Analysis of clinical characteristics of 516 patients with non-Hodgkin's lymphoma in Shanghai area

Abstract

The aim was to determine the clinical and cytogenetic characteristics of non-Hodgkin's lymphoma (NHL) in Shanghai. A retrospective analysis was conducted in 516 patients with NHL. Patient clinical data, including age, sex, diagnosis, immunophenotypes, and karyotypes, were collected. The median age was 58 years. There was a male predominance in all NHL, except extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue. Patients with B cell NHL (1.5%) expressed CD3. T cell NHL patients (11.5%) expressed CD20. Epstein–Barr virus latent integral membrane protein 1, BCL6, CD10, Bcl-2, CD68, myeloperoxidase, CD99, CD30, CD15, and CD43 were present in various types of NHL. Complex karyotypes accounted for 92.3% of the 73.7% patients with abnormal karyotypes. Immunoglobin heavy chain gene translocation was present in 60.3% of B cell and 23.7% of T/NK cell neoplasms. Understanding the complex clinicopathological and molecular features of NHL may help with prognosis and serve as targets for treatments.

Keywords:

• Lymphoma
• Non-Hodgkin
• Epidemiologic studies
• Immunophenotyping
• Cytogenetic analysis
• In situ hybridization
• Fluorescence

Introduction

At present, non-Hodgkin's lymphoma (NHL) is one of the most common neoplasms, and accounts for approximately 3% of neoplasms worldwide. Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal zone lymphoma are the most common subtypes of NHL according to the Hematological Malignancy Research Network,¹ but prevalence of the various subtypes of NHL varies in different regions of the world. At the time of writing, marginal zone lymphoma accounted for about 4% of all subtypes of NHL, but was less common in Shanghai than in western countries.² The prevalence of FL was relatively lower in Asia compared to the USA and Europe,³ but similar to that found in Iraq where Burkitt's lymphoma (BL) was the second most common subtype of NHL.⁴

Prognosis of B cell NHL has greatly improved with the introduction of rituximab as part of standard treatments, resulting in an increased 5-year overall survival rate from 45 to 58%.⁵ However, many patients relapse and have become resistant to treatments. With T/NK cell neoplasms, the therapeutic effects of chemotherapy have not been satisfactory, with only a 5-year overall survival rate of 32%.⁶

The study of NHL has undergone several developmental stages beginning with the clinical features and histopathology to that of immunopathology and molecular genetics. Currently, NHL can be further categorized by different immunophenotypes and/or cytogenetic aberrations even within the same subtype. For example, DLBCL can be divided into germinal center B cell-like (GCB) or activated B cell-like (ABC) subtypes by gene expressions of CD10, BCL6, MUM1,GCET1, and FOXP1.⁷ The ABC subtype has a poor prognosis. In addition, the presence of chromosome 17 aneuploidy predicted an inferior prognosis in DLBCL,⁸ and chromosomal instability was correlated with an unfavorable outcome in DLBCL.⁹ In mature T/NK cell neoplasms, non-random genetic alterations are less common and diagnosis relies mainly on morphology and immunophenotypes. However, effective immunostain algorithms for classifying T/NK cell neoplasms are lacking at present. Further studies of these immunophenotypes and of cytogenetic aberrance may help to better understand the biological properties of NHL and thus provide further developments for new agents to cure the disease.

Cytogenetic analysis is often performed in China on bone marrow preparations. Positive results with this technique depend largely on bone marrow involvement of NHL and the abundance of living cells for analysis, but it may produce false negative results. A more precise technique is now available using fluorescence in situ hybridization (FISH), but the use of conventional cytogenetic analysis can be used as a tool for screening abnormalities of chromosomes. The Sino-US leukemia Cooperative Group of Shanghai previously reported the results of cytogenetic analysis of NHL cases and found that chromosome aberrations were involved in nearly all chromosomes. Abnormalities in chromosomes 1, 3, 6, and 14 were frequently found in DLBCL,¹⁰ but studies with larger sample sizes are required to confirm the results.

The aim of this study was to illustrate the clinicopathological and cytogenetic features of NHL in the Shanghai area. We conducted a retrospective analysis of 516 adult patients consecutively diagnosed with NHL according to the World Health Organization (WHO) classification criteria.¹¹ Immunophenotypes and cytogenetic aberrance are presented here. We also compared parameters between the different subtypes of NHL in order to investigate the existence of any associations between certain characteristics and special subtypes of NHL.

Materials and methods

The study recruited 516 patients consecutively diagnosed with NHL in our hospital from 2003 to 2007. Cases with acquired immunodeficiency were excluded. The research proposal was approved by the Internal Review Board at Fudan University, China. Patients’ medical records were reviewed by two physicians, and the clinical data including age, sex, diagnosis, immunophenotypes, and karyotypes were collected. Diagnosis was made by experienced hematopathologists based on morphology and immunophenotypes according to the WHO classification criteria.

Biopsy sections were stained with hematoxylin–eosin. All specimens were routinely processed for immunochemistry analysis. Expression of CD3, CD79a, CD20, BCL6, CD10, Bcl-2, Lmp1, CD68, MPO, CD99, CD30, CD15, and CD43 on tumor cells were studied. For conventional cytogenetic analysis, preparations of lymph nodes were cultured for 2 days and stimulated by lipopolysaccharide on the third day. At least 20 metaphases were analyzed for each sample. Descriptions of karyotypes were made according to the International System for Human Cytogenetic Nomenclature (1995).¹² Immunoglobulin heavy chain (IGH) translocation was analyzed by FISH. Breakpoints within the IGH locus were detected using Vysis LSI IGH probe (Vysis, Downers Grove, IL, USA). The sample preparations were performed following the manufacturer's recommendation. A clonal aberration was defined when more than 3% of interphase cells carry such translocation.

Descriptive statistical analysis was carried out using STATA 12 (StataCorp, College Station, TX, USA). Continuous variables were compared using Wilcoxon rank sum test. Chi-squared test was applied to assess the association between categorical variables. Statistical significance was defined as a P value less than 0.05.

Results

Distribution of subtypes of NHL

Ninety-three cases were diagnosed as T/NK cell neoplasms and 423 cases as B cell NHL. Among cases with B cell NHL, 70.9% (300/423) were aggressive including BL, DLBCL, mantle cell lymphoma (MCL) and follicular lymphoma grade III (FL-III). The most frequent subtype of B cell NHL was DLBCL (56.3%) followed by FL-III (8.0%). The number of cases with indolent B cell NHL including follicular lymphoma grade I and II(FL-I/II), lymphoplasmacytic lymphoma, nodal marginal zone lymphoma (MZL), splenic B cell marginal zone lymphoma (SMZL), extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), and small lymphocytic lymphoma (SLL) was 29.1% (123/423). SLL was the most frequent subtype of indolent B cell NHL (14.2%), followed by FL-I/II (11.3%). All T/NK cell neoplasms were aggressive in this study; these include peripheral T cell lymphoma, not otherwise specified (PTCL), angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma, and NK/T cell lymphoma, nasal type. The PTCL subtype was the most common (55.9%) (Table 1).

Table 1. Distribution of subtypes of NHL

Gender and age distribution of NHL

The median age for all NHL cases was 58 years (Table 2). The median age was 37 years for ALCL and 45 years for BL, both of which were much younger than the other subtypes of NHL. The overall male:female gender distribution was 1.67:1, with a male predisposition for all subtypes except for MALT lymphoma. There was a considerable male predisposition for SLL (4:1) and MZL (3:1). No significant difference in age was found between male and female (z = −1.35, P = 0.18).

Table 2. Median age and sex ratio of NHL

Immunohistochemistry analysis

Although most patients with B cell NHL expressed B cell markers CD20 and CD79a, there were 0.7% (3/405) of cases with B cell NHL that did not express CD20 and 8.8% (35/397) of CD79a. Loss of CD79a expression was more common in FL-III than in the early stage of FL, but there was no significant difference between them (χ² = 2.00, P = 0.16). T cell marker CD3 was occasionally expressed in DLBCL, SLL, and FL-I/II. BCL6, as an apoptosis inhibiting factor, was present in BL, DLBCL, FL, and PTCL. It was more common in FL-III than FL-I/II (χ² = 6.70, P = 0.01). Expression of CD10 was more frequent in FL-I/II than FL-III (χ² = 4.28, P = 0.04). Lmp1, which is caused by EB virus infection, was mainly present in the T cell lymphoma subtypes AITL and PTCL, but was only expressed in 6.1% (12/197) of DLBCL and 3.6% (1/28) of FL-III. Bcl-2 was less common in FL-III than FL-I/II, but the difference was not significant (χ² = 2.99, P = 0.08). CD68 was absent in most subtypes of NHL, with only a few observed cases in DLBCL, FL-I/II, MCL, PTCL, and SLL. MPO was present in 66.7% (2/3) of patients with ALCL, but its significance was undetermined due to the limited sample size. Because the meaning of CD99 in NHL was not clear, few patients were evaluated for the expression of it. This marker was present in a high proportion of patients with the subtypes ALCL, PTCL, and MCL. The proportion of patients with ALCL that expressed CD30 was 91.7% (11/12). CD30 was also expressed in a few patients with DLBCL and PTCL. CD15 was absent in most patients with only one case observed in each of ALCL and PTCL. CD43 was widely expressed in all NHL, except for BL (Table 3).

Table 3. Immunophenotypes of NHL

Cytogenetic analysis

Cytogenetic analysis of lymph node preparations was performed in all patients. Successful analysis was performed in 71.5% (369/516) of the cases, and 73.7% (272/369) of these had abnormal karyotypes. Complex karyotypes accounted for 92.3% (251/272) of these. Normal karyotypes were found in 26.3% (97/369) of patients. Chromosomal abnormalities were observed in 79.4% (235/296) of B cell NHL and 52.1% (38/73) of T/NK cell neoplasms. Abnormal karyotypes were more frequent in B cell NHL than T/NK cell neoplasms (χ² = 22.74, P = 0.000). In addition, chromosomal abnormalities occurred more frequently in aggressive B cell NHL than in indolent B cell NHL (χ² = 7.09, P = 0.008) (Table 4).

Table 4. Conventional cytogenetic analysis

Chromosome aberrations of the most prevalent NHL in this study are shown in Table 5. Loss of the Y chromosome and the gain of an X chromosome were the most frequent chromosomal abnormalities in DLBCL, FL, and PTCL. Only 16.8% (26/155) of patients with DLBCL had normal karyotypes. Trisomy 3 was common in DLBCL. Chromosomal abnormalities of chromosome 6 including deletions of 6q23, 6p21, 6q25, 6q13, 6q15, 6q24, 6q21, 6q12, and 6p12 were observed in 5.8% (9/155) of the cases. There were 12.5% (8/64) of the FL patients with normal karyotypes, and t(14;18)(q32;q21) was observed in 21.9% (14/64) of the FL. Other common chromosomal aberrations included t(3;14) and trisomy 7. Of the patients with PTCL, 41.5% (17/41) had normal karyotypes. The most frequent chromosomal abnormalities occurred in chromosomes 3, 5, and 7 and the sex chromosomes. In patients with SLL, 32.6% (15/46) had normal karyotypes. Abnormalities of chromosome 11 were found in 17.4% (8/46) of cases including del(11)(q14), del(11)(q22), del(11)(q21;q23). Trisomy 12 was common in SLL.

Table 5. Chromosomal abnormalities of NHL

FISH analysis

Conventional cytogenetic analysis indicated that 11.8% (61/516) of cases had chromosomal translocation involving 14q32, but IGH translocation using FISH analysis showed a greater number at 53.7% (277/516) of patients. IGH translocation in B cell NHL was found in 60.3% (255/423) of cases; in T/NK cell neoplasms, 23.7% (22/93) of cases carried such translocation. Significantly more IGH translocation was present in patients with B cell NHL than in T/NK cell neoplasms. Cases with IGH translocation in aggressive B cell NHL were present in 60.0% (180/300) patients. With indolent B cell NHL, 61.0% (75/123) of cases carried IGH translocation. The difference was statistically insignificant between these two groups (χ² = 0.035, P = 0.852) (Table 6).

Table 6. FISH analysis

Discussion

This study showed that DLBCL was the most common aggressive B cell NHL in the Shanghai area, followed by FL-III. SLL was the most common indolent B cell NHL, followed by low-grade FL. These results are consistent with a previous study which indicated that DLBCL and FL were the most common subtypes of B cell NHL, accounting for more than 70% of the total.¹ PTCL was the most common T/NK cell neoplasm with 55.9%, more common than that reported by the International PTCL Project in which the prevalence was 25.9%.¹³ However, our results were similar to the study conducted in Taiwan,¹⁴ but different from those reported in Southwest China and Pakistan, where nasal type NK/T cell lymphoma¹⁵ and ALCL predominated,¹⁶ respectively. MCL accounted for 4% of B cell NHL and 3.3% of all NHL in this study, consistent with the study conducted in USA.¹⁷ MZL accounted for 1.9% of B cell NHL, less common than reported in Chile, where MZL was 10.3%.¹⁸

The median age of NHL in this study was 58 years, much younger than that reported in western countries where patients had a median age of over 70 years.¹ For BL and ALCL, the median age was younger, which is similar to the results observed in India.¹⁹ There was a general male predisposition for all subtypes of NHL, except for MALT lymphoma. All patients with MALT lymphoma were female in this study, suggesting a more female predisposition in Shanghai than in southwest China.¹⁵ There was a significant male predominance in patients with SLL, with a ratio of male to female up to 4:1. This ratio is higher than that observed in western countries, where it is about 2:1 male to female.²⁰ Patients with MZL were predominately male with a ratio of male to female of up to 3:1, but a larger sample size is required to confirm this conclusion.

Our study found that T/NK cell lymphoma could express B cell antigen. A few patients with T/NK cell neoplasms expressed CD20 and/or CD79a. PTCL and AITL have also been reported to express CD20.^21,22 Recently, CD79a was reported to be expressed in mature T cell lymphoma,²³ though the mechanism has not been elucidated. CD3 was expressed in a few cases with DLBCL, FL and SLL in our study, as previously reported in B cell lymphoma.²⁴ This result was a pan T lineage marker expressed in early thymocytes and late stages of T cells, suggesting that neoplasms may derive from early lymphoid progenitor cells.

Lmp1 is frequently expressed in latent Epstein–Barr virus infections associated with B cell proliferation, which functions in activation of cellular signaling.²⁵ High Lmp1 expression has been reported to be associated with poor prognosis in nasopharyngeal carcinoma²⁶ and NHL.²⁷ In our study, Lmp1 was expressed in AITL, DLBCL, FL-III, and PTCL and it was absent in indolent NHL. Lmp1 might be associated with the aggressiveness of NHL.

CD68 is regarded as a special biomarker for monocyte–macrophage cell lineage, but a previous study has indicated that CD68 could be expressed in human glioma with poor prognosis.²⁸ In this study, expression of CD68 can be seen in DLBCL, in the early stage of FL, MCL and PTCL. Its clinical significance needs to be further elucidated.

MPO, as a biomarker for myeloid cell lineage, has been reported to be expressed in intravascular large cell lymphoma.²⁹ In our study, it was expressed in two-thirds of cases with ALCL, but a larger sample size is needed to make any definitive conclusions. CD99, as an adhesion molecule, was present in DLBCL, MCL, PTCL, and ALCL, as reported previously in ALCL,³⁰ DLBCL,³¹ and lymphoblastic lymphoma.³² Because this marker is seldom evaluated, its expression may be underestimated. A previous study has indicated that about 43% of primary cutaneous anaplastic large cell lymphoma expressed CD15.³³ We observed only one case of ALCL that expressed CD15, and none of the ALCL in this study involved skin. CD15 may play important role in skin involvement of NHL.

Loss of the Y chromosome is known to be related with aging in elderly males, but it also plays important roles in tumor development. Loss of the Y chromosome frequently occurs in solid tumors^34,35 and hematological malignancies.³⁶ In this study, loss of the Y chromosome occurred frequently in several types of NHL, but the exact function of such aberrance is not clear. The Y chromosome is vulnerable to DNA deletion caused by oxidative stress,³⁷ and oxidative stress has been reported to be linked with tumor initiation.³⁸ Recent research indicated that oxidative stress has a strong prognostic value in DLBCL.³⁹ As loss of the Y chromosome was universal in this study, there is the possibility that oxidative stress has important roles in the development of NHL. However, the Y chromosome was once reported to suppress tumor growth in vivo⁴⁰ and that the TSPY gene on the Y chromosome may function as a tumor suppressor.⁴¹ Whether other genes on the Y chromosome serve as tumor suppressors in NHL needs to be elucidated.

Numerical and structural aberrance of the X chromosome has been found in solid tumors⁴² and acute leukemia.⁴³ Kaneko reported a trisomy X in a female with gastric NHL.⁴⁴ In our study, additions of the X chromosome were frequent in DLBCL, FL, and PTCL. X inactivation is used to make gene expression balanced between the two sexes, which occurs by the X-inactive-specific transcript (Xist) gene on the X chromosome.⁴⁵ Xist can also trigger gene silencing in blood cells.⁴⁶ There is the possibility that the additional X chromosome supplied Xist may keep anti-oncogenes silenced in NHL. Conversely, the additional X chromosome could supply oncogenes, such as the gene for encoding the X-linked inhibitor of apoptosis protein, to help the tumor cells proliferate.⁴⁷ The exact function of the additional X chromosome in NHL needs to be further elucidated.

A previous study has shown that loss of 6p21 is frequent in DLBCL transformed from FL.⁴⁸ The survival rate of primary central nervous system lymphoma with the loss of 6q was lower than in those patients without the deletion.⁴⁹ Genes on chromosome 6 play important roles in the development of DLBCL. Various aberrations of chromosome 6 were detected in DLBCL in this study. Using a more precise technique such as FISH can be used to detect genes involved in the development of DLBCL on chromosome 6. Trysomy 3 was found in DLBCL and PTCL in this study, and it occurred more frequently in DLBCL. Chromosome 3q amplification has a profound effect in the development of lung cancer.⁵⁰ Several oncogenes on chromosome 3q have been identified including PIK3CA, which has been correlated with activity of phosphatidylinositol (PI) 3-kinase.⁵¹ The PI3K/AKT pathway was reported to be activated in DLBCL.⁵² Trisomy 3 may increase the activity of the PI3K/AKT pathway in DLBCL, but this increased activity has not been reported in PTCL. Other oncogenes on chromosome 3 require further investigations.

There were 21.9% cases with FL in this study that carried the translocation t(14;18). The prevalence was much lower than in western countries,^53,54 suggesting that the cell origin of FL in China may be different from that in western countries. The use of a more precise analysis technique should be considered in order to clarify whether the prevalence of t(14;18) was not underestimated.

The translocation t(3;14) has been reported to be carried by DLBCL, FL, and MALT lymphoma, and it was found to correlate with the expression of BCL6.^55,56 In this study, about 6% of FL carried t(3;14), and it was not present in DLBCL, but expression of BCL6 was more common than the t(3;14) in FL. It is possible that this difference may be due to the different techniques employed.

Trisomy 7 was observed in FL and PTCL in this study. It was previously reported to be the marker of progression from indolent FL to aggressive FL.⁵⁷ CDK6, encoding cyclin-D-dependent kinase 6 on chromosome 7, has been proposed as the cell cycle regulator and to be important for cell proliferation and malignant transformation.⁵⁸ CDK6 on an additional chromatid may supply proliferative advantages for NHL. Other oncogenes on chromosome 7 remain to be explored.

The loss of parts of chromosome 11q was frequent in SLL in this study, comprising 17.4% of SLL, which is similar to the results reported in Germany.⁵⁹ For chromosome 6, its loss accounted for 4.3% in this study. In another study conducted in China, it was reported that loss of 6q comprised 7.5% of SLL.⁶⁰ These results are similar to studies conducted in Italy⁶¹ and Germany.⁶²

Trisomy 12 accounted for 17.0% of SLL patients in this study, consistent with a previous study in which about 10–20% of cases carried trisomy 12.⁶³ Loss of 13q14, another common cytogenetic aberrance of SLL, was infrequent in our study, but loss of chromosome 13 constituted 4.3% of SLL. Trisomy 5 was detected in about 7% of PTCL in this study. Our study also found that other common cytogenetic aberrations such as deletions of chromosome 5q or t(5;9)⁶⁴ were infrequent.

FISH analysis has certain advantages in that it can be performed on paraffin-embedded sections, resulting in reductions of false-negative results. Our results indicate that FISH is more sensitive than conventional cytogenetic analysis in detecting chromosomal abnormalities. About a quarter of patients with T/NK cell neoplasms carried IGH translocation in this study. The occurrence of IGH translocation in T cell lymphoma is known as lineage infidelity. The occurrence of lineage infidelity suggests that neoplasms are derived from precursor lymphoid cells.⁶⁵ IGH translocation may serve as an indicator of prognosis in T/NK cell neoplasms.

Conclusion

NHL has complex clinicopathological and molecular features. We evaluated the general characteristics and some special features of a large cohort of patients with NHL in Shanghai. Differences in age and sex distributions, different environmental factors, and regional diversity may be attributed to the different biological characteristics of neoplasms. The wide diversity of neoplasms is characterized by a variety of immunophenotypes and karyotypes. Identifying some of these may help with prognosis and serve as targets for treatments.