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Altered patterns of retinoblastoma gene product expression in adult soft-tissue sarcomas.

Altered expression of the retinoblastoma (RB) tumour-suppressor gene product (pRB) has been detected in sporadic bone and soft-tissue sarcomas. Earlier studies, analysing small cohorts of sarcoma patients, have suggested that these alterations are more commonly associated with high-grade tumours, metastatic lesions and poorer survival. This study was designed to re-examine the prevalence and clinical significance of altered pRB expression in a large and selected group of soft-tissue sarcomas from 174 adult patients. Representative tissue sections from these sarcomas were analysed by immunohistochemistry using a well-characterised anti-pRB monoclonal antibody. Tumours were considered to have a positive pRB phenotype only when pure nuclear staining was demonstrated, and cases were segregated into one of three groups. Group 1 (n = 36) were patients whose tumours have minimal or undetectable pRB nuclear staining (< 20% of tumour cells) and were considered pRB negative. Patients with tumours staining in a heterogeneous pattern (20-79% of tumour cells) were classified as group 2 (n = 99). The staining of group 3 (n = 39) was strongly positive with a homogeneous pRB nuclear immunoreactivity (80-100% of tumour cells). pRB alterations were frequently observed in both low- and high-grade lesions. Altered pRB expression did not correlate with known predictors of survival and was not itself an independent predictor of outcome in the long-term follow-up. These findings support earlier observations that alterations of pRB expression are common events in soft-tissue sarcomas; nevertheless, long-term follow-up results indicate that altered patterns of pRB expression do not influence clinical outcome of patients affected with soft-tissue sarcomas. It is postulated that RB alterations are primary events in human sarcomas and may be involved in tumorigenesis or early phases of tumour progression in these neoplasias.

The prognosis of patients with soft-tissue sarcomas has traditionally been determined by clinical and histopathological features of the tumour such as grade, size and depth. A number of staging systems have been developed which broadly categorise patients into groups of different survival potential (Russel et al., 1977;Beahrs et al., 1988;Shiu and Brennan, 1989). To date there is no universally accepted system for the staging of soft-tissue sarcomas, although the most widely employed is the AJCC (Beahrs et al., 1988). Recent advances in the recognition and understanding of molecular genetic events occurring in the pathogenesis of human sarcomas have resulted in attempts to define new prognostic markers.
Children affected with the hereditary form of retinoblastoma often develop a second malignancy several years after successful treatment of their primary tumour (Abramson et al., 1984). Most of these secondary neoplasms are bone or soft-tissue sarcomas (Hansen et al., 1985). In contrast, secondary neoplasms are uncommon in patients with the sporadic form of the disease. Knudson proposed that retinoblastoma developed as the result of two mutations inactivating both alleles of a single gene (Knudson, 1971). The RB gene maps to chromosome 13, band 13q14, and it encodes for a 110 kDa nuclear phosphoprotein (Friend et al., 1986;Fung et al., 1987;Lee et al., 1987). pRB is phosphorylated in a cell cycle-dependent manner; underphosphorylated pRB products are the predominant form in G,, exerting a growth-suppressive effect (Buchkovich et al., 1989;DeCaprio et al., 1989;Xu et al., 1991). Since pRB does not seem to possess sequence-specific DNA-binding activity, it is postulated that the negative regulatory effect of underphosphorylated pRB is through complex formation with DNAbinding proteins (DeFeo-Jones et al., 1991), such as the transcription factor E2F (Chepallan et al., 1991). In addition, pRB appears to be involved in the differentiation programme of certain cell lineages, since homozygous mutant RB mouse embryos die during embryonic development with major derangements of the central nervous system and erythropoiesis (Jacks et al., 1992;Lee et al., 1992). In recent years numerous lines of evidence have confirmed the concept that the RB gene is the prototypical tumour-suppressor gene (Huang et al., 1988;Sumegi et al., 1990). RB mutations and altered patterns of pRB expression have been detected in other malignancies, including soft-tissue sarcomas (Cance et al., 1990;Stratton et al., 1990;Wunder et al., 1991), acute myelogenous leukaemia (Kornblau et al., 1992), breast cancer Varley et al., 1989), bladder carcinomas (Cairns et al., 1991;Cordon-Cardo et al., 1992;Logothetis et al., 1992) and small-cell carcinoma of the lung (Harour et al., 1988;Hensel et al., 1990).
We previously studied a group of 44 primary high-grade bone and soft-tissue sarcomas of all age groups using immunohistochemistry, and observed a 70% incidence of altered pRB expression (Cance et al., 1990). Patients whose tumours had strong homogeneous expression of pRB had significantly better survival than patients with heterogeneous or absent expression of the pRB gene product. Given the potential clinical significance of these findings, we have extended our original study and analysed a larger cohort of 174 adult soft-tissue sarcomas for pRB expression. In the present analysis, all bone and paediatric tumours were excluded; however, specimens from metastatic sarcomas were included but were analysed separately. The tumour samples used in this study were obtained fresh following surgical resection, embedded in a cryopreservative solution (OCT compound, Miles, Elkhart, IN, USA), and stored at -70C until needed. Formalin-fixed sections of each specimen were stained with haematoxylin-eosin and examined microscopically. One pathologist (JMW) rereviewed the histopathological diagnosis, tumour grade and quality of the tissue of 280 sarcomas. All paediatric, bone and tumours of uncertain histopathology were excluded. A total of 174 tumours met the inclusion criteria and were used for analysis.

Patients
Clinical information on every adult patient admitted to Memorial Sloan-Kettering Cancer Center from July 1982 to the present has been maintained in a computerised prospective database. Precise clinical follow-up information was available on 172 of the 174 patients, including the type of treatment and follow-up time as well as disease status. Tumour characteristics including tumour size, site, histological subtype and grade were also recorded.
Monoclonal antibodies and immunohistochemistry Immunohistochemical analyses were performed utilising mouse monoclonal antibody Rb-PMG3-245 (IgG, subclass) as the primary antibody. This antibody was generated by immunising mice with the TrpE/Rb fusion protein and it has been shown to specifically recognise the 1100 kDa RB gene product (Cordon-Cardo and Richon, 1994). A non-specific IgG, mouse monoclonal antibody was used in all experiments as a negative antibody control. Normal tissue known to express the RB-encoded protein, as well as a pRB-positive (T24, bladder cancer) and a pRB-negative (Y79, retinoblastoma) cell line, were used as controls.
The avidin-biotin complex immunoperoxidase technique was the method used for the present study. Briefly, frozen tissue blocks were cut to 6 1tm thin sections placed on microslides and formalin fixed. The Rb-PMG3-245 antibody (final concentration, 10 g ml-') and the non-specific negative control antibody (used at the same concentration) were applied to consecutive sections of the same tumour. After extensive washing, tissues were incubated with biotinylated horse antimouse secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 30 min at a concentration of 1:100, followed by avidin-biotin-peroxidase complexes (Vector Laboratories) at 1:25 dilution. Diaminobenzidine was used as the final chromogen and ethyl green as the nuclear counterstain.

Statistical methods
Fisher's exact test was used to assess the association between clinicopathological factors and altered patterns of pRB (Mehta and Patel, 1983). The Kaplan-Meier method (Kaplan RB Meier, 1958) was used to estimate the survival functions. All non-tumour-related deaths were censored in the Kaplan-Meier analysis. The log-rank test (Peto et al., 1977) was used to compare differences in survival between groups of patients.

Results
The histopathological and relevant clinical characteristics of this group of soft-tissue sarcomas are summarised in Table I. Most of the specimens analysed were large, high-grade primary tumours. Thirty-three cases (19%) were metastatic lesions. Liposarcomas and leiomyosarcomas represented the most common histopathological subtypes.
Nuclear staining for pRB was typically observed in endothelial cells, lymphocytes and fibroblasts. We considered tumour samples studied to have a positive pRB phenotype only when pure nuclear staining was demonstrated. Cytoplasmic staining was occasionally observed in some sarcomas analysed. The intensity and pattern of pRB nuclear staining with the Rb-PMG-245 antibody was used to separate the cases into one of three groups. Group 1 (n= 36) were patients whose tumours had minimal or undetectable nuclear staining (<20% of tumour cells) and were considered pRB negative. Patients with tumours staining in a heterogeneous pattern (20-79% of tumour cells) were classified as group 2 (n = 99). The staining of group 3 (n = 39) was strongly positive with a homogeneous pRB nuclear immunoreactivity

Survival analysis
The median follow-up time for the entire study population was 37 months, with a mean of 39 months. Patients in groups 1, 2 and 3 had median follow-ups of 30, 35 and 34 months respectively. The survival of patients in these groups was compared using the Kaplan-Meier method. Figure la illustrates the tumour-specific survival stratified by pRB staining patterns. There was no difference in survival between the three groups at long term follow-up. Tumour grade is typically a dominant factor influencing survival. When we divided the 172 patients into two groups based on grade (high vs low), the survival was significantly different between these two categories (P<0.01). Survival was then analysed based on pRB status and tumour grade. Tumours of the same grade had similar survival characteristics regardless of pRB status. The log-rank results were not significant.
Survival analysis ofprimary sarcomas The demographics of this group are shown in Table III. The above analysis included 64 patients who presented with either local recurrences or distant metastases, which may potentially bias survival. The Kaplan-Meier curves in Figure lb represent the survival of patients who presented with primary tumours stratified by their pattern of Rb-PMG3-245 antibody immunostaining. Survival was very similar for each group. To eliminate the possible influence of tumour grade, survival for all primary high-grade lesions was also independently analysed. There was no statistical difference in survival between the two groups at long-term follow-up (Figure lc).

Discussion
Sarcomas often present as secondary neoplasms in patients with the hereditary form of retinoblastoma (Hansen et al., 1985;Friend et al., 1986). This early observation led to questions concerning the potential role of RB in the tumorigenesis of sporadic bone and soft-tissue sarcomas. Subsequent studies revealed that RB mutations also occur in sporadic sarcomas. Stratton et al. (1989) found homozygous RB deletions in only 3 of 63 sarcomas analysed, and loss of heterozygosity in 5 of 22 informative cases. Reissmann et al. (1989) also found RB deletions in three of nine osteosarcomas and 4 of 29 soft-tissue sarcomas. These studies were conducted utilising restriction fragment length polymorphism and Southern blot assays in order to identify RB gene mutations. Subtle alterations, sufficient to decrease or abort RB gene expression, are usually undetectable when using these techniques and may explain the lower frequency of RB alterations reported.
Methods which are guided to the analysis of gene expression are known to be more sensitive in detecting underlying RB alterations. Wunder et al. (1991), using Northern blot analysis, demonstrated an association between tumour grade and altered RB transcripts. They found low to undetectable RB mRNA levels in 10 of 25 high-grade bone and soft-tissue sarcomas analysed. In contrast, only 1 of 11 low-grade sarcomas and none of the four lipomas studied showed altered RB transcripts.
In a preliminary study from our group and for the present report, expression of pRB was assessed by the Rb-PMG3-245 monoclonal antibody and immunohistochemistry. This technique allows correlation of expression with microanatomic features and can identify the degree of heterogeneity and range of intensity of expression within a tumour. In our earlier series of 56 non-selected soft-tissue sarcomas, we showed that pRB was undetectable or heterogeneously expressed in 70% of the primary high-grade sarcomas, as well as in all metastatic lesions analysed (Cance et al., 1990). In the present study similar results were obtained. Briefly, primary high-grade lesions showed pRB alterations in 67 of 88 (76%) cases, while local recurrent and metastatic tumours had altered expression in 27 of 31 (87%) and 27 of 33 (82%) cases respectively. Nevertheless, we also observed that 22 of 32 (70%) low-grade sarcomas studied displayed either heterogeneous or undetectable pRB expression.
The basis for the heterogeneous pattern of pRB immunostaining observed on tumour cells in clinical samples may be interpreted as follows. First of all, it could be a genuine biological phenomenon of identifying two distinct tumour clones, one having the wild-type protein and another harbouring a mutation. On the other hand, it may be caused by limitations of the assay and reagents utilised. For example, it could reflect pRB variations that occur during cell cycle, either intercellular differences in the level of bound vs unbound pRB or its degree of phosphorylation. However, this last issue is unlikely since the antibody used (Rb-PMG3-245) detects both hypo-and hyperphosphorylated pRB products. In addition, while pRB expression in normal cells has been felt to be ubiquitous, recent analyses in normal tissue have documented low to undetectable pRB levels in association with specific cell types, including fibroblasts (Szekely et al., 1992;Cordon-Cardo and Richon, 1994). It is then reasonable to postulate that well differentiated soft-tissue sarcomas with a low proliferative index have low to undetectable pRB expression, below the threshold of immunohistochemistry. It is interesting to note that the number of low-grade tumours displaying a homogeneous pRB phenotype decreased with disease progression from 30% in primary sarcomas to 18% in local recurrences. This change was paralleled with a concurrent increase in the percentage of pRB heterogeneous lesions. This inverse relationship between tumour progession and pRB expression was not demonstrated in the high-grade tumours. In those cases, the extent of heterogeneous and undetectable pRB levels was similar in primary and metastatic lesions.
In our original study we analysed the survival of 44 patients with primary high-grade bone and soft-tissue sarcomas from all age groups. We found that survival of patients with altered pRB levels was significantly shorter at 24 months than patients whose tumours had homogeneous pRB staining. In the present study we observed similar differences at 18 and 24 months of follow-up between survial and pRB-positive vs pRB-negative cases, as in our initial study. However, beyond 24 months pRB expression did not appear to influence the survival of adult patients with primary high-grade soft-tissue sarcomas. Given the current survival results, we updated the follow-up of our previous data and reanalysed the results to test the durability of our early differences (Figure 2). At approximately 24 months the three curves begin to come together and the differences were no longer observed. It is not an unknown biological observation to see early differences lose their significance over time. For example, the contribution of various clinicopathological features of patients with sarcoma can vary according to duration of follow-up. Tumour grade is the dominant factor in early metastases, whereas the influence of this variable on the development of late metastases is equivalent (Gaynor et al., 1993). The early survival advantage of the patients with pRB positive sarcomas may reflect a delay in metastatic potential which diminishes with time. Other important differences between the two studies are the exclusion of osteosarcomas and paediatric sarcomas, as well as the more than doubling of the sample size. Bone and paediatric sarcomas are known to have high response rates to chemotherapy. Consequently, the prognosis of these patients has been dramatically improved by the addition of adjuvant chemotherapy (Link et al., 1991). This is not the case in adults with extremity soft-tissue sarcomas (Mazanet and Antman, 1991). As for the number of patients studied, the potential for 'type II' statistical error is now reduced compared with our original study, in which conclusions were, in essence, based on the survival of 13 patients. In summary, based on data from this study we cannot ascribe prognostic significance to alterations in RB gene expression in adult soft-tissue sarcomas. Given the high percentage of abnormal RB gene expression in primary low-and high-grade sarcomas, it appears that alterations in the expression of this gene occur both frequently and early in the pathogenesis of soft-tissue sarcomas. Our data suggest that, in adult soft-tissue sarcomas, alterations of the RB gene may be more important in tumorigenesis or early tumour progression than in late disease stages. More studies are required comparing RB and other tumour-suppressor gene abnormalities developing in well-characterised groups of patients affected with soft-tissue sarcomas in order to evaluate their critical role as tumour markers able to stratify patients in prognostic categories.