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(American Journal of Pathology. 2000;157:1063-1070.)
© 2000 American Society for Investigative Pathology

Short Communications

Histological and Immunoglobulin VH Gene Analysis of Interfollicular Small Lymphocytic Lymphoma Provides Evidence for Two Types

David W. Bahler^*, Nadine S. Aguilera, Carolyn C. Chen^*, Susan L. Abbondanzo and Steven H. Swerdlow^*

From the Division of Hematopathology,^*
Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania and the Armed Forces Institute of Pathology,
Washington, District of Columbia

	Abstract

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Interfollicular small lymphocytic lymphoma (I-SLL) has not been well characterized and its relationship to small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL) is uncertain. Moreover, two different proliferation center growth patterns have been described with respect to reactive germinal centers. In this study, we evaluate the histological and immunophenotypic features of 13 cases of I-SLL and immunoglobulin heavy chain variable (VH) gene sequences from 10 cases. Immunophenotypic analyses indicate that cases showing either growth pattern have the same CD5-positive B cell phenotype typical of SLL or CLL. Sequence analysis revealed the use of VH, D, and J gene segments often found in CLL, although there may be more frequent use of J6. Similar to recent studies of CLL, there were approximately equal numbers of cases with either mutated or unmutated VH genes without evidence of ongoing mutation, consistent with I-SLL having either a naïve or memory B cell origin. Interestingly, the mutational status of the I-SLL VH genes seemed to correlate with the two different histological growth patterns. These studies support the proposal that I-SLL represents SLL/CLL and suggest the recently proposed two types of CLL originating from either memory or naïve B cells may have different histological patterns of growth in lymph nodes that show architectural preservation.

	Introduction

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Interfollicular small lymphocytic lymphoma (I-SLL) as described by Ellison et al³ is an indolent malignancy of small B lymphocytes present in the interfollicular areas of lymph nodes that histologically resembles SLL. Unlike typical SLL, however, the normal lymph node architecture is not completely effaced because reactive follicles and open sinuses are also present. Similar to SLL, I-SLL have proliferation centers that in some cases are present around the reactive follicles (perifollicular) and in others localized only between reactive follicles. Proliferation centers, which have also been termed pseudofollicles, are characteristic histological features of CLL or SLL and represent pale areas composed of cells that cytologically resemble prolymphocytes and paraimmunoblasts (intermediate-sized lymphocytes with central prominent nucleoli) and more mitotic figures.⁴ Whether I-SLL does indeed represent SLL/CLL or some other type(s) of mature B cell neoplasm, however, has not been well established. Studies to confirm I-SLL cells have the characteristic immunophenotype of SLL have been reported for only a few cases.⁵ Moreover, finding perifollicular proliferation centers is a histological pattern that suggests the possibility of a marginal zone B cell neoplasm that would not be expected to express CD5 or even an atypical mantle cell lymphoma.¹

The possibility that I-SLL may represent two distinct neoplasms, one with proliferation centers organized around reactive follicles and the other with proliferation centers found only between reactive follicles, is particularly interesting in light of recent studies of immunoglobulin VH genes that suggest CLL represents two distinct neoplasms.^6-9 Sequence analysis of VH genes can provide valuable information about the developmental stage of a lymphoma cell of origin, because somatic hypermutation is thought to occur as B cells pass through the germinal center.^10,11 Similar to their normal B cell counterparts, neoplasms of pregerminal center B cells seem to mostly express unmutated VH genes, neoplasms derived from postgerminal center B cells express mutated VH genes, whereas neoplasms of germinal center B cells typically show evidence of active hypermutation and have mutated VH genes.^11,12 Recent studies suggest that CLL can have either a pregerminal or postgerminal center cell of origin and that patients with these two different types of CLL have markedly different responses to treatment.^8,9 Whether these two proposed different types of CLL show histological differences in involved lymph nodes that still show significant architectural preservation is not known.

To further characterize I-SLL, 15 biopsies from 13 patients were analyzed in this study, and the VH genes were cloned and sequenced from 10 of the cases. These studies confirmed that I-SLL has an immunophenotype similar to SLL/CLL and are consistent with I-SLL representing SLL/CLL that shows preservation of lymph node architecture. They also suggest that the proposed two different types of pregerminal center or postgerminal center CLLs may have different histological patterns of growth.

	Materials and Methods

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Case Selection

The 13 cases studied were obtained from the hematopathology files of the University of Pittsburgh Medical Center and Armed Forces Institute of Pathology. Selection was based on having histological features that resembled SLL on review of hematoxylin and eosin-stained tissue sections where there was also partial preservation of normal lymph node architecture with at least some intact sinuses and reactive follicles.

Immunohistochemical Staining

The various antibodies used and suppliers are listed in Table 1 . Staining was done on 5-µm deparaffinized tissue sections using the avidin-biotin complex technique of Hsu et al.¹³ Various enhancement procedures were used including microwave treatment, protease treatment, or tyramide treatment (Dupont-New England Nuclear Research Products, Boston, MA). Hyperplastic human tonsil tissue sections were used as controls. The slides were also counterstained with hematoxylin before evaluation.

DNA was isolated from unstained 5-µm tissue sections obtained from paraffin-embedded material as described.¹⁴ All PCR reactions were performed in 50-µl volumes under standard conditions using Taq polymerase as described.¹⁵ Initially, B cell clones were identified by amplifying a small proportion of the DNA preparation for 37 cycles with a 5' consensus framework region 3 (FW3) and 3' JH1 primer. The resultant products, which correspond mostly to CDR3 sequences, were subsequently electrophoresed in 8% acrylamide gels, stained with ethidium bromide, so that prominent bands indicative of monoclonal B cell populations could be easily identified.^14,15 The methods used for obtaining more complete length VH gene PCR products from paraffin-extracted DNA specimens have been previously described.¹⁶ Briefly, DNA is first amplified for 37 cycles using a variety of 5' primers specific for the framework region one (FW1) sequences of the various VH families with the consensus 3' JH1 primer. To increase the sensitivity and obtain sufficient DNA for subsequent cloning, secondary amplification reactions were performed for 15 cycles using 0.5 µl of the initial reaction as template under the same conditions only substituting an internal JH primer (JH2 for JH1) or in two cases (case 1 and 13) an internal FW1 primer where it seemed the JH2 primer failed to work. Sequences for the various FW1 and JH primers used have been previously published.¹⁶ The more full-length VH gene PCR products were analyzed by electrophoresis in 1.5% agarose and ethidium bromide staining.

Cloning and Sequencing of PCR Products

PCR products corresponding to the mostly complete VH genes were isolated from 1.5% low-melt agarose gels and the DNA purified using Wizard DNA preps (Promega, Madison, WI). Approximately one-third of the purified DNA was cloned using the PCR-Script kit (Stratagene, La Jolla, CA). Plasmid DNA used for sequencing reactions was obtained from overnight cultures of randomly selected bacterial colonies using Wizard minipreps (Promega). Dideoxy sequencing in both directions was performed using Sequenase (Amersham, Cleveland, OH) with approximately one-third of the DNA following the manufacture’s plasmid protocol. Isolation and purification of the clonal FW3-JH1 PCR products from 8% acrylamide gels was performed as described.¹⁶ The small FW3-JH1 PCR products were directly sequenced from both ends as described.¹⁶

Analysis of VH Sequences

Sequences were analyzed using version 4.0 MacVector software (IBI, New Haven, CT) and the VBASE database.¹⁷ Germline D segments were assigned following the recommendations of Corbet et al¹⁸ with the exception that one difference was allowed within a stretch of 10 consecutive nucleotides. Expected numbers of replacement (R) and silent (S) mutations in the CDRs and FWRs were calculated by considering all possible mutations as described by Chang and Casali.¹⁹

	Results

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Clinical and Histological Findings

The 13 patients ranged in age from 39 to 78 years (median, 57 years) with six men and seven women (Table 2) . Although only limited clinical information was available, three of five patients studied were known to have widespread lymphadenopathy with at least three known sites of involvement above and below the diaphragm. The peripheral blood lymphocyte counts at diagnosis were reported to be normal in six of seven patients for whom this information was known and mildly elevated in one patient (12,800 per µl). Bone marrow involvement was documented in two of four patients examined. Six patients with follow-up information were alive with disease at 9 to 46 months.

View larger version (127K): [in this window] [in a new window]   Figure 1. Histological features of I-SLL. A: A low-power view of an I-SLL (case 4) that demonstrated extensive sinus preservation, reactive follicles, and only scattered pale proliferation centers. B: A low-power view of an I-SLL (case 9) with perifollicular proliferation centers around reactive follicles. C: A higher magnification of case 9 showing a reactive follicle (upper left) with its thin mantle zone surrounded by cells which closely resemble those of a proliferation center. D: A higher magnification of an I-SLL (case 13) that demonstrated reactive follicles partially colonized by proliferation center type I-SLL cells. The residual reactive follicular center cells are best seen in the lower left corner associated with tingible body macrophages.

The neoplastic cells were L26 (CD20)-positive in all cases. Relative to the reactive follicles which demonstrated the strongest staining, the proliferation centers had intermediate CD20 intensity levels and the small lymphocytes, which made up the bulk of neoplastic population, were the most weakly stained (Figure 2) . The lymphoma cells in 10 of 13 cases were clearly CD5+-positive, and in three cases the staining was indeterminate (Table 4) . The three cases lacking definite CD5 positivity could only be studied by paraffin-section immunohistochemistry which has a lower sensitivity compared to flow cytometry of 80%.²⁰ The lymphoma cells were positive for CD43 in all 12 cases tested. Positive staining for CD23 was seen in 11 of 12 cases tested, whereas one case showed equivocal CD23 positivity. Staining for cyclin D1 was clearly negative in 12 of 13 cases tested and equivocal in one case. Surface immunoglobulin was weak to absent in the one case where staining intensity was known by flow cytometry (case 12).

View larger version (142K): [in this window] [in a new window]   Figure 2. Representative immunohistochemical staining studies. Positive staining of I-SLL cells from case 1a with CD20 (right) and CD5 (left). Note the more intense CD20 staining of the follicular center cells relative to the more weakly staining I-SLL cells. Scattered CD5-positive T cells are present in the reactive follicle.

Rearranged VH genes were amplified, cloned, and sequenced from the 10 cases where DNA could be obtained. Clonally related repetitive VH sequences that had identical CDR3s were identified in all 10 cases (Figure 3 , Table 5 ). The same CDR3 sequences were also independently obtained by directly sequencing the clonal bands identified in each case using a standard heavy-chain PCR technique. This was done to confirm that the repetitively isolated VH sequences indeed represented B cell clones and were not repetition artifacts that can potentially arise with very poor quality DNA that is sometimes obtained from paraffin-embedded tissue specimens. All of the VH genes seemed to be functional because no stop codons or frame shifts were identified. In one case (case 2) two apparently functional VH genes were obtained which could represent the lack of allelic exclusion that has been described in occasional cases of CLL.²¹ Comparing these clonal I-SLL VH genes to the known germline gene segments revealed that six of 11 used VH gene segments from the VH3 family, four from the VH1 family, and one from the VH4 family. Although most of the identified germline VH gene segments were found in only one clone, the V1-69 and V3-9 gene segments were both used by two different VH genes. Germline D segments could be assigned to all but two of the 11 VH genes (Figure 3) . As summarized in Table 5 , D segments from the D3 family were used most often being found in six of 11 (55%) VH genes. The D3-3 gene segment was the most frequently used member of the D3 family being found in four of 11 (36%) of cases and always in the same hydrophobic reading frame. The J6 gene segment was used by eight of 11 VH genes which may account for the average CDR3 length of 18.7 being slightly longer than normal (Figure 3) . Inspection of the deduced CDR3 amino acid sequence did not reveal conserved motifs between the VD or DJ junctions (Figure 3) .

View larger version (30K): [in this window] [in a new window]   Figure 3. CDR3 sequences of I-SLL VH genes. The deduced amino acid sequences are shown along with the proposed D and J segment assignments. Nucleotide identity from the proposed D and J sequences is indicated with a dash. NA indicates that D segments could not be assigned with the described criteria. The complete VH gene nucleotide sequences have been deposited in the GenBank database (accession numbers AF299096-AF299106).

Four of the VH sequences (cases 1, 8, 11, and13) showed significant numbers of apparent point mutations from the proposed germline gene sequences that cannot easily be explained by germline polymorphisms or Taq polymerase errors. All of these four mutated VH gene segments had homology values of 97% or less to the proposed germline genes (Table 5) , whereas the seven VH gene segments considered to be unmutated all had homology values of 99% or more. Information concerning the types of mutations, either replacement or silent, and locations in the VH gene segments, either CDR or FWR, is summarized in Table 6 for the four mutated VH genes. Two of these VH gene segments seem to have more replacement than silent mutations in CDR1 and CDR2 than would be expected by chance alone (cases 11 and 13). This finding suggests that at least some of the replacement mutations in the CDRs may have been positively selected through antigen binding.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ellison et al³ proposed that I-SLL represented an indolent primarily lymph node-based neoplasm of interfollicular B cells that could display two histological growth patterns. All cases had proliferation centers but in approximately half of the described cases, the proliferation centers surrounded reactive germinal centers, whereas in the other cases, the proliferation centers were present only between reactive follicles. It was also suggested based on morphological analysis that I-SLL represented early or in situ SLL.

To further study whether I-SLL represents a single entity and how it may be related to SLL/CLL, 15 biopsies from 13 different cases were obtained from our tissue archives. The cases selected for study were histologically and clinically similar to those described by Ellison.³ They were approximately equally divided between those that had proliferation centers located around reactive follicles (perifollicular) and those where the proliferation centers were present only between reactive follicles. The majority of lymphoma cells in the proliferation centers, even when perifollicular, were cytologically indistinguishable from the prolymphocytes and paraimmunoblasts present in SLL/CLL. In two cases with perifollicular proliferation centers, the prolymphocyte-like cells were present within reactive follicles, so called "follicular colonization." Although this histological feature had not been previously described for I-SLL, it does not help elucidate the cellular origin because follicular colonization has been described in postgerminal center marginal zone B cell lymphomas²² as well as pregerminal center mantle cell lymphomas.²³

Immunophenotypic studies indicate that I-SLL with either pattern of growth is a B cell neoplasm that resembles SLL/CLL in being positive for CD5, CD23, and CD43.¹ Although mantle cell lymphomas also typically express CD5, the observed cyclin D1 negativity effectively rules this out from consideration and the positive staining with CD23 would also be very unusual for mantle cell lymphoma.¹ In addition, cases with perifollicular proliferation centers also differed cytologically from typical mantle cell lymphomas where prolymphocytes and paraimmunoblasts are not usually found.¹ The presence of proliferation centers surrounding reactive germinal centers with absent or thinned mantle zones gives these I-SLL lymphomas an appearance that can resemble marginal zone lymphomas.^1,5 However, I-SLL differs immunophenotypically from marginal zone lymphomas that are only rarely CD5- or CD23-positive.¹ In addition, the malignant monocytoid appearing perifollicular cells in marginal zone lymphomas also usually differ cytologically from the prolymphocytes and paraimmunoblasts present in I-SLL.

The use of specific VH gene segments that we observed further supports I-SLL being closely related to SLL/CLL. For example, seven of the nine different I-SLL VH gene segments we identified have been reported to be frequently used by CLL being found in 5% or more of cases.^6,8 Two I-SLL cases used the V1-69 gene segment that seems to be overrepresented in CLL relative to normal CD5-positive B cells being found in |mf10 to 20% of cases.^6,8,24 The V4-34 gene segment, which also seems to be overrepresented in CLL relative to normal CD5-positive B cells^6,8 was identified in one I-SLL case. The frequent use of D3-3 (formally called DXP4) which was present in four of 11 VH genes (36%) is similar to what has been described for CLL^6,8 and may be related to the high frequency this D segment is used by normal CD5-positive B cells.²⁵ Additional I-SLL cases will need to be examined to determine whether the use of J6 which was identified in eight of 11 genes (76%) is significantly different from that observed in CLL or normal CD5-positive B cells.

Similar to what has been described for CLL, the VH genes used by I-SLL are either mutated (homology 98% or less from germline) or nonmutated (homology 99% or greater) and do not show evidence of ongoing mutation.^6,8 Because VH gene hypermutation is thought to occur as B cells pass through the germinal center, these findings are consistent with I-SLL, like CLL/SLL, representing two malignancies, one derived from naïve unmutated pregerminal center B cells and one of mutated postgerminal center memory B cells.^8,9 It may be significant that two of the four mutated I-SLL VH genes identified in this study use the V4-34 and V3-23 gene segments which seem to be almost always mutated when found in CLL.^6,8 In addition, all seven of the nonmutated I-SLL VH genes used the J6 joining segment, which may be related to J6 being overrepresented by |mf10-fold in nonmutated relative to mutated productive VH rearrangements in normal CD5-positive B cells.²⁶ These findings further support the possibility that I-SLL with mutated and unmutated VH genes represents two distinct malignancies and also suggest they may recognize different antigens.

An especially interesting finding of this study is that the VH gene mutation status seems to correlate with the two different patterns of proliferation center growth seen in I-SLL. Specifically, three of the five sequenced I-SLL with perifollicular proliferation centers had highly mutated VH genes, whereas five of six VH genes from I-SLL with proliferation centers located only between reactive follicles were unmutated. Although the correlation was not perfect, the mutated VH gene from the single case where perifollicular proliferation centers were not identified (case 1) had approximately half as many mutations as the three other mutated cases suggesting it may be different from the others. Also, reexamination of the histology of this case after unmasking the VH gene mutation status revealed several follicles that may have been associated with subtle perifollicular proliferation centers. Conversely, the VH gene in one of the two cases with perifollicular proliferation centers that was classified as nonmutated (case 10) had three mutations and was most homologous to a germline gene termed HHG4.²⁷ However, HHG4, which is thought to be a variant of V3-21 differing at three nucleotide positions, has yet to be identified in a genomic clone.¹⁷ It is possible, therefore, that the VH gene from case 10 may have additional mutations where V3-21 and HHG4 differ which would place it in the mutated category (<99% homologous). Although most memory B cells have mutated VH genes, it is also possible that the two cases with perifollicular proliferations centers without mutated VH genes display other markers of memory B cells such as CD148.²⁸

Because most lymph node biopsies of CLL/SLL show complete architectural effacement, our data are consistent with I-SLL representing early CLL/SLL as initially suggested by Ellison.³ Indeed, one the two follow-up biopsies analyzed in this study showed greater effacement and less of an interfollicular growth pattern than the initial biopsy obtained several months earlier in agreement with this sequence of events (case 1). After complete architectural effacement, it is unlikely that cases with perifollicular proliferation centers will be histologically distinguishable from those with only scattered proliferation centers although they may appear more vaguely nodular. Therefore, only lymph node biopsies where there is less architectural effacement may be predictive of the VH mutational status.

In summary, the immunophenotypic and VH gene findings from this study support I-SLL being closely related, if not identical, to CLL/SLL. It is still possible, however, there are some biological differences between I-SLL and typical SLL/CLL that remain to be identified. From a diagnostic standpoint, I-SLL is important to recognize because it can be confused histologically with reactive follicular hyperplasia, marginal zone lymphoma, or occasionally follicular lymphoma. The clinical features of our patients and those described by Ellison³ support I-SLL being primarily a nodal-based disease, although bone marrow involvement may be a frequent finding. In addition, the frequency of subtle peripheral blood involvement in I-SLL is not settled because immunophenotypic or genotypic studies would be required to document involvement in the absence of a lymphocytosis, although it seems peripheral blood involvement does sometimes occur.⁵ Our studies also suggest that I-SLL represents two distinct neoplasms, one derived from naive pregerminal center B cells and one from postgerminal center memory B cells, with the later being more often associated with the presence of perifollicular proliferation centers. The recent studies of CLL relating VH mutation status with survival^8,9 suggest that I-SLL cases with perifollicular proliferation centers may have a better prognosis.

	Acknowledgements

 
We thank John Miklos for technical assistance.

	Footnotes

 
Address reprint requests to David W. Bahler, M.D., Ph.D., Department of Pathology, University of Utah School of Medicine, Room 5C124, Salt Lake City, UT 84132. E-mail: bahlerdw{at}aruplab.com

Supported by the Pathology Education and Research Foundation (University of Pittsburgh) and a Translational Research Grant from the Leukemia and Lymphoma Society (D. W. B.).

Accepted for publication July 1, 2000.

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