Clonal cytogenetic abnormalities are predictor in developing non-Hodgkin lymphomas?

    Clonal cytogenetic abnormalities are predictor in developing non-Hodgkin lymphomas? Ying Wang, Yi Xiao, Xiangyu Meng, Heng Zhang, Qinlu Li, Fankai Meng, Lifang Huang, Chunrui Li, Jianfeng Zhou PII: DOI: Reference:

S0014-4800(16)30266-0 doi: 10.1016/j.yexmp.2017.01.007 YEXMP 4000

To appear in:

Experimental and Molecular Pathology

Received date: Accepted date:

18 September 2016 9 January 2017

Please cite this article as: Wang, Ying, Xiao, Yi, Meng, Xiangyu, Zhang, Heng, Li, Qinlu, Meng, Fankai, Huang, Lifang, Li, Chunrui, Zhou, Jianfeng, Clonal cytogenetic abnormalities are predictor in developing non-Hodgkin lymphomas?, Experimental and Molecular Pathology (2017), doi: 10.1016/j.yexmp.2017.01.007

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ACCEPTED MANUSCRIPT Clonal cytogenetic abnormalities are predictor in developing non-Hodgkin lymphomas? Ying Wang, MD*, Yi Xiao, MD*, Xiangyu Meng, MD&, Heng Zhang, MD*, Qinlu Li,

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MD*, Fankai Meng, MD*, Lifang Huang, MD*, Chunrui Li, MD*, Jianfeng Zhou,

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MD*.

*Department of Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie-Fang Avenue, Wuhan 430030, Hubei,

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P. R. China; &

Center for Evidence-based and Translational Medicine, Zhongnan Hospital of

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Wuhan University, Wuhan 430030, Hubei, P. R. China;

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Abstract word count: 244 Total word count: 1968 Reference count: 24

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Running title: Clonal cytogenetic abnormalities of NHLs Abbreviations: PCR = polymerase chain reaction; DLBCL = diffuse large B-cell lymphoma; SMZL= splenic marginal zone lymphoma; WHO = World Health

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Organization; NHL = non-Hodgkin lymphoma; RLH = reactive lymphoid hyperplasia; CT= computed tomography

Correspondence: Chunrui Li, MD, Department of Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie-Fang Avenue, Wuhan 430030, Hubei, P. R. China. Tel: +86-27-63639810; Fax: +86-27-63639830; E-mail: [email protected]

ACCEPTED MANUSCRIPT Abstract： Pathological analysis is the cornerstone for diagnosing malignant lymphoma. Status of cytogenetic abnormalities is frequently left unexamined if no evidence of

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malignancy is found in pathological analysis. In this study, we presented 3 cases in which clonal cytogenetic abnormalities were detected but morphological alterations of

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the same tissue did not support malignant non Hodgkin lymphoma at the first lymph node biopsy. Case 1 is a 55-year-old female with lymphadenopathy neoplastic process confirmed by flow cytometry and polymerase chain reaction (PCR). Chromosome revealed

47,XX,t(3;22)(q27;q11),+del(9)(p12)[16]/46,XX[4].

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analysis

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pathological analysis of subsequent lymph node biopsy indicated diffuse large B-cell

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lymphoma (DLBCL). Case 2, a 74-year-old female, for whom the pathological analysis, molecular studies and flow ctyometric analysis of the first lymph node

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biopsy found no evidence of clonal cell. Cytogenetic analysis demonstrated a terminal

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deletion of chromosome 7 and 1, and the patient received a second lymph node biopsy and splenectomy. A pathological diagnosis of splenic marginal zone lymphoma

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(SMZL) was made. In case 3 who was a 66-year-old female with right cervical and axillary lymph node enlargement. Cytogenetic analysis showed clonal karyotypic abnormalities: 48，XX, t(14;18)(q32;q21) [13]/46, XY [7]. The diagnosis of follicular

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lymphoma was rendered by the second biopsy of axillary lymph node according to the analysis of morphology and immunohistochemistry. We propose that clonal cytogenetic abnormalities may be a high potential risk for developing non-Hodgkin lymphomas. Follow-up and rebiopsy must be performed in patients who are cytogenetically abnormal but morphologically benign.

Key Words: cytogenetics; lymph node; lymph node pathology; lymphoma

ACCEPTED MANUSCRIPT INTRODUCTION The

current

World

Health

Organization

(WHO)

classification

of

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hematolymphoid malignancies is based on a multidisciplinary approach that integrates

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morphologic, molecular, immunophenotypic and cytogenetic findings, among which

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the morphology is considered as the ‘gold standard’ in making a diagnosis of malignant NHL. Numerous recurrent and clonal cytogenetic abnormalities described in non-Hodgkin lymphoma (NHL) subsets provide the characteristic information for

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diagnosing specific entities, such as the t(14;18) which is highly associated with follicular lymphoma and t(11;14) with mantle cell lymphoma

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. However, does it

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make sense that there is clonal chromosome abnormality in reactive lymphoid hyperplasia？

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A few small series and single case reports have described clonal cytogenetic

diseases

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abnormalities in lymph nodes from healthy individuals or patients with non-neoplastic . Some studies showed that prior clonal cytogenetic abnormalities might

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be associated with subtle neoplasia preceding the presence of notable morphological evidence of neoplasm.11-13

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In this study, we performed cytogenetic analysis on 163 diagnostic lymph node and extra-nodal tissue biopsy specimens submitted for hematopathologic evaluation from 2012 to 2015, of which, with details three cases in which clonal cytogenetic abnormalities of lymph node were observed, but no evidence of malignancy was revealed in pathological analysis. We also summarized the clinical features of 16 reactive lymphoid hyperplasia (RLH) literature cases with clonal karyotypic abnormalities finally diagnosed as NHL. According to the results, the clonal chromosomal rearrangements in lymph node might be linked to subsequent development of malignancy.

CASE REPORTS Case 1. A 55-year-old female presented with enlargement of right axillary and

ACCEPTED MANUSCRIPT laterocervical lymph nodes and fever for one month. A right axillary LN biopsy showed reactive follicular hyperplasia. No evidence of abnormal clonal populations was detected in flow cytometric analysis of LN. Ig/TCR clonality test was negative

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according to the EuroClonality (BIOMED-2) guidelines and consensus reporting

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system.14

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Chromosome analysis at the first lymph node biopsy showed a complex karyotype with clonal abnormalities. Based on G-banding, the karyotype was interpreted as 47,XX,t(3;22)(q27;q11),+del(9)(p12)[16]/46,XX[4]（ISCN，2014）(Fig.

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1A). FISH studies with BCL6 showed BCL6 rearrangement (Fig. 1B & 1C). A repeated chromosome analysis of the second LN biopsy displayed the same clonal

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abnormalities.

PET-CT scan which was performed one week after the first lymph node biopsy

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revealed multiple enlarged lymph nodes, the location of which included bilateral

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axillary, portal, retroperitoneal, left internal iliac, bilateral external iliac and inguinal, with increased metabolism, resembling lymphoma. Malignant lymphoma cannot be

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excluded. A subsequent lymph node biopsy demonstrated that the lymph node was affected by diffuse infiltration of large lymphoid cells. The immunohistochemical analysis demonstrated typical characteristics of DLBCL that expression of CD20、

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CD45、CD79a and Ki-67 were positive and CD3 were negative.（Fig.2）

Case 2. A 74-year-old female presented with fatigue and intermittent fever for 2 months. After admitted to our hospital, the patient received physical examination by which generalized superficial lymph node and spleen enlargement was revealed. Lymphadenopathy in the axilla, mediastinum and hilum was displayed in chest computed tomography (CT) scan and B ultrasound also showed splenomegaly. Due to persistent lymphoadenomegaly, she underwent a first biopsy of a left submaxillary lymph node that was 2 cm in diameter. Infiltration by a large number of non-clonal lymphocytes amid salivary tissue was observed and there was no sufficient evidence for the diagnosis of malignant NHL. Molecular studies showed absence of clonal products. No obvious clonal cells with a typical phenotype could be identified in

ACCEPTED MANUSCRIPT lymph node specimens using flow cytometry. One month later, a second lymph node biopsy and splenectomy were performed, and this time a pathological diagnosis of splenic marginal zone lymphoma (SMZL) was made on the basis of morphological

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(Fig.3) and immune phenotype results (Fig.4), which was further confirmed by a

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clonal rearrangement of the immunoglobulin light chain κ gene identified by PCR

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analysis. (Fig.5)

Cytogenetic studies of the first lymph node biopsy demonstrated the terminal deletion

of chromosome 7 and 11. The karyotype was

interpreted as

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46,XY,del(7)(q32),del(11)(q32)[13]/46,XY[7] （ISCN，2014）(Fig.6A). FISH studies with probe of 7q- showed the long arm deletion of chromosome 7(Fig. 6B & 6C). A

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follow-up lymph node karyotype examination showed consistent results.

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Case 3. A previously healthy 66-year-old woman came to our hospital with a

night

sweats.

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2-month history of right cervical and axillary lymph node enlargement, fever, and Physical

examination

displayed

generalized

superficial

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lymphadenopathy and chest CT scan showed swollen mediastinal or hilar lymph nodes. Sections of the biopsy from the right cervical lymph node showed distorted architecture with follicular hyperplasia. It was unclear whether this process is

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neoplastic or reactive. The immunohistochemical analysis did not support the existence of malignant clonal lymphocytes. On the basis of morphology and immunohistochemistry, a diagnosis of "reactive follicular hyperplasia" was made. There were no positive findings in PCR arrangement and FCM analysis. The patient was continuously followed for six months after the treatment of anti-infection and the enlarged superficial lymph nodes did not shrink and clinical symptoms of the patient were progressive. A right subaxillary lymph node biopsy was performed afterwards; the diagnosis of “follicular lymphoma” was finally rendered according to the results of morphology and immunohistochemistry analysis (Fig.7) Cytogenetic analysis of both lymph node biopsies showed identical clonal karyotype abnormalities: 48，XX, t(14;18)(q32;q21)[13]/46, XY [7].（ISCN，2014） (Fig.8)

ACCEPTED MANUSCRIPT Written informed consent was obtained in accordance with the Declaration of Helsinki.

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Discussion

There are a few previously reported cases of bone marrow specimen showing

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that cytogenetic abnormalities often precede morphological evidence in a developing neoplasm.15-17 Clonal cytogenetic abnormality is a hallmark of malignancy, and can be traced in cases of reactive lymphoid hyperplasia evolving to malignant

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lymphoma.18 As we know, the development of neoplasia is currently considered as resulted from accumulation of multiple genetic mutations that give afflicted cells a

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proliferative advantage rather than only one genetic event which dominates the evolution toward lymphoma.12 So far, Sevilla DW et al.11 retrospectively analyzed a

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large series of RLH cases and found that BCL6 gene rearrangements which play a key

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role in the pathogenesis of lymphomas by leading to the activation of oncogene expression were the second largest cytogenetic abnormality seen in reactive lymph

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nodes in their study. The BCL6 gene rearrangements themselves appear insufficient for generating lymphomas and may represent the “first hit” providing mild proliferative advantage, requiring additional genetic or epigenetic alterations. Symons

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WA et al. 19 also provided a case report of a patient who had morphological evidence consistent with DLBCL six months after the discovery of a t (3; 14) with BCL6 gene rearrangement at the first lymph node biopsy. Similarly, our patient in case 1 who had karyotypic abnormality of a t(3;22) with BCL6 gene rearrangement at the first lymph node biopsy was also diagnosed as DLBCL two months later by pathological analysis. The karyotype of case 1 also showed a deletion of chromosome 9(9p-) in addition to BCL6 rearrangements, supporting aforementioned views. In our case 2, chromomsomal analysis demonstrated the terminal deletion of chromosome 7 (7q-), an abnormality reported in up to 40% of SMZL cases but in less than 10% of other B-cell lymphomas, 20 which forced clinical doctors to take a second lymph node biopsy from armpit. Finally, pathologist made a definite diagnosis of SMZL according to the morphology and immunohistochemical results of the second

ACCEPTED MANUSCRIPT biopsy. It is reasonable to speculate that clonal abnormality revealed in cytogenetic analysis not only facilitates a more accurate discovery of a subtle neoplasm, especially in the early stage of the disease, but also provides specific information for

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the classification of malignant NHL. Cytogenetic findings in this case provided important evidence of malignant NHL and complete remission was achieved after six

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cycles of R-CHOP chemotherapy.

Pathological analysis of involved lymph nodes or lymphoid tissue, recognized as a gold standard in the diagnosis of lymphoma, is a challenging field where

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considerable knowledge, experience and skills are required to make the correct diagnosis. Moreover, pathological diagnosis of lymphoma is easily affected by

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sampling issues, such as tissue section submitted being very small and the structure of lymph node being incomplete which would both result in few abnormal cells, or

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sampling artifact of reactive lymphoid tissue adjacent to concurrent lymphoma. The 21

demonstrated that clinically meaningful diagnostic

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report by Mastasar MJ et al.

revision occurs frequently in the second-opinion pathology review for a diagnosis of

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lymphoma and rate of major diagnostic revision even arrived at about 20%. Indeed, sometimes it is difficult for pathologists to give an accurate diagnosis only by external information (morphologic analysis and immunohistochemistry), especially when

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facing an ambiguous case. Due to the existence of t (14; 18), the typical cytogenetic alteration of follicular lymphoma, Case 3 was followed for six months and finally diagnosed as FL at the second lymph node biopsy. The result of chromosome analysis and FISH played a crucial assistant role in providing the evidence of malignant clonal cell and gave the strong support to pathologists to correct their diagnosis. What is worthy of our attention, to the same lymph node or extra-nodal lymphoid tissue, cytogenetic analysis which is a traditional examination method focuses on revealing the internal change, in contrast to pathological analysis which pays more attention to external information of the malignant disease. So combining both detection methods will be more beneficial for the diagnosis of malignant NHL. Review of the literature revealed a handful of reported cases with morphologically normal but cytogenetically abnormal results, which were finally

ACCEPTED MANUSCRIPT diagnosed as NHL at the second lymph node biopsy (Table Ⅰ). Although the clinical relevance of clonal abnormalities in morphologically normal lymph nodes remains controversial, there is evidence that cytogenetic abnormalities help in predicting the

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clinical outcome. Clinical follow-up of patients reported by Sevilla DW et al.11 showed only one in all cases developed NHL, compared to all 10 patients with clonal 13

to whom a diagnosis of

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genetic abnormalities in the series of Au WY et al.

malignant lymphoma were made with a second biopsy performed after 0.3-37.1 months from the first one. Sevilla DW et al.

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made a conclusion that the median

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duration of follow-up of their cases (5 years) was insufficient to determine if, and how many of, the RLH cases could progress to low grade lymphomas and longer follow-up

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evaluation was needed. Other literatures or case reports

19, 22-24

suggest that clonal

cytogenetic abnormalities often indicate underlying neoplasia, even in the absence of

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morphologic and immunocytochemical evidence and follow-up monitoring is needed.

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Although flow cytometry and PCR analysis of IG/TCR gene rearrangements are valuable tools for the diagnosis of NHL, clonal abnormal cells or positive IGH/TCR

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gene rearrangements results were not usually presented at the first biopsy of lymph node, as shown in our study and other reports. Therefore, cytogenetic evaluation of lymph node is necessary due to the aberrant clones are often small and difficult to be

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detected by flow cytometry or PCR analysis. In summary, we presented three cases with clonal cytogenetic abnormalities at the first lymph node biopsy which showed no evidence of malignancy on the pathological analysis. All three cases were finally diagnosed as NHL at the second biopsy of lymph node by pathological analysis. In our view, cases with clonal cytogenetic abnormality are at increased potential risk of developing malignant NHL. Therefore, to make a precise diagnosis of malignant lymphoma, we recommend the following process: first, for patients with clinical features suggesting malignant lymphoma, pathological and cytogenetic studies should be both performed. Second, rebiopsy must be performed in patients who are cytogenetically clonal abnormal but morphologically benign. Third, in order to answer the question that for cases where invasive progression is noted but repeated biopsy is not feasible, whether findings of

ACCEPTED MANUSCRIPT cytogenetic evaluation can be used as the only criterion based on which subsequent management strategy (lymphoma treatment or long term watchful waiting) is decided, more experience from studies with large sample size and clinical practice should be

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warranted.

ACCEPTED MANUSCRIPT Acknowledgements This work was supported by grants from the National Natural Science Foundation of

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China (No. 81570110, 81500082).

Conflicts of Interest and Source of Funding: The authors have disclosed that they

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have no significant relationships with, or financial interest in, any commercial

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companies pertaining to this article.

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ACCEPTED MANUSCRIPT Figure legends

Figure 1.cytogenetic analysis from the first lymph node biopsy sample in case 1. A.

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G-banded karyotype. The arrows point to the clonal abnormalities including the t (3; 22) and the del (9p). B. Interphase nuclei showing 1F/1O/1G signal pattern indicating

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BCL6 gene rearrangement by using BCL6 break-apart probe. Arrows mark the abnormal cells. C. Metaphase FISH showing rearrangement of BCL6 gene. Note the

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split orange and green signals (arrows).

Figure 2. Histopathology of lymph node from case 1. Initial diagnostic biopsy

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showing lymph node reactive hyperplasia. A. 10× magnification B. 40× magnification. Subsequent biopsy showing a diffuse infiltrate of large lymphoid cells with anaplastic

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features. C. 10× magnification D. 40× magnification. E. Lymphocytic infiltrate

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staining positive for CD20. F. Increased proliferation rate demonstrated by Ki-67 positive staining, focally up to 80%. G. Lymphocytic infiltrate staining negative for

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CD3. H. Lymphocytic infiltrate staining negative for CD21.

Figure 3. Histopathology of lymph node from case 2. A. Initial diagnostic biopsy

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showing lymph node reactive hyperplasia. (H&E stain) B. Subsequent biopsy showing spleen marginal zone lymphoma. (H&E stain) C. lymphocyte were positive for CD20. D. lymphocyte were positive for IgD. E. About 20% of the neoplastic cells display nuclear Ki67 staining.

Figure 4. Flow cytometric analysis of the second lymph node biopsy from case 2. Abnormal cells were positive for CD20, partly positive for CD23, CD25, kappa, Ki-67 and negative for CD5, CD10, CD103, CD11c, Bcl2 and lamda.

Fig.5 Ig κ rearrangement was monoclonal at 290 bp by capillary electrophores from case 2 at the second lymph node biopsy.

ACCEPTED MANUSCRIPT Figure 6. The cytogenetic analysis from the first lymph node biopsy sample in case 2. A. G-banded karyogram. The arrows point to clonal abnormalities including the del (7q) and del (11q). B. Interphase nuclei showing 1O/2G signal pattern indicating the

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partial deletion of long arm of chromsome 7 by using a probe for D7S486/CEP7. Arrows mark the abnormal cells. C. Metaphase FISH showing partical deletion of

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chromosome 7. Note the absence of the orange signal indicating the deletion of the D7S486 locus at 7q32 (arrow)

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Fig 7 Histopathology of lymph node from case 3. A. Initial diagnostic biopsy showing lymph node reactive hyperplasia. (H&E stain) B. Second biopsy showing follicular

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lymphoma. (H&E stain) C. Lymphocytic infiltrate staining positive for CD10. D. Lymphocytic infiltrate staining positive for BCL2. E. Lymphocytic infiltrate staining

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positive for CD20. F. Lymphocytic infiltrate staining positive for CD21. G. About 20%

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of the neoplastic cells display nuclear Ki67 staining.

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Fig.8 cytogenetic analysis from the first lymph node biopsy sample in case 3. A. G-banded karyotype. The arrows point to the clonal abnormalities including the t (14; 18). B. Interphase nuclei showing 2F/1O/1G signal pattern indicating IgH/BCL2

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fusion gene positive by using IgH/BCL2 dual color and dual fusion probe. Arrows mark the abnormal cells.

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Highlights 1. Three cases with morphologically normal but clonal cytogenetic abnormalities were finally diagnosed as NHL at the second lymph node biopsy. 2. Cytogenetic abnormalities may precede morphological alteration in developing NHL. 3. Follow-up and rebiopsy must be performed in patients who are cytogenetically abnormal but morphologically benign.