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Molecular Oncology, Markers, Clinical Correlates |
Departments of Haematology [P. S., T. R., E. K., M. B-M.], Pathology [A. P., E. C., O. Z.], and Chemotherapy [H. N.], Medical University of Lodz, 93-513 Lodz, Poland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the last several years there has been a marked tendency to search for new, more specific and sensitive prognostic factors in HD. Among a number of laboratory parameters considered valuable in the prediction of outcomes in neoplastic patients, the alterations of some oncogenes involved in the pathogenesis of malignancies have become a particularly interesting problem. In solid tumors, as well as in non-Hodgkin’s lymphomas, the prognostic value of expression of PCNA and genes such p53 and bcl-2 on neoplastic cells has already been suggested (5) . There are only few, rather inconsistent reports concerning this problem in HD.
On the basis of a retrospective study of 327 patients with HD, we have analyzed the prognostic significance of several factors with regard to response to treatment and survival time. Apart from a number of well known and traditionally used clinical and laboratory parameters, we have also examined the pretreatment expression of some oncoproteins on Reed-Sternberg cells and their mononuclear variants, Hodgkin’s cells (R-S/H cells), as a potentially important prognostic factor in HD.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 2
. In all patients, a uniform scheme
of chemotherapy was applied: between 1970 and 1981, COPP or MOPP
regimens were used, and since 1982, alternating MOPP and ABVD
regimens were used. After that, appropriate radiotherapy was usually
performed (Table 3)
. The data discussed
below were analyzed.
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Clinical parameters were evaluated on the basis of individual history
of the disease in combination with physical examination, X-ray results,
ultrasonography, CT methods, and, if necessary, exploratory laparotomy
(Table 4)
. Clinical staging was performed
in compliance with the Ann Arbor Conference principles.
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5 cm (6)
.
Morphological and Biochemical Parameters.
These parameters were histological type; ESR; number of leukocytes,
erythrocytes, and platelets; absolute number of lymphocytes, monocytes,
and eosinocytes; serum LDH activity; alkaline phosphatase activity;
serum iron concentration; level of albumin; serum fractions of
globulins (
, ß-1, ß-2, and 
-globulins); and renal and
hepatic sufficiency tests. All morphological and biochemical parameters
were estimated using standard laboratory methods.
Immunohistochemical Parameters.
These parameters included the expression of PCNA, as well as the
protein products of the p53 and bcl-2 genes, on
R-S/H cells. This retrospective examination could be performed in 194
patients on suitably retained, paraffin-embedded lymph nodes specimens
(stored routinely in our center from the 1970s). Immunohistochemical
examinations using monoclonal antibodies (DAKO A/S, Glostrup,
Denmark) with PCNA and the protein products of the p53 and
bcl-2 genes were performed in the lymph nodes obtained from
nontreated HD patients. In all cases, the index of positively stained
R-S/H cells was determined. The color reaction for standard
immunohistochemical examination in the avidin-biotin complex system was
obtained by 3,3-diaminobenzidine.
Morphological and biochemical factors analyzed in this study were considered according to commonly established cut-off points, determined by normal laboratory ranges. However, to take into account some biochemical parameters and immunohistochemical factors, as well as to estimate the treatment properly, the cutoff points were established according to statistical principles. Namely, on the basis of histograms (not shown) and frequency distribution tables for those factors examined, the middle value of the class comprising the median number of cases was chosen as a cutoff value.
In case of PCNA, p53, and BCL-2 expression, on the basis of frequency distribution for each antigen, the following cutoff values were chosen: for PCNA, 40%; for p53, 20%; and for BCL-2, 10%.
All of those parameters were analyzed for the OS and DFS and were correlated with response to the first-line therapy. DFS was defined as the time interval between the end date of the first-line treatment, if CR was obtained, and the date of eventual relapse.
Assessment of Response to Treatment.
Two categories of response to the first-line therapy were noted: CR and
resistance to first-line treatment, i.e., 
PR, which
included partial remission, no change, or progressive disease.
According to standard criteria, CR was defined as a disappearance of all evidence of the disease without development of new lesions. Partial remission was defined as a greater than 50% decrease of all of the measurable pathological lesions, also without a development of new lesions. No change was defined as a decrease in pathological lesions less than 50% or an increase less than 25%. Progressive disease was defined as increase in the size of measurable lesion by at least 25% or appearance of any new lesion.
Follow-up Evaluation.
The whole panel of follow-up procedures and tests for HD
patients used routinely in our center comprised physical examination,
complete blood count and sedimentation ratio, blood chemistry, chest
X-ray (or CT scan if necessary), and abdomen ultrasonography (or CT
scan if necessary). Frequency of those examinations was as follows:
every 3 months for the first 2 years after completing the whole
treatment required and achieving CR; next, every 6 months for 3–5
years after the end of successful treatment; and finally, once a year
after the 5th year after the treatment. Despite of the panel of
follow-up tests, we have also controlled the CR patients with only
physical and blood examination (if no clinical symptoms of relapse are
present) during every month’s visits (1st and 2nd years after
treatment) and then every 2–3 months up to 5 years, and every 6 months
after the end of treatment.
DI.
Because the results of therapy may be significantly affected by the
real doses of cytostatics given, the DI was assessed for all of the
patients in this study. DI was calculated on the basis of the model of
Hryniuk and Bush (7)
and was expressed as the ratio of
actually received average dose to the planned average dose over the
same time frame as prescribed in the original protocols. The received
actual DI was calculated on a per-patient/per-cycle basis and averaged
across the patients who received the same regimen of chemotherapy. The
total number of mg/m2 was determined for each
drug given throughout cycle of the treatment, for each patient.
Cumulative dose of drug was calculated as its summary dose in all
cycles applied to patient. Then, the total number of days between the
first day of chemotherapy and one cycle time after the day of last
treatment was divided by 28 (recommended time frame of one cycle) to
give the divisor to determine the actually received average dose for
each drug/for cycle.
The programmed cumulative dose of cytostatics was calculated in the same way. This differed in particular patients, depending on stage of the disease. The following optimal intensity of treatment (i.e., number of cycles and/or summary dose of radiotherapy planned) was stated for statistical calculations: for early stages (IA–IIA), radiotherapy, with a total dose 40 Gy (30 Gy for extended fields and 10 Gy for involved regions); for intermediate stages (IIB–IIIA), four cycles of chemotherapy and complementary radiotherapy (40 Gy); and for advanced stages (IIIB–IV), eight cycles of chemotherapy, with optional local radiotherapy for bulky sites (8) . However, in the analyzed group were six patients in stage I B, with either bulky mediastinal tumor mass or infradiaphragmatic disease, who received 2–3 cycles chemotherapy as a complementation to mantle radio-therapy.
Statistics.
Statistical analysis was carried out on the basis of logistic forward
stepwise multivariate regression test, as well as proportional hazard
Cox regression model and Kaplan-Meier survival analysis, tested by the
log-rank test. For the analysis of differences between means/medians,
the following nonparametric tests were used when necessary:
Mann-Whitney U test, Kruskal-Wallis ANOVA, and median test.
Results were considered significant if P was less than 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). CR was achieved in 257 patients (78.6%) after the first-line therapy. Eighty-seven of these patients (37.0%) subsequently relapsed: 40 of the relapses occurred during the first 12 months from the end of the treatment, whereas 47 relapsed after 1 year from CR. Of those 87 relapsed patients, 49 achieved a second long-lasting CR after salvage therapy (median DFS, 6.0 years), 16 entered CR but subsequently again relapsed (median DFS, 3.0 years), and remaining 22 either did not respond or only partially responded to the treatment (median DFS, 1.5 years).
Seventy (21.4%) patients did not achieve CR after the first-line
treatment (PR, i.e., patients with only partial remission,
no response, or even progression of the disease during therapy); in 36
of them (52.1%) a CR after second-line therapy was obtained.
The mean time of survival in the whole examined group of HD
patients was as follows: for OS, 8.5 years; for DFS, 7.0 years (Fig. 1)
. The longest time of observation was
22.5 years. At the time of submitting the manuscript (in July
1997), 244 patients were alive and free of disease, 58 died or
were lost from observation, and 25 had the symptoms of an active
disease and were receiving treatment. In patients who achieved
continuous CR after the first-line treatment (CR group), the time of
survival, both OS and DFS, was significantly longer than in PR
(P < 0.001).
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and 2)
, was verified by multivariate
regression tests. Only 6 of 32 factors analyzed showed their
independent statistical significance. Table 5
shows the data from regression
analysis. Pretreatment factors adversely correlating with OS were as
follows: the presence of systemic symptoms (P = 0.002),
poor clinical status according to Karnofsky’s scale (score < 70;
P = 0.003), age at diagnosis of >45 years
(P = 0.005), advanced (III/IV) clinical stage
(P = 0.007), multisite nodal localization of disease
(more than three sites; P = 0.01), and huge primary
tumor mass (bulky disease; P = 0.03).
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The following parameters showed their important, statistically
significant value for the prediction of poor response to the first-line
treatment (PR patients): bulky disease (P = 0.02),
advanced clinical stage (III/IV; P = 0.03),
Karnofsky’s scale score <70 (P = 0.03), and age at
diagnosis of >45 years (P = 0.04; Table 5
).
Pretreatment serum LDH activity in the PR ranged from 143 to 592 IU
(mean, 323.4 ± 110.1 IU; normal range, 120–230 IU). These
results were significantly higher than in CR patients, in whom the
level of LDH was 110–458 IU (mean, 204.6 ± 73.8 IU;
P = 0.02). However, serum LDH levels did not show
statistical significance as an independent prognostic factor. It
did not influence either the response to the treatment in the logistic
multiple regression test or the time of survival in the Cox regression
analysis.
Other morphological and biochemical parameters that were analyzed in
this study (Table 2)
, such as pretreatment ESR; hemoglobin level;
absolute number of leukocytes, lymphocytes, monocytes, eosinocytes,
erythrocytes, and platelets; alkaline phosphatase activity; serum iron
levels; the concentration of serum proteins in electrophoresis; and
renal and hepatic sufficiency tests, did not influence the survival
time or the response to treatment in multivariate analysis.
Additional Clinical Predictive Parameters.
Clinical analysis enabled us to detect an additional predictive factor,
i.e., the intensity of treatment. The DI of COPP or MOPP,
and ABVD regimens is shown in Table 6
.
The majority of therapeutic agents in these regimens were applied in
satisfactory dosages, comparable to those of the original protocols.
However, sometimes it was necessary to modify the drug dosages
(especially adriamycin) due to age, performance status, side effects of
chemotherapy, or lack of cooperation with the physician. For these
reasons, in the whole analyzed group, 49 (15.0%) patients did not
receive adequate therapy (DI ratio under 70% of planned total doses).
The differences in the time of survival between those patients and the
rest of the group were statistically significant (for OS,
P = 0.009; for DFS, P = 0.02).
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Because both a low DI of cytostatics and a lack of or too-low dose of
radiotherapy influenced the time of survival and response to the
first-line treatment in similar statistical strength, we have compared
all those cases in the same NAIT group. The total number of NAIT
patients was 68, because in 14 cases, both radiotherapy and
chemotherapy doses were reduced. The cut-points (for both 70% of
planned doses) were established on the basis of DI values distribution
in histogram (not shown). The percentage of CR achieved after the
first-line therapy in this NAIT patients was 57.4, which was
significantly lower than in patients with an adequately intensive
treatment (84.2%; P = 0.01). NAIT correlated
negatively also with the time of survival (for OS, P =
0.02; for DFS, P = 0.03; Fig. 2
).
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).
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summarizes the results of PCNA,
p53, and BCL-2 expression on R-S/H cells obtained in the whole group of
patients. Positive PCNA reaction was observed on R-S/H cells in 138
(71.1%) cases, with the proliferate index in whole group ranging from
0 to 95% (mean, 51.5%). Tumor cells were p53-specifically
labeled in 112 HD patients (57.7% %), with the index ranging from 0
to 90% (mean, 26.2%) in the whole group. BCL-2 protein expression was
demonstrated in the cytoplasm of R-S/H cells in 91 (46.9%) cases; the
index in the whole group ranged from 0 to 60% (mean, 11.0%). The
coexpression of two antigens was observed in 37 patients (19.1%), and
coexpression of all three antigens was observed in 59 patients
(30.4%).
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5
6)
.
Significant negative correlation was found to occur between the indexes
of PCNA and both OS (P = 0.02) and DFS
(P = 0.01). Much stronger correlation with survival
time was recorded for p53 (OS, P = 0.001; DFS,
P = 0.0002) and BCL-2 expression (OS, P = 0.009; DFS, P = 0.002).
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). In PR indexes of PCNA, p53 and
BCL-2 were much higher than in CR patients and were 10–95%
(mean, 78.2%), 20–90% (mean, 49.6%), and 0–60% (mean, 20.5%),
respectively (Table 7)
.
The best prognostic pretreatment pattern was lack of the estimated
protein expression, whereas the worst prognosis involved patients with
the coexpression of PCNA, p53, and BCL-2 on R-S/H cells (Fig. 7)
.
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.
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Among all of the pretreatment factors analyzed in regard to poor
response to the first-line treatment (PR) only four factors had
statistically independent, predictive strength: high p53 index
(P = 0.0007), poor performance status (Karnofsky’s
scale < 70; P = 0.009), multisite nodal
localization of disease (more than three sites; P =
0.03), and high PCNA index (P = 0.04; Table 8
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the last several years, scientists have been searching for new, more specific and sensitive prognostic factors. For example, aneuploidy and S-phase fraction are well-known prognostic factors in solid tumors and non-Hodgkin’s lymphomas. Erdkamp et al. (17) , analyzing these parameters in relation to clinical characteristics of HD patients, concluded that the S-phase fraction could be a good indicator in cases in which the outcomes may be worse. However, DNA aneuploidy did not correlate either with time of survival or with other known prognostic factors.
A number of other laboratory parameters, such as serum ß-2-microglobulin level (18) , interleukin-2 (19) , CD-8 antigen (20) , or urinary excretion of pseudouridine (21) , have been also considered as valuable for the prognosis in HD. According to some authors, one of the most important prognostic factors in HD is histological type of the disease (22) . Additionally, Alavaikko et al. (23) suggested that the presence of follicular dendritic cells in neoplastic areas of lymph nodes may predict a favorable outcome in the HD patients.
In non-Hodgkin’s lymphoma patients, as well as in pediatric HD, a valuable prognostic factor seems to be the pretreatment serum LDH activity (24 , 25) . In our study, high LDH activity significantly correlated with worse response to treatment, and it was the only biochemical parameter investigated that appeared to have an independent prognostic importance.
Abnormal expression of p53 and BCL-2 oncoproteins on neoplastic cells has been recently shown in carcinomas, sarcomas, and lymphoid neoplasms. The expression of PCNA, a marker of cell proliferation, can also correlate with the progression of some of human malignancies. However, data from the studies concerning HD are thus far scarce and controversial.
Lack of p53 expression on R-S/H cells in the lymphocyte-predominant type of HD and positive staining in the remaining histopathological types of the disease were reported by Lauritzen et al. (26) . Overexpression of the BCL-2 was demonstrated in a wide range of HD cases, and it can be speculated that this abnormality may play a role in the pathogenesis of HD (5) . PCNA expression without any statistical significant differences between the different types of HD was found by Hell et al. (27) and Kordek et al. (28) .
Therefore, thus far, only Xerri et al. (29) have investigated the correlation between the oncoprotein expression in R-S/H cells and some clinical parameters in HD, such as clinical staging, B-symptoms, probability of relapse, and DFS. The authors were not able to find any statistical correlation between the p53 expression on R-S/H cells and these parameters. Additionally, Trümper et al. (30) found that the activity of soluble p53 in serum of 21 of 33 HD patients bears no statistical correlation with other clinical prognostic factors.
Our immunohistochemical examinations of PCNA and the protein products of the p53 and bcl-2 genes, performed in the lymph nodes obtained from patients with HD, showed that the high expression of all these antigens correlated with poor response to the treatment and short time of survival. The lowest pretreatment indexes of PCNA, p53, and BCL-2 were found in the patients who achieved CR and were alive and free from disease during the whole period of our observation. Statistical analysis of survival time led us to the conclusion that PCNA index, as well as the expression of the p53 and BCL-2 proteins on R-S/H cells, can be taken into consideration as a new prognostic factor in HD.
Treatment modality is an important factor influencing the rate of relapses (1) . Simultaneously, we found that the early CR, achieved by the fourth cycle of chemotherapy, can be used as an independent positive prognostic factor. Similar results were reported by Somers et al. (31) . An inadequate treatment (usually due to lack of radiation therapy) can be also a cause of failure in the treatment in a number of patients with HD.
The data on the real predictive value of many factors may not be fully consistent, mainly because the number of examined patients is still too small and because of differences in statistical tests used for the analysis. Therefore, the standardization of methods and multicenter collaborative studies on large groups of patients (2 , 3 , 31 , 32) is necessary to establish the set of factors with a real prognostic value, with the view of selecting the most adequate therapeutic modality in HD.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Haematology, Medical University of Lodz,
Copernicus Hospital, Pabianicka 62, 93-513 Lodz, Poland. Phone/Fax:
48-42-684-68-90. 
2 The abbreviations used are: HD, Hodgkin’s
disease; ABVD, Adriamycin, bleomycin, vinblastine, dacarbazine; COPP,
cyclophosphamid, vincristine, procarbazine, prednisone; CR, complete
remission; CT, computed tomography; DFS, disease-free survival time;
DI, dose intensity; ESR, erythrocyte sedimentation rate; LDH, lactate
dehydrogenase; MOPP, nitrogranulogen, vincristine, procarbazine,
prednisone; NAIT, not adequately intensive treatment; OS, overall
survival time; PCNA, proliferating cell nuclear antigen; PR, poor
response group.
Received 7/30/97; revised 12/ 3/99; accepted 12/ 6/99.
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niak L., Domagal W., Osborn M. Percentage of proliferating cell nuclear antigen (PCNA)-positive Reed-Sternberg cells in nodular sclerosis Hodgkin’s disease. Patol. Pol., 44: 129-131, 1993.[Medline]
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