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Keywords:

  • diffuse large B-cell lymphoma;
  • hepatocyte growth factor;
  • c-MET;
  • prognosis

Summary

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References

The expression and prognostic significance of hepatocyte growth factor (HGF) and its receptor c-MET (MET proto-oncogene) was analysed in 96 cases of diffuse large B-cell lymphoma (DLBCL). Tissue sections were immunohistochemically stained for HGF and c-Met. The prognosis of HGF-positive and c-Met-positive cases was significantly worse than negative cases (HGF: P = 0·0036; c-Met: P = 0·0002). In addition, in the low-risk international prognostic index group, HGF-negative and c-Met-negative cases had a significantly better prognosis than positive cases (HGF: P = 0·0009; c-Met: P < 0·0001). Our results suggest that HGF/c-MET is a useful clinical marker of prognosis for patients with DLBCL.

Hepatocyte growth factor (HGF) is a multifunctional factor of mesenchymal origin, which acts as a mitogen, motogen and morphogen, depending on the cell target and cellular context (Matsumoto & Nakamura, 1996). HGF can also modulate haematopoiesis (Kimiecik et al, 1992). The receptor for HGF is the product of c-Met, which has the characteristics of a tyrosine kinase receptor (Bottaro et al, 1991) and the HGF/c-Met receptor system is important for the multifunctions of HGF.

Several studies have shown that c-Met is overexpressed in aggressive cancers, such as prostate, thyroid and gastric carcinomas and malignant lymphoma (Di Renzo et al, 1995; Humphrey et al, 1995; Yonemura et al, 1996). Furthermore, HGF/c-Met have been reported to be associated with tumour invasion (Weimar et al, 1999). High serum HGF levels occur in patients with malignant lymphoma.

The diffuse large B-cell lymphoma (DLBCL) constitutes approximately 50% of all B-cell lymphomas and survival can be predicted by clinical parameters, as recently established by the International Non-Hodgkin's lymphoma Prognostic Factors Project. However, there is no reliable biological marker for the prediction of prognosis for patients with DLBCL. The aim of the present study was to examine the expression of HGF and c-MET in DLBCL and determine the relationship between these expressions and the prognosis of 96 patients with DLBCL.

Tissue specimens

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References

All 96 tumour tissues specimens were obtained by biopsy and/or surgical resection from patients with DLBCL and had been filed in the Department of Pathology, Fukuoka University, from 1980 to 2000. Some of the cases in the present study were reported previously (Ohshima et al, 2001). The specimens were fixed in formalin buffer and embedded in paraffin. The cases were retrospectively diagnosed with DLBCL according to the World Health Organization (WHO) classification system. All clinical and laboratory data, along with the follow-up data, were obtained from the hospital medical records and patients’ charts. All relevant data were available, including individual risk factors of the international prognostic index (IPI), such as age, clinical stage, performance states, lactate dehydrogenase level and number of extranodal sites (Shipp et al, 1993). The IPI was scored as 0, 1, 2, 3, 4 or 5. Cases were classified into two groups based on the IPI: the low-risk group (IPI = 0, 1, 2) and the high risk group (IPI = 3, 4, 5).

Immunostaining

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References

Sections from the paraffin-embedded tissue blocks were immunohistochemically stained for HGF (IBL, Gunma, Japan), and its receptor, c-MET (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The percentages of positive cells were averaged to yield an immunohistological score of 0–100%. The following categories were defined: negative (positively stained tumour cells ≤20%; HGF, Fig 1A and cMet, Fig 1B) and positive (positively stained tumour cells >20%; HGF, Fig 1C and c-Met, Fig 1D). The staining pattern was classified as either cytoplasmic or nuclear. At least 1000 cells were counted in the representative area to determine the positivity.

image

Figure 1. Representative results of immunohistochemical staining. Lymphoma cells are negative for HGF (A), negative for c-Met (B), positive for HGF (C) and positive for c-Met (D). Both c-Met and HGF are expressed mainly in the cytoplasm. Original magnification ×400. Survival rates according to HGF (E), c-MET (F), HGF in low-risk IPI group (G), c-MET in low-risk IPI group (H), HGF in high-risk IPI group (I) and c-MET in high-risk IPI group (J).

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Statistical analysis

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References

Survival curves were calculated according to the Kaplan and Meier method (SAS System, SAS Institute Inc., Cary, NC, USA). Survival analysis was performed using the generalized Wilcoxon test. Differences were considered significant if the P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References

The age of the 96 patients ranged from 16 to 87 years (median, 35 years). They comprised 45 males and 51 females (male:female = 1:1·1). All patients received CHOP therapy (cyclophosphamide, hydroxydaunomycin, oncovin, prednisone) or CHOP-modified chemotherapy. Of the 96 cases, 18 were HGF-positive and 78 were HGF-negative. Furthermore, 66 cases were c-Met-positive and 30 were c-Met-negative.

The prognosis of patients with HGF/c-MET-positive DLBCL was significantly poorer than those with HGF/c-MET-negative DLBCL (HGF, P = 0·0036 and c-Met, P = 0·0002; Fig 1E and F).

Thirty-seven of 96 cases (38·5%) presented with a high-risk IPI, and 59 cases (61·4%) exhibited a low-risk IPI. HGF expression was detected in nine of 59 cases (15·2%) of the low-risk group, and in nine of the 37 cases (24·3%) of the high-risk group. c-Met expression was detected in 16 of 59 cases (27%) of the low-risk group, and in 14 of 37 cases (37·8%) of the high-risk group. In the low-risk group, cases that were negative for HGF expression had a significantly better prognosis than HGF-positive cases (P = 0·0009; Fig 1G). In addition, c-Met-negative cases also had a significantly better prognosis than those positive for c-Met (P < 0·0001; Fig 1H). In the high-risk group, neither c-Met (P = 0·3404; Fig 1J) nor HGF (P = 0·5994; Fig 1I) expression significantly influenced the survival rate.

Discussion

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References

HGF causes the spread of epithelial cells, a property that led to the use of the original name ‘scatter factor’ (SF), a term still used synonymously with HGF. HGF receptor was identified as the gene product of c-Met (Bottaro et al, 1991). HGF and c-Met are coexpressed in many epithelial neoplasms. Umeki et al (1999) analysed the clinical significance of the c-Met oncogene in colorectal cancer. They found that the expression of this oncogene did not influence the clinical stage, histopathological differentiation, tumour markers or overall survival. In contrast, other studies indicated that c-MET expression was associated with poor clinical outcome. Similar results have been reported on the association of HGF and epithelial cancer. In the present study, HGF/c-Met expression was associated with poor survival of patients with DLBCL. Weimar et al (1997) noted that c-Met was expressed on immature, activated and certain malignant B cells. Otherwise HGF/SF increased the adhesion of c-Met positive B-cell lymphoma cells to fibronectin and collagen, mediated via β1-integrins α4β1 and α5β1, and furthermore, promoted migration and invasion. These findings might indicate why HGF/c-Met positive lymphomas have a poorer prognosis.

Our results also suggested HGF/c-MET as a potentially useful marker of clinical outcome. However, Hsiao et al (2003) analysed serum levels of HGF in malignant lymphoma and reported the presence of high HGF serum levels in cases of advanced Ann Arbor stage with extranodal sites of lymphomatous involvement, but HGF level did not influence the survival rate. Further studies are warranted to clarify the relationship between serum HGF and immunohistochemical staining for HGF.

In conclusion, HGF/c-Met protein overexpression is an important parameter for survival in patients with DLBCL, and the expression of this factor is independent of IPI.

References

  1. Top of page
  2. Summary
  3. Material and methods
  4. Tissue specimens
  5. Immunostaining
  6. Statistical analysis
  7. Results
  8. Discussion
  9. References
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  • Weimar, I.S., Weijer, K., van den Berk, P.C.M., Muller, E.J., Miranda, N., Bakker, A.Q., Heemskerk, M.H.M., Hekman, A., de Gast, G.C. & Gerritsen, W.R. (1999) HGF/SF and its receptor c-MET play a minor role in the dissemination of human B-lymphoma cells in SCID mice. British Journal of Cancer, 81, 4353.
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