oncology, and double daggerBiostatistics Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Am J Surg Pathol. 2005 Nov;29(11):1490-1496. Abstract quote  

Histologic grading has been used as a guide for clinical management in follicular lymphoma (FL). Proliferation index (PI) of FL generally correlates with tumor grade; however, in cases of discordance, it is not clear whether histologic grade or PI correlates with clinical aggressiveness.

To objectively evaluate these cases, we determined PI by Ki-67 immunostaining in 142 cases of FL (48 grade 1, 71 grade 2, and 23 grade 3). A total of 24 cases FL with low histologic grade but high PI (LG-HPI) were identified, a frequency of 18%. On histologic examination, LG-HPI FL often exhibited blastoid features. Patients with LG-HPI FL had inferior disease-specific survival but a higher 5-year disease-free rate than low-grade FL with concordantly low PI (LG-LPI). However, transformation to diffuse large B-cell lymphoma was uncommon in LG-HPI cases (1 of 19; 5%) as compared with LG-LPI cases (27 of 74; 36%).

In conclusion, LG-HPI FL appears to be a subgroup of FL with clinical behavior more akin to grade 3 FL. We propose that these LG-HPI FL cases should be classified separately from cases of low histologic grade FL with concordantly low PI.

Follicular Lymphoma with Marginal Zone Differentiation: Microdissection Demonstrates the t(14;18) in Both the Follicular and Marginal Zone Components

Subramanian Yegappan, etal.

Mod Pathol 2001;14:191-196 Abstract quote

On occasion, follicle center lymphomas (FCL) may contain a marginal-zone (MZ) component in which the interfollicular lymphoid cells take on an MZ cell morphology. In the past, these have been termed composite lymphomas. However, recent studies suggest that the two components are clonally related. It is unknown whether the bcl-2 translocation present in most FCLs is present in the cells that demonstrate MZ cell morphology.

We have identified three cases of low-grade FCL with a MZ component suitable for laser capture microdissection (LCM) of the two components. Cases were immunophenotyped in paraffin section with antibodies to CD10, CD20, bcl-2, and bcl-6. LCM was done to isolate cells from each component. Polymerase chain reaction for t(14;18) using primers to the major breakpoint region was performed on DNA extracts. The sensitivity of the PCR assay was decreased to 5%–10% follicle center cells in a background of reactive tonsil cells. All three cases showed different phenotypes in each component. The FCL component was positive for all four of the above markers, whereas the MZ component expressed only CD20 and bcl-2. Both components showed t(14;18) amplicons of identical size, with the MZ component signal being stronger than the 5%–10% sensitivity control, suggesting that the signal was not from rare, contaminating FCL cells.

These results confirm that both components are clonally related and support the theory that these are indeed FCLs with MZ differentiation (that retain the t(14;18)) rather than the reverse, MZ lymphoma with follicle center differentiation.


Decreased CD10 Expression in Grade III and in Interfollicular Infiltrates of Follicular Lymphomas

Camellia Eshoa, MD, Sherrie Perkins, MD, PhD, Bal Kampalath, MD, Vinod Shidham, MD, MIAC, MRCPath, Mark Juckett, MD, and Chung-Che Chang, MD, PhD

Am J Clin Pathol 2001;115:862-867 Abstract quote

CD10 expression in various grades and interfollicular infiltrates of follicular lymphoma (FL) has not been well documented. Immunohistochemical staining for CD10 (clone 56C6) was performed on paraffin-embedded tissue from 26 cases of classic FL. Negative or weak expression of CD10 was more frequent in grade III (5/6 [83%]) than in grade I FLs (3/15 [20%]). CD10+ interfollicular infiltrates were present in 16 cases. Six (38%) of 16 cases showed that CD10 expression was strong or moderate in follicular areas but weak or negative in interfollicular infiltrates.

Our results suggest that CD10 expression is frequently weak to negative in grade III and in interfollicular infiltrates of FLs. Therefore, lack of CD10 expression on small specimens, such as from needle core biopsy or fine-needle aspiration, does not preclude the possibility of a diagnosis of FL. Furthermore, lack of CD10 expression in diffuse large B-cell lymphoma does not exclude the possibility that the neoplastic lymphocytes are of follicle center cell origin.

Clinicopathologic Analysis of CD10+ and CD10– Diffuse Large B-Cell Lymphoma Identification of a High-Risk Subset With Coexpression of CD10 and bcl-2

Yin Xu, MD, PhD, Robert W. McKenna, MD, Kyle H. Molberg, MD, and Steven H. Kroft, MD

Am J Clin Pathol 2001;116:183-190 Abstract quote

We analyzed 53 cases of diffuse large B-cell lymphoma (DLBCL) to determine whether expression of CD10 is a relevant biologic parameter. Tumor morphologic features were assessed semiquantitatively. Bcl-2 protein expression was studied by immunohistochemical analysis. The presence or absence of CD10 by flow cytometry was correlated with clinical and pathologic characteristics. CD10+ (23 cases) and CD10– (30 cases)

DLBCLs were indistinguishable based on age, sex, extranodal presentation, B symptoms, clinical stage, morphologic features, or bcl-2 expression. However, cases with a CD10+ phenotype showed a significantly lower rate of complete remission. Cases expressing bcl-2 showed trends toward a lower rate of complete remission and poorer overall survival. Examination of CD10 and bcl-2 interaction revealed that the prognostic effects for both of these antigens were due to a subset of CD10+ bcl-2–positive cases. Compared with cases expressing one or neither of these markers, patients with dual-positive tumors had a poorer complete response rate to initial therapy and strikingly worse overall survival.

While CD10+ and CD10– DLBCLs are similar with regard to a variety of clinical and pathologic features, CD10 and bcl-2 coexpressing tumors are an extremely high-risk subset based on response to therapy and overall survival.


CD30 Expression in Follicular Lymphoma

Laura J. Gardner, MD, Jacek M. Polski, MD, H. Lance Evans, MD, Sherrie L. Perkins, MD, PhD, and Cherie H. Dunphy, MD

From the Division of Hematopathology, Department of Pathology, St Louis University Health Sciences Center, St Louis, Mo (Drs Gardner, Evans, and Dunphy); the Department of Pathology, University of South Alabama, Mobile (Dr Polski); and the Section of Hematopathology, University of Utah Health Sciences Center, Salt Lake City (Dr Perkins)

Arch Pathol Lab Med 2001;125:1036–1041 Abstract quote

Context.—CD30+ anaplastic large cell lymphomas were originally described as being of T-cell, null cell, and B-cell origin. CD30, however, is not a specific marker of anaplastic large cell lymphoma and has been found to be expressed in reactive as well as neoplastic populations as a probable activation marker. In addition, CD30+ cells have also been described in both diffuse large B-cell and follicular lymphomas (FLs), resembling the pattern seen in reactive tonsils and lymph nodes.

Objective. —We report an index case of FL with CD30 expression, which on initial touch preparations and flow cytometric immunophenotyping revealed a prominent population of CD30+ cells with marked cellular pleomorphism (anaplasia) in a background of typical FL. Immunohistochemistry of the paraffin section for CD30 in our index case confirmed unequivocal CD30+ pleomorphic cells in the malignant nodules in occasional clusters. This case prompted a study of additional cases of FL for pattern of immunoreactivity with CD30 on paraffin sections.

Design.—Twenty-two additional cases of FL (grades 1–3) were retrieved for CD30 immunoperoxidase staining as in the index case.

Results.—This study demonstrated 32% of the additional cases of FL had definitive CD30+ , large, pleomorphic malignant cells by paraffin immunohistochemistry. In 2 cases (9%), the pattern of immunoreactivity with CD30 showed clustering and variable staining of large cells, as our index case.


GENERAL The following findings will support a diagnosis of follicular lymphoma over reactive follicular hyperplasia:

Germinal center cells positive in 80-85% of follicular lymphomas

Reactive follicles can occasionally harbor an appreciable number of T lymphocytes which are bcl-2 positive, leading to false positive interpretation of staining; comparison with a corresponding section stained with a T-cell marker should obviate such a problem

bcl-2 negativity in follicular lymphomas are lower in grade I lymphomas and higher in grade III lymphomas

BCL-2 Is Consistently Expressed in Hyperplastic Marginal Zones of the Spleen, Abdominal Lymph Nodes, and Ileal Lymphoid Tissue.

Meda BA, Frost M, Newell J, Bohling SD, Huebner-Chan DR, Perkins SL, Lim MS, Medeiros LJ, Elenitoba-Johnson KS.


Am J Surg Pathol. 2003 Jul;27(7):888-94 Abstract quote

BCL-2 is an antiapoptotic protein overexpressed in follicular lymphomas, principally as a result of the t(14;18)(q32;q21), and useful in distinguishing follicular lymphoma (usually BCL-2 positive) from follicular hyperplasia (BCL-2 negative). BCL-2 is also overexpressed in other lymphoma types without the t(14;18), including marginal zone B-cell lymphoma, because of other, poorly understood mechanisms. It has been suggested that BCL-2 immunoreactivity can distinguish between malignant (BCL-2 positive) and reactive (BCL-2 negative) marginal zone B cells.

In this study, we evaluated 26 spleen, 10 abdominal lymph node, and 3 ileum specimens with marginal zone B-cell hyperplasia for BCL-2 expression immunohistochemically.

We also analyzed these cases using polymerase chain reaction methods to evaluate for the presence of clonal rearrangements of the immunoglobulin heavy chain gene (IgH) using consensus V FRIII and J region primers, and the t(14;18) involving both the major breakpoint and the minor cluster regions of the bcl-2 gene. All (100%) cases of splenic, abdominal lymph node, and ileal marginal zone hyperplasia displayed strong BCL-2 reactivity in the marginal zone B cells. In all cases analyzed, IgH polymerase chain reaction demonstrated a polyclonal pattern, and bcl-2/JH DNA fusion sequences were not detected.

Our results indicate that BCL-2 is consistently expressed by reactive marginal zone B cells of the spleen, abdominal lymph nodes, and ileal lymphoid tissue and should not be used as a criterion for discriminating between benign and malignant marginal zone B-cell proliferations involving these sites.

CD20 or CD79a
If the interfollicular zone contains a large number of B cells, this is indicative of invasion of the interfollicular areas by follicular lymphoma (invasive features). In reactive follicular hyperplasia, the interfollicular zone only contains scattered B cells
Normal nor or few CD10+ lymphoid cells outside the germinal centers
If a significant number of CD10+ cells are found in the interfollicular zone, this feature is indicative of interfollicular invasion. NOTE that granulocytes are CD10 positive, but they can be easily distinguished from lymphoid cells
Germinal center cells stain aberrantly positive in ~50% of follicular lymphoma
A Useful Marker to Distinguish Florid Follicular Hyperplasia From Follicular Lymphoma by Flow Cytometry

Xinjian Chen, MD, PhD, Peter E. Jensen, MD, and Shiyong Li, MD, PhD

Am J Clin Pathol 2003;119:842-851 Abstract quote

HLA-DO expression is regulated during B-cell development and activation.

To determine whether the level of HLA-DO expression is a helpful marker in the differential diagnosis between florid follicular hyperplasia and follicular lymphoma, we analyzed single-cell suspensions from 29 lymph node specimens by 4-color flow cytometry. Of the 29 specimens, 7 were from patients with florid follicular hyperplasia, in which the clonality of the germinal center B cells (GCBCs) was indeterminate by flow cytometric immunophenotyping. The remaining 22 cases were follicular lymphomas; neoplastic cells in 2 of these cases lacked expression of surface immunoglobulin light chains.

The level of HLA-DO expression in the reactive CD10+ GCBCs in florid follicular hyperplasia was markedly down-regulated compared with the CD10– polytypic B cells of the same specimens. In contrast, the level of HLA-DO expression in the CD10+ neoplastic cells in all cases of follicular lymphomas was similar to that of the CD10– polytypic B cells, thus higher than the reactive CD10+ GCBCs. This difference was confirmed by immunohistochemical staining.

Our results suggest that HLA-DO is a useful marker to differentiate florid follicular hyperplasia from follicular lymphoma by flow cytometry, particularly when clonality of the CD10+ B cells is in question.
Ig Light chain restriction
See in germinal centers of follicular lymphomas
Reactive conditions have a network pattern and polytypic rather than discrete membrane staining
Lack of polarity in follicular lymphoma
Reactive follicles show polarity with a substantial proportion of the germinal center cells in the dark zone being stained up
Low Ki-67 index, if found, is supportive of a diagnosis of follicular lymphoma
CD10 and BCL-6 Expression in Paraffin Sections of Normal Lymphoid Tissue and B-Cell Lymphomas

Ahmet Dogan, M.D., Ph.D., MRCPath; Eniko Bagdi, M.D.; Philippa Munson, M.Sc.; Peter G. Isaacson, M.B., Ch.B., D.M., FRCPath, D.Sc.

From the Department of Histopathology, Royal Free and University College Medical School, London, U.K.

Am J Surg Pathol 2000;24:846-852 Abstract quote

In this study the authors explored the value of immunostaining for follicular center B-cell markers, BCL-6 and CD10, in paraffin sections as a tool for the differential diagnosis of B-cell lymphomas.

The cases studied comprised reactive lymphoid hyperplasia (RLH; n = 19), follicular lymphoma (FL; n = 50), low-grade mucosa-associated lymphoid tissue (MALT) lymphoma (n = 24), mantle cell lymphoma (n = 19), splenic marginal zone lymphoma (n = 13), diffuse large B-cell lymphoma (DLBCL; n = 54), Burkitt's lymphoma (BL; n = 20), nodular lymphocyte predominance Hodgkin's disease (NLPHD; n = 16), and classic Hodgkin's disease (CHD; n = 13).

In RLH, CD10 and BCL-6 were expressed almost exclusively by the follicular center cells. In contrast in FL, the expression of CD10 (39/50) and BCL-6 (34/36) was seen in both follicular and interfollicular neoplastic B cells. Marginal zone/MALT lymphomas and mantle cell lymphoma were always negative. In DLBCL the expression was variable for both CD10 (21/54) and BCL-6 (39/47), with some tumors, including cases of transformed follicular lymphoma (9/10), coexpressing CD10 and BCL-6, and others expressing only BCL-6, and a small group expressing neither marker, possibly reflecting the underlying primary pathogenetic events such as the rearrangement of BCL-2 or BCL-6 genes. BL was always both CD10 and BCL-6 positive. In NLPHD the L&H cells expressed BCL-6 (11/13) but not CD10, whereas in CHD BCL-6 expression was seen in half of the cases.

This study demonstrates that both CD10 and BCL-6 are reliable markers of follicular center B-cell differentiation. CD10 and BCL-6 immunostaining have an important role in differential diagnosis of FL from RLH and other low-grade B-cell lymphomas. The results also suggest that a CD10/BCL-6 expression pattern may be helpful in identifying main subsets of DLBCL. However, additional studies comparing genotype with immunophenotype are required.

The usefulness of immunohistochemistry in the diagnosis of follicular lymphoma in bone marrow biopsy specimens.

West RB, Warnke RA, Natkunam Y.

Department of Pathology, Stanford University Medical Center, CA 94305-5302, USA.

Am J Clin Pathol 2002 Apr;117(4):636-43 Abstract quote

We used a panel of paraffin antibodies to determine whether neoplastic and nonneoplastic lymphoid aggregates in the bone marrow can be distinguished reliably. Formalin-fixed, paraffin-embedded bone marrow core biopsy specimens with lymphoid aggregates were stained using primary antibodies directed against bcl-2, bcl-6, CD5, CD10, CD20, and CD23.

We studied 61 cases (26 follicular lymphoma and 35 benign or atypical aggregates). We found that no single stain is sufficient for identification of neoplastic lymphoid aggregates. However, this distinction was made possible by using a panel of antibodies.

Under the conditions we tested, the most useful antibodies were CD10, bcl-2, CD5, and CD20. Most benign or atypical aggregates do not express CD10 and CD23. In addition, nonneoplastic aggregates had a large population of T cells. bcl-2 was useful in an architectural context for distinguishing neoplastic aggregates. bcl-6 often was expressed in both neoplastic and nonneoplastic aggregates and, thus, poorly discriminated between these processes. We studied the expression of CD10 and bcl-6 in selected lymph nodes in some cases.


Expression of bcl-6 and CD10 Protein Is Associated With Longer Overall Survival and Time to Treatment Failure in Follicular Lymphoma

Nurija Bilalovic, MD, Anne Kirsti Blystad, MD, Rastko Golouh MD, PhD, Jahn M. Nesland, MD, PhD,etal.
Am J Clin Pathol 2004;121:34-42 Abstract quote

Follicular lymphomas (FLs) are a heterogeneous group of tumors, but prognostic factors are evaluated insufficiently in this common hematologic neoplasm.

While bcl-6 and CD10 are expressed characteristically in FLs, their significance for biologic behavior of FL has not been studied previously. Samples from 73 patients with FL and clinical follow-up from 7 to 231 months were evaluated by immunohistochemical analysis. Patients with high levels of bcl-6 expression had favorable overall survival (OS) ( P = .003), disease-specific survival (DSS) ( P = .033), and time to treatment failure ( P = .003) compared with patients with low levels of bcl-6 expression.

Multivariate analysis showed that the results for OS, DSS and time to treatment failure were independent of the international prognostic index. Patients with CD10+ FLs also had longer OS ( P = .001), DSS ( P = .007), and time to treatment failure ( P = .004), and grade 1 FL was associated with better OS ( P = .01) and a statistical trend for longer DSS ( P = .05) and time to treatment failure ( P = .05), but these results were not independent of bcl-6 expression or the international prognostic index in multivariate analysis.

Higher-grade transformation of follicle center lymphoma is associated with somatic mutation of the 5' noncoding regulatory region of the BCL-6 gene.

Lossos IS, Levy R.

Division of Oncology, Department of Medicine, Stanford University Medical Center, Stanford, CA 94305-5306, USA.

Blood 2000 Jul 15;96(2):635-9 Abstract quote

Follicle center lymphoma (FCL) is an indolent low-grade B-cell non-Hodgkin's lymphoma (NHL) that frequently transforms to aggressive diffuse large B-cell lymphoma (DLBCL). Histologic transformation of FCL is commonly associated with accumulation of secondary genetic alterations. The BCL-6 gene is altered by chromosomal rearrangements and mutations clustering in its 5' noncoding regulatory region in up to 70% of primary DLBCL, but in a significantly smaller subset of FCL. Previous studies have shown that both chromosomal rearrangements and mutations could deregulate BCL-6 expression.

To evaluate the association between progressive accumulation of BCL-6 regulatory region mutations and the histologic transformation of FCL, we analyzed by extensive cloning and sequencing paired biopsy specimens obtained at the time of FCL diagnosis and transformation (6 patients) or FCL relapse (3 patients). In an additional patient, biopsy specimens obtained at the time of diagnosis, FCL relapse, and subsequent transformation to DLBCL were evaluated. The presence of identical mutations in the paired diagnosis and posttransformation DLBCL specimens confirmed the common clonal origin of both the pretransformation and the posttransformation lymphomas. No new mutations in the 5' noncoding regulatory region of the BCL-6 gene were detected in any of the specimens evaluated at the time of FCL relapse. In contrast, 5 of the 7 transformed specimens contained new mutations not found in the paired original biopsy specimens obtained at the time of FCL diagnosis or relapse. The number of these new mutations ranged from 1 to 6 per specimen. Some of the new mutations tended to cluster in certain areas of the 5' noncoding regulatory region of the BCL-6 gene.

Our results show that transformation of FCL to DLBCL is associated with accumulation of new mutations in the 5' noncoding regulatory region of the BCL-6 gene, that by deregulation of the BCL-6 gene expression may play a role in lymphoma transformation.


Follicular lymphoma with a burkitt translocation--predictor of an aggressive clinical course: a case report and review of the literature.

Voorhees PM, Carder KA, Smith SV, Ayscue LH, Rao KW, Dunphy CH.

Department of Internal Medicine, Division of Hematology and Oncology, University of North Carolina, Chapel Hill, NC 27599-7525, USA.
Arch Pathol Lab Med. 2004 Feb;128(2):210-3. Abstract quote  

Follicular lymphoma is an indolent lymphoma characterized by the (14;18) translocation, which leads to aberrant expression of Bcl-2. Translocations involving 8q24 are most commonly associated with Burkitt lymphoma and result in c-Myc overexpression.

We report a case of follicular lymphoma of predominant small cleaved-cell type (grade 1) associated with both a t(14;18)(q32;q21) and a t(8;22)(q24;q11). The 8q24 translocation predicted an aggressive clinical course, as the lymphoma transformed into acute lymphoblastic leukemia within a year of initial diagnosis.

Routine cytogenetic analysis is recommended at initial diagnosis of follicular lymphoma to better identify abnormalities that may predict prognosis and influence therapy.
  Count large nucleolated cells
Use x10 eyepiece and x40 objective
At least 10 fields of neoplastic follicles are counted (not selected for those with most numerous large cells) and the mean count per HPF is obtained
Original study was based on 18 mm eyepiece: adjust figure accordingly if 20 mm or 22 mm eyepiece is used instead
CRITERION (number of large cells/hpf)
Predominately small cleaved cells
Mixed small and large cells

Predominately large cells

NOTE: If discrete areas of Grade 3 follicular lymphomas are found in Grade 1 or Grade 2 cases, this should be explicitly stated

Still some admixed small cleaved cells (centrocytes)
Exclusively large nucleolated cells
Presence of preserved reactive germinal centers in follicular lymphoma is a strong histopathologic indicator of limited disease stage.

Adam P, Katzenberger T, Eifert M, Ott MM, Rosenwald A, Muller-Hermelink HK, Ott G.

From the Pathologisches Institut, Universitat Wurzburg, Germany.

Am J Surg Pathol. 2005 Dec;29(12):1661-4. Abstract quote  

Follicular lymphoma (FL) typically presents as a systemic disease (stages III/IV).

We repeatedly observed in cases with conserved reactive follicle structures (so-called partial infiltration) an association with a limited clinical stage (I/II). In this study, we analyzed 53 lymph node biopsies of FL with conserved reactive follicle structures. In 44 cases (83%) of the patients with partial infiltration, a limited stage of disease (Ann Arbor stage I/II) was found, whereas only 9 of 53 cases (17%) suffered from a systemic disease. In those cases with at least one follicle totally spared by lymphoma, 95% of the patients (38 of 40 cases) presented with a limited stage (I/II) of disease, compared with only 20% (10 of 49 cases) in a control group with full-blown infiltration (P < 0.001).

Analyzing systematically all 321 FL cases sent to the Reference Center Wurzburg in the year 2001, reactive follicle remnants were detected in 34 of 321 (10.6%) cases with 26 of 34 (76%) tumors showing limited stage (I/II) disease, including all 18 cases with at least one totally spared follicle.

Our results therefore show a clear association between the occurrence of preserved reactive follicles in FL and limited disease stage.

Taken from Chan JKC. Practical Lymphoma Diagnosis: A Simplified Approach. Presented at the 111th Semi-Annual California Tumor Tissue Registry. December 2001.
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Rosai J. Ackerman's Surgical Pathology. Ninth Edition. Mosby 2004.
Sternberg S. Diagnostic Surgical Pathology. Fourth Edition. Lipincott Williams and Wilkins 2004.
Robbins Pathologic Basis of Disease. Seventh Edition. WB Saunders 2005.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 6th Edition. McGraw-Hill. 2003.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.

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