# Immunohistochemical Estimates of Angiogenesis, Proliferative Activity, p53 Expression, and Multiple Drug Resistance Have No Prognostic Impact in Osteosarcoma: A Comparative Clinicopathological Investigation

Authors: Flemming Brandt Sorensen; Kenneth Jensen; Michael Vaeth; Henrik Hager; Anette Mariane Daa Funder; Akmal Safwat; Johnny Keller; Mariann Christensen
Journal: Sarcoma
 (2008)

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## Abstract

Purpose. To investigate angiogenesis, multiple drug resistance (MDR) and proliferative activity as prognostic variables in patients suffering from osteosarcoma.
Methods. Histologic biopsies from 117 patients treated in the period from 1972 through 1999 were immunohistologically investigated regarding angiogenesis (CD34), proliferative activity (MIB-1), and the expression of p53 and MDR (P-glycoprotein (Pgp); clones JSB-1, C494, and MRK16). Quantitative and semiquantitative scores of immunoreactive cells were analyzed statistically along with retrospectively obtained clinicopathologic variables.
Results. Chemotherapy reduced the rate of amputations (P=.00002). The Pgp was overexpressed  (score ≥2) in 48% of the primary, diagnostic biopsies, and high Pgp correlated with high Pgp in postsurgical specimens (P=.003). In contrast, no such associations were disclosed for estimates of angiogenesis (P=.64) and p53 (P>.32), whereas the MIB-1 index was reduced in the post-chemotherapy specimens  (P=.02).
The overall, disease-specific survival was 47%, increasing to 54% in patients receiving pre-operative chemotherapy. Statistical analyses showed prognostic impact exclusively by patient age and type of osteosarcoma.
Discussion. The studied series of patients documented already prior to the chemotherapy era, a rather excellent survival and estimates of angiogenesis, proliferation, p53, and Pgp expressions, did not demonstrate sufficient power to serve as predictors of treatment response or survival.


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## Body

## 1. Introduction

The treatment of patients with osteosarcomas has during the last two
decades changed from a solely surgical approach to a highly complex multimodal
therapy with preoperative chemotherapy as the dominating innovation. Prior to the chemotherapy era, the studies addressing survival of patients with
osteosarcoma have, among others, identified morphological type of tumor, site
of tumor, tumor stage and size, and duration of symptoms as prognostic
variables of relevance [1, 2]. Investigations including large patient
populations treated after the introduction of preoperative chemotherapy
protocols have evidenced a shift toward histological response to chemotherapy as the most
informative prognostic parameter [3–5], a conclusive finding also corroborated by meta-analysis [6] and large-scale studies [7].Although the treatment outcome for
patients with osteosarcoma has improved significantly regarding survival after
the introduction of preoperative chemotherapy, it is still difficult to optimize
the therapy offered to the individual patient in that the most important
prognostic variable cannot be evaluated precisely until after surgical
intervention. The extent of tumor necrosis can be guided by various image
scans, but first after completion of preoperative chemotherapy, objective
measures in the histopathological examination of the surgical specimen are able
to estimate the true extent of chemotherapy-induced tumor necrosis [8, 9].Attempting to get around this
obstacle, researchers have focused their investigations on the characteristics
of the primary, diagnostic biopsy from patients with osteosarcomas. Features
like proliferation rate [10], P53 gene alteration [11], and especially, multiple drug resistance (MDR) [12] have been examined. Findings regarding prediction of chemotherapeutically
induced tumor necrosis have, however, been rather contradicting, and thus
difficult to implement in a routine diagnostic setting of osteosarcoma.
Moreover, recent results from a randomized phase III trial of the European Osteosarcoma Intergroup suggest
that intensified chemotherapy may improve histologic response but not survival
in osteosarcoma patients [13]. This questions the idea of using the
extent of chemotherapeutically induced osteosarcoma necrosis as a surrogate
measure of clinical outcome.The development of new microvessels
in tumors, the so-called neoangiogenesis, has for some years been investigated
in a number of different types of neoplasms, and has indeed been shown to be of
prognostic significance in some. Estimating the level of angiogenesis in a
malignant neoplasm is attractive for several reasons, and may reflect the
capacity for a malignant tumor to metastasize, and from the therapeutic point
of view, for developing antiangiogenetic drugs that may lead to starvation and
ultimately death of the tumor in question. The angiogenetic level may also be
of interest in planning systemic chemotherapy, as it is the case for patients
with osteosarcomas, and it has accordingly also been investigated in such
neoplasms [14–18].In this retrospective study, we have
investigated the prognostic value of angiogenesis, proliferation rate, alterations
in p53 and MDR/P-glycoprotein (Pgp) expressions in osteosarcomas from patients treated
at the Sarcoma Center,
Aarhus University Hospital, Denmark from
1972 through 1999, using an immunohistochemical approach. The study thus
includes patients treated both before and after the introduction of preoperative
chemotherapy, and provides an update of the treatment results in patients with
osteosarcoma at Aarhus University Hospital.


## 2. Patients

The cohort of patients studied was retrospectively
retrieved from the files of the University Institute of Pathology, Aarhus University Hospital, Denmark in the period from 1972 through 1999. This database was combined with the clinical database at the Department of Oncology, Aarhus University Hospital, Denmark, and a total of 134 consecutively admitted patients treated for primary,
nonmetastatic osteosarcoma of the extremities and pelvis were identified. Most
of the patients had been referred to the Sarcoma Center of Aarhus University
Hospital. Retrieval of clinical follow-up information and/or histological
material was unsuccessful in 17 cases, leaving a total of 117 patients for inclusion
into the investigation with at least 5 years of follow-up after primary
treatment.The clinicopathological data of the
studied patients are summarized in Table1. Prior to 1984, the treatment of
osteosarcoma at the Sarcoma Center, Aarhus University Hospital
was limited to surgery, but after 1984, preoperative chemotherapy was offered
to all patients, followed by surgical treatment which has increasingly focused on
limb-salvage resections. In the early chemotherapy era, the MAP-regimen
(mitomycin, adriamycin, and cisplatin) [19] was
used, based on Mayo Clinic experience.
From 1991, the chemotherapy was changed to the European Osteosarcoma Intergroup Study approach, based on
doxorubicin and cisplatin [13].
A few patients received chemotherapy and/or radiation therapy alone or in
combination (Table 1), mostly as an adjuvant postoperative therapy at recurrent
disease, in that only very few patients refused surgery at primary treatment.Table 1
Clinicopathological data of 117 patients with primary osteosarcoma.
NSexWomen41Men76AgeMean (years)23.8SD (years)16.6Range (years)4–74Tumor siteHumerus and radius12Pelvis10Femur66Tibia21Fibula6Tarsal bones2Extent of tumorIntraosseous6+ Cortical breakthrough16+ Soft tissue extension95Type of osteosarcomaClassical high grade98Other types19Largest tumor diameteraMean (cm)10.1SD (cm)3.9Range (cm)3–20Diagnostic delaybMean (months)5.3SD (months)3.9Range (months)1–36Type of surgical treatmentNone5Tumor resection26Amputation86Histological radicality of surgerycIntralesional resection4Marginal resection5Wide resection(radical)49Amputation (radical)54Chemotherapyd+ Preoperative chemotherapy46− Preoperative chemotherapy63+ Other kind of chemotherapy8Radiation therapye+ Radiation therapy7− Radiation therapy110Clinical response to primary treatmentComplete clinical response107Persistent disease10Histological response to preoperative chemotherapy100% tumor necrosis6>90% tumor necrosis1150–90% tumor necrosis14<50% tumor necrosis10No necrosis5Recurrent diseasefNo recurrence51+ Recurrence56Persistent disease10Survival statusgSurvivors58Death caused by OS58Death from unrelated disease1Tumera diameter not known for 3 patients.Diagnosticb delay was estimated from the anamnestic informations given in the clinical records and was unknown for 19 patients.Fivec patients did not receive surgical treatment, but were by own wish treated by either chemotherapy and/or radiation therapy alone/combined or refused any treatment.Oned patient received chemotherapy as the only treatment modality and did not wish the subsequent surgery, whereas 7 other patients received chemotherapy as adjuvant to other treatment.Twoe patients received radiation therapy as the only treatment.Rfecurrence status at the closure of the study.Survivalg status at the closure of the study.The clinical data were retrieved from
the medical records. Patients
were seen at regular intervals as outpatients at the Department of Oncology, Aarhus University Hospital, or at admission
to this Department or the Department of Orthopaedic Surgery in the case of recurrence.
Thus, any type of recurrence was added to the clinical records and to the
clinical database, and in the case of death, the cause of death was established
from autopsy records or from the death certificates. The investigation was
approved by the Ethical Committee of Aarhus County (Project no. 1999/4601) and by the Danish
Data Registration Authorities (Danish Data Protection Agency, Project no. 2000-41-0174).


## 3. Material and Methods

Primary, diagnostic tumor biopsies,
formalin fixed, and paraffin embedded, from all 117 patients and tumor
specimens from the 112 patients, who underwent surgical treatment, were
available for investigation. One representative tissue block was selected from
each case by screening all histological sections. In this way, 117 tissue
blocks from primary, diagnostic biopsies, and 46 tissue blocks from surgical
resection specimens of patients receiving preoperative chemotherapy were
selected for the investigation. When possible, the tissue blocks were selected
from the soft tissue extensions of the osteosarcomas, in that this tissue had
underwent none or only a short period of decalcification in either nitric acid
or EDTA.The surgical specimens from patients
treated with preoperative chemotherapy had been processed according to
international guidelines of estimating the extent of chemotherapy-induced tumor
necrosis [8, 9]. Briefly, the resected osteosarcoma was sawed along the
largest diameter of the tumor, and a slice of the surgical specimen was
subsequently decalcified and embedded in paraffin blocks in toto. All histological sections cut from each tissue block were
examined and a semiquantitative estimate of overall tumor cell necrosis in
percent was reported. The tissue blocks used for further studies were carefully
selected to ensure vital tumor tissue, but in 6 cases with 100% tumor necrosis,
no further investigations were performed.


### 3.1. Immunohistochemistry

Histologically,
4μm thick sections were cut from each tissue block, placed on coated slides, and
deparaffinized in xylene followed by ethanol. Endogenous peroxidase was blocked
in methanol with H2O2 (v/v 35%). Several experiments were
carried out to unmask epitopes, including various enzymatic treatments and
buffer baths in micro-oven, but the fragile, decalcified tissue sections mostly
detached from the histologic slides. The best results were obtained by
immersing the histologic slides in TEG buffer (pH 9.0) at 60C° in a
heating cupboard for 72 hours, followed by sensibilization in TRIS buffer (pH
7.6) and normal horse serum (DAKO, Glostrup, Denmark).Incubation with primary antibodies
for detecting proliferation (clone MIB-1, DAKO, Glostrup, Denmark), p53 (clone DO-7, DAKO, Glostrup, Denmark), and CD34 (clone QBend10, Immunotech, Quebec, Canada), diluted1:200,1:200, and 1:100, respectively, in antibody diluent (DAKO, Glostrup, Denmark) was performed overnight (18 hours), followed by rinsing in TRIS buffer. Secondary biotinylated antibody
(PK-6200, Vector) was added for 1 hour, followed by TRIS buffer added 0.5% blocking
reagent (DuPont TSA
Kit). To intensify the staining, the histologic sections were treated with
tyramide (in TRIS buffer added H2O2) and rinsed in TRIS buffer.
Avidin-biotin complex served as the tertiary antibody (PK-6200, Vector), with
incubation for 1 hour followed by rinsing in TRIS buffer. Diaminobenzidine
(DAB) was used as chromogen, and finally the histologic sections were counter
stained in Mayer’s hematoxyline and cover slipped in Aquatex. The tyramide
signal amplification (TSA) showed excellent results for staining with MIB-1 and
p53, whereas it had no improvement on the staining for CD34, which was overall
of rather poor quality with only 95 assessable biopsies out of a total of 163.The immunohistological staining for
MDR (Pgp) was carried out by employing three different monoclonal antibodies,
clones JSB-1, C494 (Zymed Laboratories Inc., Calif, USA), and MRK16 (Alexis Biochemical, Lausen, Switzerland), diluted1:20,1:500, and 1:400, respectively. Visualization was
performed by the Envision technique (DAKO, Glostrup, Denmark). Preliminary trials showed rather different
staining results of individual biopsies with the 3 different antibodies, and accordingly
it was decided to perform the staining procedure of all cases with a primary
antibody cocktail containing all 3 clones, which has also been recommended by
an international consensus report regarding detection of Pgp [20]. All
immunohistochemical procedures were followed by negative controls, omitting the
primary antibody. For MIB-1, p53, and CD34, the tumor sections had an inborn
positive control, and sections from normal human kidney were used as positive
control for the Pgp staining.There were no differences in the
quality or scorability of the immunohistochemical stains, when comparing
osteosarcomas included early or late in the nearly 30-year uptake period of the study.


### 3.2. Scoring of MIB-1 and p53

The immunoreactions for both MIB-1
and p53 are nuclear, but a few cases showed in addition a weak, cytoplasmic
reaction, which did not, however, interfere with the morphometric
quantification of the sections, in that the tyramide signal amplification
technique highly intensified the nuclear staining. The quantification of the
MIB-1 and p53 immunoreactivity was carried out, using the same morphometric
approach, applying the computer-assisted CAST-grid system (Olympus, Denmark).
At low magnification, the histological sections were projected onto a computer
screen, using a video camera. The computer software enables drawing around the
tumor area (the so-called meander), and within this area, the computer
generates fields of vision at high magnification (1425X), with the first field
generated at random, and the subsequent fields distributed systematically
within the meander area. A counting frame was superimposed on the histological
image, and using an unbiased, two-dimensional counting rule [21], the
positive and negative nuclear profiles were counted (Figure 1). The ratio of
positive to negative nuclear profiles was calculated in percent, and, moreover,
the density index of positive nuclear profiles pr. mm2 tumor area
was calculated. On the average, 50 fields of vision (range: 18–103) were
investigated in each tumor (corresponding to a mean tumor area of about 80 mm2),
with a mean of 140 counted nuclear profiles per case (range: 5–542). The sampling
fraction within the studied tumor sections ranged between 0.7% and 6.7%.Figure 1
Field of vision showing an
osteosarcoma stained immunohistochemically by MIB-1 to demonstrate cells in
proliferation cycle (brown nuclear stain). A counting frame has been
superimposed onto the histologic section for estimation of the rate and index
of neoplastic proliferation. Using anunbiased two-dimensional counting rule, nuclei in focus inside the frame or on the
hatched, blue edges are counted, as long as they do not intersect with the
fully drawn, red exclusion edges of the frame or their extensions. In this
example, 6 nuclei in cycle (brown) and 10 “resting” nuclei (blue) are counted (original
magnification: 400X).


### 3.3. Estimation of Angiogenesis

The histological sections were
scanned at low magnification to localize the three so-calledhot spots with increased
microvascularity (Figure 2(a)). In each of these hot spots, the neoangiogenesis
within the tumor tissue was quantified using the Chalkley technique [22],
as recommended earlier [23]. This method uses a graticule, mounted in
the ocular of the microscope, with 25 randomly distributed points. At high
magnification (250X), the ocular was rotated until the highest number of these
25 points superimposed on microvascular profiles in the tumor tissue (Figure 2(b)).
The mean of the three hot spot counts was reported as the angiogenetic Chalkley
number. In cases with no obvious hot spots, the mean of three randomly selected
fields of vision within the tumor area represented the Chalkley number. As
mentioned above, the angiogenesis could not be scored in a large number of
cases because of a false negative immunostain. This is probably caused by an
inherent defect in the studied tissue sections, may be due to acid exposure
during decalcification at the primary tissue processing, in that a lot of
microvascular profiles could be demonstrated morphologically.(a) Low-power view of a histologic section from an
osteosarcoma, showing a microvascular hot spot in the lower left corner (original
magnification: 40X). (b) Histologic section with the Chalkley graticule
superimposed. The graticule, equipped with 25 stochastically spaced points, is
rotated until the highest number of these 25 points coincides with immunohistochemically
stained microvascular profiles in the tumor tissue. The mean of three hot spot
counts is reported as the angiogenetic Chalkley number (immunohistochemical
stain by CD34 to highlight endothelial cells and vascular profiles; original
magnification: 200X).
(a)(b)


### 3.4. Scoring of MDR/Pgp

The scoring of Pgp immunostaining
was carried out on a semiquantitative scale, in accordance with earlier studies
performed at theRizzoli Clinic, Italy [12]: score 0 = complete absence of immunoreactive tumor cells; 1 = scattered, weakly immunoreactive tumor cells in less than 10% of the
histological specimen; 2 = diffuse, weakly immunoreactive tumor cells in more
than 10% of the tumor area; 3 = diffuse, intense immunopositivity in more than
10% of the tumor cells. A Pgp score ≥2 was considered to represent resistance
to chemotherapy [12, 17]. The heterogeneity of the Pgp immunostain
within one tumor section, and among different tumor sections, was only
moderate, with just a few cases showing marked variability in staining intensity,
and the scoring was based on the highest degree of immunoreactivity within the
examined histological slides. Mostly, the Pgp positive cells showed a diffuse
cytoplasmic immunoreactivity, but in some cases, an enhanced membrane staining
was visualized (Figure 3), as was reinforced immunoreactivity of the Golgi
region. Complete absence of Pgp-staining was only recorded in 13 specimens, and
in one specimen the Pgp-reactivity could not be estimated because of recurrent
detachment of the histological section from the slide during processing.Figure 3
Histologic section of an
osteosarcoma stained by a “cocktail” of antibodies against P-glycoprotein (MDR).
In this case, the stain shows intense membranous accentuation with a sparse
cytoplasmic reaction (MDR score = 3; original magnification 200X).


## 3.1. Immunohistochemistry

Histologically,
4μm thick sections were cut from each tissue block, placed on coated slides, and
deparaffinized in xylene followed by ethanol. Endogenous peroxidase was blocked
in methanol with H2O2 (v/v 35%). Several experiments were
carried out to unmask epitopes, including various enzymatic treatments and
buffer baths in micro-oven, but the fragile, decalcified tissue sections mostly
detached from the histologic slides. The best results were obtained by
immersing the histologic slides in TEG buffer (pH 9.0) at 60C° in a
heating cupboard for 72 hours, followed by sensibilization in TRIS buffer (pH
7.6) and normal horse serum (DAKO, Glostrup, Denmark).Incubation with primary antibodies
for detecting proliferation (clone MIB-1, DAKO, Glostrup, Denmark), p53 (clone DO-7, DAKO, Glostrup, Denmark), and CD34 (clone QBend10, Immunotech, Quebec, Canada), diluted1:200,1:200, and 1:100, respectively, in antibody diluent (DAKO, Glostrup, Denmark) was performed overnight (18 hours), followed by rinsing in TRIS buffer. Secondary biotinylated antibody
(PK-6200, Vector) was added for 1 hour, followed by TRIS buffer added 0.5% blocking
reagent (DuPont TSA
Kit). To intensify the staining, the histologic sections were treated with
tyramide (in TRIS buffer added H2O2) and rinsed in TRIS buffer.
Avidin-biotin complex served as the tertiary antibody (PK-6200, Vector), with
incubation for 1 hour followed by rinsing in TRIS buffer. Diaminobenzidine
(DAB) was used as chromogen, and finally the histologic sections were counter
stained in Mayer’s hematoxyline and cover slipped in Aquatex. The tyramide
signal amplification (TSA) showed excellent results for staining with MIB-1 and
p53, whereas it had no improvement on the staining for CD34, which was overall
of rather poor quality with only 95 assessable biopsies out of a total of 163.The immunohistological staining for
MDR (Pgp) was carried out by employing three different monoclonal antibodies,
clones JSB-1, C494 (Zymed Laboratories Inc., Calif, USA), and MRK16 (Alexis Biochemical, Lausen, Switzerland), diluted1:20,1:500, and 1:400, respectively. Visualization was
performed by the Envision technique (DAKO, Glostrup, Denmark). Preliminary trials showed rather different
staining results of individual biopsies with the 3 different antibodies, and accordingly
it was decided to perform the staining procedure of all cases with a primary
antibody cocktail containing all 3 clones, which has also been recommended by
an international consensus report regarding detection of Pgp [20]. All
immunohistochemical procedures were followed by negative controls, omitting the
primary antibody. For MIB-1, p53, and CD34, the tumor sections had an inborn
positive control, and sections from normal human kidney were used as positive
control for the Pgp staining.There were no differences in the
quality or scorability of the immunohistochemical stains, when comparing
osteosarcomas included early or late in the nearly 30-year uptake period of the study.


## 3.2. Scoring of MIB-1 and p53

The immunoreactions for both MIB-1
and p53 are nuclear, but a few cases showed in addition a weak, cytoplasmic
reaction, which did not, however, interfere with the morphometric
quantification of the sections, in that the tyramide signal amplification
technique highly intensified the nuclear staining. The quantification of the
MIB-1 and p53 immunoreactivity was carried out, using the same morphometric
approach, applying the computer-assisted CAST-grid system (Olympus, Denmark).
At low magnification, the histological sections were projected onto a computer
screen, using a video camera. The computer software enables drawing around the
tumor area (the so-called meander), and within this area, the computer
generates fields of vision at high magnification (1425X), with the first field
generated at random, and the subsequent fields distributed systematically
within the meander area. A counting frame was superimposed on the histological
image, and using an unbiased, two-dimensional counting rule [21], the
positive and negative nuclear profiles were counted (Figure 1). The ratio of
positive to negative nuclear profiles was calculated in percent, and, moreover,
the density index of positive nuclear profiles pr. mm2 tumor area
was calculated. On the average, 50 fields of vision (range: 18–103) were
investigated in each tumor (corresponding to a mean tumor area of about 80 mm2),
with a mean of 140 counted nuclear profiles per case (range: 5–542). The sampling
fraction within the studied tumor sections ranged between 0.7% and 6.7%.Figure 1
Field of vision showing an
osteosarcoma stained immunohistochemically by MIB-1 to demonstrate cells in
proliferation cycle (brown nuclear stain). A counting frame has been
superimposed onto the histologic section for estimation of the rate and index
of neoplastic proliferation. Using anunbiased two-dimensional counting rule, nuclei in focus inside the frame or on the
hatched, blue edges are counted, as long as they do not intersect with the
fully drawn, red exclusion edges of the frame or their extensions. In this
example, 6 nuclei in cycle (brown) and 10 “resting” nuclei (blue) are counted (original
magnification: 400X).


## 3.3. Estimation of Angiogenesis

The histological sections were
scanned at low magnification to localize the three so-calledhot spots with increased
microvascularity (Figure 2(a)). In each of these hot spots, the neoangiogenesis
within the tumor tissue was quantified using the Chalkley technique [22],
as recommended earlier [23]. This method uses a graticule, mounted in
the ocular of the microscope, with 25 randomly distributed points. At high
magnification (250X), the ocular was rotated until the highest number of these
25 points superimposed on microvascular profiles in the tumor tissue (Figure 2(b)).
The mean of the three hot spot counts was reported as the angiogenetic Chalkley
number. In cases with no obvious hot spots, the mean of three randomly selected
fields of vision within the tumor area represented the Chalkley number. As
mentioned above, the angiogenesis could not be scored in a large number of
cases because of a false negative immunostain. This is probably caused by an
inherent defect in the studied tissue sections, may be due to acid exposure
during decalcification at the primary tissue processing, in that a lot of
microvascular profiles could be demonstrated morphologically.(a) Low-power view of a histologic section from an
osteosarcoma, showing a microvascular hot spot in the lower left corner (original
magnification: 40X). (b) Histologic section with the Chalkley graticule
superimposed. The graticule, equipped with 25 stochastically spaced points, is
rotated until the highest number of these 25 points coincides with immunohistochemically
stained microvascular profiles in the tumor tissue. The mean of three hot spot
counts is reported as the angiogenetic Chalkley number (immunohistochemical
stain by CD34 to highlight endothelial cells and vascular profiles; original
magnification: 200X).
(a)(b)


## 3.4. Scoring of MDR/Pgp

The scoring of Pgp immunostaining
was carried out on a semiquantitative scale, in accordance with earlier studies
performed at theRizzoli Clinic, Italy [12]: score 0 = complete absence of immunoreactive tumor cells; 1 = scattered, weakly immunoreactive tumor cells in less than 10% of the
histological specimen; 2 = diffuse, weakly immunoreactive tumor cells in more
than 10% of the tumor area; 3 = diffuse, intense immunopositivity in more than
10% of the tumor cells. A Pgp score ≥2 was considered to represent resistance
to chemotherapy [12, 17]. The heterogeneity of the Pgp immunostain
within one tumor section, and among different tumor sections, was only
moderate, with just a few cases showing marked variability in staining intensity,
and the scoring was based on the highest degree of immunoreactivity within the
examined histological slides. Mostly, the Pgp positive cells showed a diffuse
cytoplasmic immunoreactivity, but in some cases, an enhanced membrane staining
was visualized (Figure 3), as was reinforced immunoreactivity of the Golgi
region. Complete absence of Pgp-staining was only recorded in 13 specimens, and
in one specimen the Pgp-reactivity could not be estimated because of recurrent
detachment of the histological section from the slide during processing.Figure 3
Histologic section of an
osteosarcoma stained by a “cocktail” of antibodies against P-glycoprotein (MDR).
In this case, the stain shows intense membranous accentuation with a sparse
cytoplasmic reaction (MDR score = 3; original magnification 200X).


## 4. Statistics

Associations between categorical
variables were assessed byχ2-test or Fisher’s exact test. Student’s t-test was used for comparison of
continuous variables between groups of patients. A rang sum test was used if
the variation in the data was poorly described by a normal distribution. A
paired t-test or McNemar’s test was used for pre versus post
comparisons. Associations between continuous variables were assessed by
Pearson’s correlation coefficient and Spearman’s correlation coefficient for
nonnormal data.Survival times and time to relapse
were described by Kaplan-Meier plots and the prognostic evaluation of the
variables was performed by log rank tests; the continuous variables were
dichotomized at the median. Patients were followed until death from disease, or
until the latest clinical control at the closure of the study. A 5% level of
significance was used for all statistical tests.


## 5. Results

### 5.1. Clinicopathologic Data

The obtained clinicopathological data are summarized in
Table1. Seventy one (61%) and 46 patients (39%) date from the time before and
after the introduction of preoperative chemotherapy, respectively.On the average, the diagnostic delay
was estimated to nearly half a year, but with a wide range, with 11 patients
having a delay for more than one year. This may explain the fact that the vast
majority of osteosarcomas evidenced soft tissue extension at diagnosis, with
only 19% of the patients presenting with a tumor limited by cortical bone.
Nineteen tumors (16%) represented other types of osteosarcomas, than the conventional
high-grade neoplasm. This group of patients showed a heterogeneous range of
morphological types, including both small cell and telangiectatic
osteosarcomas, and osteosarcomas of lower grade malignancy.Five patients did not receive
surgical treatment, whereas 26 patients underwent limb-salvage surgery in
combination with preoperative chemotherapy. The majority of the patients had
amputation which represents a reflection of secularity related to the retrospective design with patients
treated within a period of 27 years. Although histological evaluation of the
surgical specimens showed intralesional resection in 4 cases, the clinical
response to treatment was judged incomplete (persistent disease) in 10
patients. However, the latter group includes also 5 patients, who did not
receive primary surgical treatment, whereas one patient was found to have
metastatic disease shortly after surgical treatment.In Table2, the relationship between
the type of surgical treatment, documented by the histological examination of
the surgical specimen, and the administration chemotherapy has been listed.
There is a significant shift from amputations to surgical treatment by wide
resections with the introduction of preoperative chemotherapy (P=.00002).
Only 35% of the patients receiving this treatment modality had, however,
excellent histologic response (>90% tumor necrosis).Table 2
Histological
radicality of surgery in relation to preoperative chemotherapycoursea. Pvalueb=.00002.
Type of histological radicality of surgical specimen:Preoperative chemotherapy not givenPreoperative chemotherapy given∑Intralesional or marginal resection549Wide resection193049Amputation431154∑6745112Fivea patients
did not receive surgical treatment, but were by own wish treated by either
chemotherapy and/or radiation therapy alone/combined or refused any treatment.bχ2-test.


### 5.2. Quantitative Variables

Results regarding angiogenesis,
proliferation, and p53 expression are summarized in Table3. Separate analysis of
these variables in the group of nonconventional osteosarcomas showed that these
parameters, on the average, were not statistically different from the results
harvested from the investigated conventional osteosarcomas (.06<P<.20).
However, in this group, also including low-grade osteosarcomas, the angiogenesis,
proliferation rate and index showed a tendency toward lower values (5.94, 43.2%,
and 1565, resp.),
whereas the p53 expression and p53 index showed a tendency toward higher values
(74.17 and 3277, resp.).
The elevated p53 scores were specifically caused by extremely high values of
this parameter in a few round cell osteosarcomas included in this limited group
of patients.Table 3
Angiogenesis, tumor cell
proliferation, and nuclear p53 expression in primary, diagnostic biopsies from
osteosarcomas.
NMeanSDRangeAngiogenesisa956.792.502.00–14.00Proliferation rate (%)11750.618.85.1–90.2Proliferation index (mm−2)1171990116414–8388p53 expression (%)11768.024.45.5–100.0p53 index(mm−2)1172615143715–10556Ita was
technically impossible to obtain evaluable immunohistochemical stains for CD34
in 12 cases.Data on angiogenesis in both
pretreatment and posttreatment histological specimens were available in 25
patients, and no statistically significant difference in the degree of
microvascularity was disclosed in the two series of biopsies (P=.64). Analyses
among the other quantitative variables investigated showed mostly no
statistically significant associations (P>.10), apart from various
associations between estimates of proliferation and the p53 expression in the
studied osteosarcomas. Thus, estimates of the pre-chemotherapy p53 expression
were inversely associated with both pre- and post-chemotherapy estimates of
proliferation (P<.04). On the other hand, estimates of the
post-chemotherapy p53 expression were positive associated with the same
estimates of tumor cell proliferation (P<.02). These associations,
however, also are reflections of the decreased proliferation in
post-chemotherapy osteosarcomas (Table 4), which was statistically significant regarding estimates of proliferation index (P=.02). No
associations were detected between the quantitative variables and the largest
tumor diameter (P>.12) or clinical parameters (P>.10)Table 4
The relationships between estimates
of tumor cell proliferation and nuclear p53 expression in 46 osteosarcomas, before
and after preoperative chemotherapy.
Pre-chemotherapyPost-chemotherapyNMeanNMeanPvalueaProliferation rate (%)4654.4839b42.8.86Proliferation index(mm−2)c46223039b1327.02p53 expression (%)4661.039b64.8.85p53 index(mm−2)c46257339b2190.32Satudent’s unpaired t-test.Oneb case could not be evaluated, whereas 6 cases were associated with 100% tumor
necrosis.Icndex reflects the number of positive nuclear profiles pr. mm2 tissue.


### 5.3. MDR/P-Glycoprotein

A total of 60 primary, diagnostic
biopsies (52%) showed a Pgp score less than 2, and 56 biopsies (48%) expressed
a score higher than 2, whereas one biopsy could not be evaluated. In the group
of nonconventional osteosarcomas, the cases considered to be chemotherapy
sensitive constituted 63% of the cases. The Pgp expression showed no correlation
with the investigated quantitative and semiquantitative parameters
(angiogenesis,P>.15; proliferation rate, P>.54;
proliferation index, P>.51; p53 expression, P>.13; p53
index, P>.22).Six out of 46 osteosarcomas were
associated with 100%, chemotherapeutically induced tumor necrosis, and in 3 cases
the post-chemotherapy specimens could not be immunohistochemically evaluated.
In the former 6 osteosarcomas, the corresponding Pgp expression in the
pre-chemotherapy, diagnostic biopsies showed a score of 1 (i.e., chemotherapy sensitivity) in 4 cases, score 2 (chemotherapy
resistance) in one case, and for the remaining case the pretreatment biopsy
could not be evaluated for technical reasons.In the 37 osteosarcomas, where
post-chemotherapy biopsies were available, there was no statistically
significant difference in Pgp expression in the pre- and post-chemotherapy
biopsies, respectively (P=.10), although the MDR-score changed from
resistance to chemotherapy sensitive status in 4 cases (Table 5). High values
of the MDR-score in the pre-chemotherapy biopsies were positively correlated
with high values of the same parameter in the paired, post-chemotherapy tumor
specimens (P=.003). Moreover, the expression of Pgp in the primary,
diagnostic biopsies was not predictive of the degree of chemotherapy-induced tumor cell necrosis (P=.53;
Table 6).Table 5
Multiple
drug resistance (Pgp expression) in 37 osteosarcomas in pre- and
post-chemotherapy biopsies,respectivelya. Pvaluec=.10.
Pre-chemotherapy MDR-scorebPost-chemotherapy MDR-scoreb∑<2≥2<220121≥251116∑251237Tahree cases
could not be evaluated in the post-chemotherapy specimens, and 6 cases were
associated with 100% tumor necrosis.MbDR-score ≥2
indicates resistance to chemotherapy.MccNemar’s test.Table 6
Relationships between multiple drug resistance (Pgp expression) in pre-chemotherapy
biopsies of 46 osteosarcomas and histological response (necrosis) in surgicalspecimensa. Pvalueb=.53.
Pre-chemotherapy MDR-scoreaTumor necrosis∑<90%≥90%≥212719<2161127∑281846MaDR-score ≥2 indicates resistance to chemotherapy.Fbisher’s exact test.


### 5.4. Prognostic Evaluation

The overall disease-specific
survival in the entire series of patient, from inclusion to closure of the
study period, was 50%, with the group of patients receiving chemotherapy
showing a survival of 54%, and in the cohort of patients prior to the era of
chemotherapy the overall survival was 47%. Regarding recurrence-free survival,
the same figures were 48%, 53%, and 45%, respectively. The group of patients
with nonconventional osteosarcomas showed no statistical differences regarding
the quantitative variables investigated, when compared to the whole series of
patients investigated (.06<P<.20), and accordingly, patients with
all morphological tumor types were aggregated into one prognostic analysis. Moreover,
separate statistical analysis of patients treated before or after the
introduction of chemotherapy showed no individual differences for the
investigated quantitative variables. Accordingly, the results of the prognostic
evaluation are presented for the whole series of patients.Patient sex, tumor site and extent,
diagnostic delay, and type of surgical treatment were without prognostic
impact, whereas patient age and histological type of osteosarcoma showed
prognostic value regarding survival (Table7; Figures 4(b), 4(d)). Thus, high-grade
osteosarcomas and age > 25 years were associated with poorer survival. Large
tumor diameter may carry a tendency to shorter survival, however,
nonsignificant (Table 7; Figure 4(c)). Interestingly, neither the administration
of preoperative chemotherapy nor the histological response to chemotherapy (i.e., the extent of tumor necrosis)
provided any prognostic impact (Table 7).Table 7
Analyses
of prognostic impact of clinicopathological, categorical variables, grouped as
shown in Table1.
SurvivalaRecurrencebNumber of patients116c107dP-valueseP-valueseSex.16.33Agef.03.79Extent of tumor.91.90Type of osteosarcoma.02.06Largest tumordiameterf.11.36Diagnosticdelayf.30.13Type of surgical treatment.16.05Preoperative chemotherapy.78.31Histological response tochemotherapyg.33.48Saurvival
analysis based on disease-specific mortality.Abll kinds of
recurrences, but patients with persistent, progressive disease after primary
therapy have been deleted from the analyses.Onec patient,
who did not receive preoperative chemotherapy, died from unrelated disease, and
has been censored from the statistical analysis.Sixd and 4
patients, who did not or did receive preoperative chemotherapy, respectively,
had persistent, progressive disease after primary treatment, and are excluded
from the statistical analysis.Pe-values as reported from log-rank tests.Afnalysis
based on a 3-group comparison, divided on the tertiles.Agnalysis
based on a comparison between patients (showing ≥90% versus <90% tumor necrosis, resp.).Kaplan-Meier plots of survival in
patients with osteosarcoma regarding (a) multiple drug resistance score (P=.12; log rank test), (b) histological type of osteosarcoma (P=.02),
(c) tumor diameter (P=.11), (d) and patient age at diagnosis (P=.03).
(a)(b)(c)(d)The data obtained regarding
angiogenesis, proliferation rate and index, p53 expression and index, and Pgp
expression (Table8 and Figure 4(a)) were without any prognostic value, neither
regarding patient survival (P>.12) nor recurrence of osteosarcoma (P>.11).Table 8
Analyses
of prognostic impact of quantitative and semiquantitative immunohistological
estimates, as obtained from the pretreatment biopsies (number of patients
analyzed shown in brackets).
SurvivalaRecurrencebno pre-chemotherapy+pre-chemotherapyno pre-chemotherapy+pre-chemotherapyP-valuescP-valuescAngiogenesisd.90.92.23.76(65)(30)(65)(35)Proliferation rate (%).56.52.82.54(70)(46)(65)(42)Proliferation index (mm−2).47.81.43.89(70)(46)(65)(42)p53 expression (%).18.29.43.11(70)(46)(65)(42)p53 index (mm−2).73.99.90.90(70)(46)(65)(42)MDR/Pgp.43.12.23.16(70)(46)(65)(42)Aanalysis
based on disease-specific mortality, and for continuous parameters divided on
the median, whereas for MDR/Pgp divided in a chemotherapy-sensitive group
(score <2) and a chemotherapy-resistant group (score ≥2).Abll kinds of
recurrences, but patients with persistent, progressive disease after primary
therapy have been deleted from the analyses.Pc-values as reported from log-rank tests.Adngiogenesis could not be estimated in 21 cases due to technical, immunohistological problems,
see text.


## 5.1. Clinicopathologic Data

The obtained clinicopathological data are summarized in
Table1. Seventy one (61%) and 46 patients (39%) date from the time before and
after the introduction of preoperative chemotherapy, respectively.On the average, the diagnostic delay
was estimated to nearly half a year, but with a wide range, with 11 patients
having a delay for more than one year. This may explain the fact that the vast
majority of osteosarcomas evidenced soft tissue extension at diagnosis, with
only 19% of the patients presenting with a tumor limited by cortical bone.
Nineteen tumors (16%) represented other types of osteosarcomas, than the conventional
high-grade neoplasm. This group of patients showed a heterogeneous range of
morphological types, including both small cell and telangiectatic
osteosarcomas, and osteosarcomas of lower grade malignancy.Five patients did not receive
surgical treatment, whereas 26 patients underwent limb-salvage surgery in
combination with preoperative chemotherapy. The majority of the patients had
amputation which represents a reflection of secularity related to the retrospective design with patients
treated within a period of 27 years. Although histological evaluation of the
surgical specimens showed intralesional resection in 4 cases, the clinical
response to treatment was judged incomplete (persistent disease) in 10
patients. However, the latter group includes also 5 patients, who did not
receive primary surgical treatment, whereas one patient was found to have
metastatic disease shortly after surgical treatment.In Table2, the relationship between
the type of surgical treatment, documented by the histological examination of
the surgical specimen, and the administration chemotherapy has been listed.
There is a significant shift from amputations to surgical treatment by wide
resections with the introduction of preoperative chemotherapy (P=.00002).
Only 35% of the patients receiving this treatment modality had, however,
excellent histologic response (>90% tumor necrosis).Table 2
Histological
radicality of surgery in relation to preoperative chemotherapycoursea. Pvalueb=.00002.
Type of histological radicality of surgical specimen:Preoperative chemotherapy not givenPreoperative chemotherapy given∑Intralesional or marginal resection549Wide resection193049Amputation431154∑6745112Fivea patients
did not receive surgical treatment, but were by own wish treated by either
chemotherapy and/or radiation therapy alone/combined or refused any treatment.bχ2-test.


## 5.2. Quantitative Variables

Results regarding angiogenesis,
proliferation, and p53 expression are summarized in Table3. Separate analysis of
these variables in the group of nonconventional osteosarcomas showed that these
parameters, on the average, were not statistically different from the results
harvested from the investigated conventional osteosarcomas (.06<P<.20).
However, in this group, also including low-grade osteosarcomas, the angiogenesis,
proliferation rate and index showed a tendency toward lower values (5.94, 43.2%,
and 1565, resp.),
whereas the p53 expression and p53 index showed a tendency toward higher values
(74.17 and 3277, resp.).
The elevated p53 scores were specifically caused by extremely high values of
this parameter in a few round cell osteosarcomas included in this limited group
of patients.Table 3
Angiogenesis, tumor cell
proliferation, and nuclear p53 expression in primary, diagnostic biopsies from
osteosarcomas.
NMeanSDRangeAngiogenesisa956.792.502.00–14.00Proliferation rate (%)11750.618.85.1–90.2Proliferation index (mm−2)1171990116414–8388p53 expression (%)11768.024.45.5–100.0p53 index(mm−2)1172615143715–10556Ita was
technically impossible to obtain evaluable immunohistochemical stains for CD34
in 12 cases.Data on angiogenesis in both
pretreatment and posttreatment histological specimens were available in 25
patients, and no statistically significant difference in the degree of
microvascularity was disclosed in the two series of biopsies (P=.64). Analyses
among the other quantitative variables investigated showed mostly no
statistically significant associations (P>.10), apart from various
associations between estimates of proliferation and the p53 expression in the
studied osteosarcomas. Thus, estimates of the pre-chemotherapy p53 expression
were inversely associated with both pre- and post-chemotherapy estimates of
proliferation (P<.04). On the other hand, estimates of the
post-chemotherapy p53 expression were positive associated with the same
estimates of tumor cell proliferation (P<.02). These associations,
however, also are reflections of the decreased proliferation in
post-chemotherapy osteosarcomas (Table 4), which was statistically significant regarding estimates of proliferation index (P=.02). No
associations were detected between the quantitative variables and the largest
tumor diameter (P>.12) or clinical parameters (P>.10)Table 4
The relationships between estimates
of tumor cell proliferation and nuclear p53 expression in 46 osteosarcomas, before
and after preoperative chemotherapy.
Pre-chemotherapyPost-chemotherapyNMeanNMeanPvalueaProliferation rate (%)4654.4839b42.8.86Proliferation index(mm−2)c46223039b1327.02p53 expression (%)4661.039b64.8.85p53 index(mm−2)c46257339b2190.32Satudent’s unpaired t-test.Oneb case could not be evaluated, whereas 6 cases were associated with 100% tumor
necrosis.Icndex reflects the number of positive nuclear profiles pr. mm2 tissue.


## 5.3. MDR/P-Glycoprotein

A total of 60 primary, diagnostic
biopsies (52%) showed a Pgp score less than 2, and 56 biopsies (48%) expressed
a score higher than 2, whereas one biopsy could not be evaluated. In the group
of nonconventional osteosarcomas, the cases considered to be chemotherapy
sensitive constituted 63% of the cases. The Pgp expression showed no correlation
with the investigated quantitative and semiquantitative parameters
(angiogenesis,P>.15; proliferation rate, P>.54;
proliferation index, P>.51; p53 expression, P>.13; p53
index, P>.22).Six out of 46 osteosarcomas were
associated with 100%, chemotherapeutically induced tumor necrosis, and in 3 cases
the post-chemotherapy specimens could not be immunohistochemically evaluated.
In the former 6 osteosarcomas, the corresponding Pgp expression in the
pre-chemotherapy, diagnostic biopsies showed a score of 1 (i.e., chemotherapy sensitivity) in 4 cases, score 2 (chemotherapy
resistance) in one case, and for the remaining case the pretreatment biopsy
could not be evaluated for technical reasons.In the 37 osteosarcomas, where
post-chemotherapy biopsies were available, there was no statistically
significant difference in Pgp expression in the pre- and post-chemotherapy
biopsies, respectively (P=.10), although the MDR-score changed from
resistance to chemotherapy sensitive status in 4 cases (Table 5). High values
of the MDR-score in the pre-chemotherapy biopsies were positively correlated
with high values of the same parameter in the paired, post-chemotherapy tumor
specimens (P=.003). Moreover, the expression of Pgp in the primary,
diagnostic biopsies was not predictive of the degree of chemotherapy-induced tumor cell necrosis (P=.53;
Table 6).Table 5
Multiple
drug resistance (Pgp expression) in 37 osteosarcomas in pre- and
post-chemotherapy biopsies,respectivelya. Pvaluec=.10.
Pre-chemotherapy MDR-scorebPost-chemotherapy MDR-scoreb∑<2≥2<220121≥251116∑251237Tahree cases
could not be evaluated in the post-chemotherapy specimens, and 6 cases were
associated with 100% tumor necrosis.MbDR-score ≥2
indicates resistance to chemotherapy.MccNemar’s test.Table 6
Relationships between multiple drug resistance (Pgp expression) in pre-chemotherapy
biopsies of 46 osteosarcomas and histological response (necrosis) in surgicalspecimensa. Pvalueb=.53.
Pre-chemotherapy MDR-scoreaTumor necrosis∑<90%≥90%≥212719<2161127∑281846MaDR-score ≥2 indicates resistance to chemotherapy.Fbisher’s exact test.


## 5.4. Prognostic Evaluation

The overall disease-specific
survival in the entire series of patient, from inclusion to closure of the
study period, was 50%, with the group of patients receiving chemotherapy
showing a survival of 54%, and in the cohort of patients prior to the era of
chemotherapy the overall survival was 47%. Regarding recurrence-free survival,
the same figures were 48%, 53%, and 45%, respectively. The group of patients
with nonconventional osteosarcomas showed no statistical differences regarding
the quantitative variables investigated, when compared to the whole series of
patients investigated (.06<P<.20), and accordingly, patients with
all morphological tumor types were aggregated into one prognostic analysis. Moreover,
separate statistical analysis of patients treated before or after the
introduction of chemotherapy showed no individual differences for the
investigated quantitative variables. Accordingly, the results of the prognostic
evaluation are presented for the whole series of patients.Patient sex, tumor site and extent,
diagnostic delay, and type of surgical treatment were without prognostic
impact, whereas patient age and histological type of osteosarcoma showed
prognostic value regarding survival (Table7; Figures 4(b), 4(d)). Thus, high-grade
osteosarcomas and age > 25 years were associated with poorer survival. Large
tumor diameter may carry a tendency to shorter survival, however,
nonsignificant (Table 7; Figure 4(c)). Interestingly, neither the administration
of preoperative chemotherapy nor the histological response to chemotherapy (i.e., the extent of tumor necrosis)
provided any prognostic impact (Table 7).Table 7
Analyses
of prognostic impact of clinicopathological, categorical variables, grouped as
shown in Table1.
SurvivalaRecurrencebNumber of patients116c107dP-valueseP-valueseSex.16.33Agef.03.79Extent of tumor.91.90Type of osteosarcoma.02.06Largest tumordiameterf.11.36Diagnosticdelayf.30.13Type of surgical treatment.16.05Preoperative chemotherapy.78.31Histological response tochemotherapyg.33.48Saurvival
analysis based on disease-specific mortality.Abll kinds of
recurrences, but patients with persistent, progressive disease after primary
therapy have been deleted from the analyses.Onec patient,
who did not receive preoperative chemotherapy, died from unrelated disease, and
has been censored from the statistical analysis.Sixd and 4
patients, who did not or did receive preoperative chemotherapy, respectively,
had persistent, progressive disease after primary treatment, and are excluded
from the statistical analysis.Pe-values as reported from log-rank tests.Afnalysis
based on a 3-group comparison, divided on the tertiles.Agnalysis
based on a comparison between patients (showing ≥90% versus <90% tumor necrosis, resp.).Kaplan-Meier plots of survival in
patients with osteosarcoma regarding (a) multiple drug resistance score (P=.12; log rank test), (b) histological type of osteosarcoma (P=.02),
(c) tumor diameter (P=.11), (d) and patient age at diagnosis (P=.03).
(a)(b)(c)(d)The data obtained regarding
angiogenesis, proliferation rate and index, p53 expression and index, and Pgp
expression (Table8 and Figure 4(a)) were without any prognostic value, neither
regarding patient survival (P>.12) nor recurrence of osteosarcoma (P>.11).Table 8
Analyses
of prognostic impact of quantitative and semiquantitative immunohistological
estimates, as obtained from the pretreatment biopsies (number of patients
analyzed shown in brackets).
SurvivalaRecurrencebno pre-chemotherapy+pre-chemotherapyno pre-chemotherapy+pre-chemotherapyP-valuescP-valuescAngiogenesisd.90.92.23.76(65)(30)(65)(35)Proliferation rate (%).56.52.82.54(70)(46)(65)(42)Proliferation index (mm−2).47.81.43.89(70)(46)(65)(42)p53 expression (%).18.29.43.11(70)(46)(65)(42)p53 index (mm−2).73.99.90.90(70)(46)(65)(42)MDR/Pgp.43.12.23.16(70)(46)(65)(42)Aanalysis
based on disease-specific mortality, and for continuous parameters divided on
the median, whereas for MDR/Pgp divided in a chemotherapy-sensitive group
(score <2) and a chemotherapy-resistant group (score ≥2).Abll kinds of
recurrences, but patients with persistent, progressive disease after primary
therapy have been deleted from the analyses.Pc-values as reported from log-rank tests.Adngiogenesis could not be estimated in 21 cases due to technical, immunohistological problems,
see text.


## 6. Discussion

Among the traditional
clinicopathological variables, only the type of osteosarcoma and patient age showed
prognostic impact regarding survival which is in agreement with other studies
of patients with conventional high-grade sarcomas [1, 2, 5], but in
contrast to earlier investigations. The size [2, 4, 5, 7] of the tumor,
patient sex [7], and diagnostic
delay [1, 2, 7] were nonsignificant regarding prognostic power. In this
study, older age, surgical treatment by amputation, no surgical treatment and
conventional high-grade osteosarcoma were indicative of an unfavorable
prognosis. This may be a reflection of the characteristics of the database in
that the patients have been collected over a period of about 30 years.
Moreover, the group of nonconventional osteosarcomas also contained tumors of
lower-grade malignancy.On the other hand, it is surprising
that neither preoperative chemotherapy nor histologic response to that
treatment showed prognostic impact. These results disagree with most large
studies of patients treated by preoperative chemotherapy for osteosarcoma [3, 4, 6, 7],
except for a recent randomized phase III study by the European Osteosarcoma Intergroup [19]. The latter
multicenter investigation could not document any relationship between the
degree of histologic response to chemotherapy and patient outcome regarding
survival [19]. The results of our study may reflect matters of
secularity related to the database investigated in that the rarity of
osteosarcoma necessitate collecting patients for study over decades,a time frame within which minor or even major
changes in treatment may occur. An overall, disease-specific patient survival
of 47% before the introduction of preoperative chemotherapy is rather high,
when compared to earlier studies [1, 2], and considering the very long
follow-up presented in the present study. With such a high survival rate before
the introduction of preoperative chemotherapy, the true value of the treatment
may not penetrate from the statistical analysis. One has, however, to consider
the retrospective design of the present study, which also implies possible bias
due to patient selection and data confounding, especially related to treatment
variables. Nevertheless, the benefit of chemotherapy was reflected by the shift
from domination of amputation to preferential limb-sparing and wide surgical resection
before and after the introduction of chemotherapy, respectively.The fraction of proliferating tumor
cells in the investigated series of osteosarcomas ranged from 5% through 90%.
This represents a larger range than that obtained in some studies [10, 24–26],
but corresponds to the figures reported by German investigators [27, 28].
We used the TSA technique to improve the often rather weak immunohistological
staining for MIB-1, which may explain our higher range of osteosarcoma cells in
proliferation cycle. The discrepancies may, however, also be caused by a large
number of confounding parameters, associated with the different
immunohistological techniques and antigen retrieval methods, and dealing with
retrospective, paraffin-embedded archival tissue, that is, the standardization of fixation methods is
questionable. Furthermore, it has been shown that the Ki-67/MIB-1 antibody may
stain noncycling cells that overexpress p53 [29], which may indeed be
the case in osteosarcomas [26].There was no significant reduction
in the proliferation rate in post-chemotherapy specimens, but a statistically
significant decrease in the proliferation index was demonstrated. This is,
however, just a reflection of the used arithmetic, in that the proliferation
index is related to the area of tissue investigated, and not the number of
MIB-1 negative cells, and the area without tumor cells increases when
chemotherapy makes its effect.It has been suggested that MIB-1
could be used as an adjuvant variable in the morphological classification of
primary bone tumors because low-grade osteosarcomas have been shown to have a
lower proliferation rate than conventional high-grade sarcomas [24],
and low-grade osteosarcomas have a higher MIB-1 rate than fibrous dysplasia [30].
With the problems associated with immunohistochemical techniques, mentioned
above, and our finding of nonsignificant, lower values of MIB-1 rate in
nonconventional osteosarcomas, we would advise against employing MIB-1
estimates for guidance in classification of osteosarcomas.An increased rate of proliferation
in metastases, when compared with the MIB-1 rate in the corresponding primary
osteosarcomas, has been published [31], and thus adds evidence to the
suggested role of MIB-1 rate to act as an indicator of biological behavior of
osteosarcomas [25]. Prognostic impact of the proliferation rate has
been reported in patients with both skeletal [32, 33] and extraskeletal [34] osteosarcomas, in contrast to our findings, which, however, are corroborated by
other investigators [10, 26].Most studies addressing the
immunohistochemical expression of p53 in osteosarcomas have used a
semiquantitative scoring approach of the staining results [35–40],
often with a 10% or 20% threshold for scoring a tumor as p53 positive. Using
this approach, the immunoreactivity for p53 has been used in attempts to
classify bone tumors [39, 41]. We have used a strictly quantitative,
morphometric technique, in accordance with an earlier editorial on this issue [42],
which explains a frequency of 100% of p53 positivity in our series of
osteosarcomas. The staining pattern, and thus the difference in p53 reactivity,
was highly different among individual tumors, showing a range from 5% through
100% p53-positive tumor cells. Our reservations in interpreting the
immunohistochemical results for MIB-1 are, however, also valid for p53. In
addition, we experienced a rather dramatic heterogeneity in p53 staining within
the same slide from one tumor, and will accordingly recommend a
random-systematic sampling approach in immunohistochemical studies of immunohistochemical
cellular p53-expression.In accordance with our results,
immunohistochemical investigations of p53 in patients with osteosarcomas have
not demonstrated prognostic impact [26, 31, 43]. Changed p53 protein
expression may not convey mutation in the P53 gene [36], and accordingly, genetic studies of P53 gene alteration may be of value. However, a few studies based
on molecular technology in evaluating the P53 gene in osteosarcomas have not been able to document prognostic value in
osteosarcomas [11, 44, 45]. Nevertheless, it may, from the theoretical
point of view, still be valuable to study various suppressor genes like P53 and their protein expressions, in
that osteosarcomas are known to occur with higher frequency in persons with impaired
or defect suppressor genes like in the case of inherited retinoblastoma [46, 47].
Indeed, a recent study has shown prognostic impact of the combined information
regarding protein expression of markers of apoptotic cell death, such as p53,
bax, and bcl-2, as compared to missing prognostic value displayed by each of
these molecules individually [48]. Also, the combination of p53 with
information regarding Pgp has been documented to contain stronger prognostic
information than the two variables separately [49]. Thus, the true
prognostic value of p53/ P53 on either
the protein or gene expression level, respectively, awaits further
clarification.The rate of Pgp positive
osteosarcomas in the present investigation was comparable to findings in other
studies [50–52], but on the average, often a little higher than
reported by other investigators [12, 53, 54]. Again, these discrepancies
can be caused by differing techniques employed, and may not reflect differences
related per se to the patient populations studied. Missing
correlations between Pgp expression and the level of proliferation and p53
expression are in agreement with earlier findings [55].Although showing a tight
correlation, the Pgp expression was not different in primary, diagnostic
biopsies and post-chemotherapy specimens in the present series of patients with
osteosarcomas, and the Pgp level in the primary biopsy could not predict the
extent of chemotherapy-induced necrosis. The latter findings have been reported
by other investigators [12, 51, 52], and a meta-analysis including 631
patients has confirmed the missing predictive power of Pgp expression with
regard to the extent of chemotherapy-induced tumor necrosis [56]. Our
methodological approach cannot, however, provide reliable answers to whether
Pgp expression is an accurate measure to predict chemotherapy resistance, in
that tumor cell necrosis in itself may not be an accurate measure for this
resistance.The incidence of Pgp overexpression
has been shown to increase after chemotherapy [52], but our findings
cannot be compared reliably with this study, in that the two investigations use
quite different scoring schemes for evaluating the MDR-status. Although the
incidence of Pgp expression has been shown to be increased in metastatic as
compared to the corresponding primary osteosarcomas [53, 54], this does
probably not imply the development of a more aggressive phenotype [57],
and experimental data suggest that MDR is not upregulated in the course of
tumor progression [58].The prognostic impact of MDR/Pgp
expression in osteosarcomas is highly debatable. Some studies have shown Pgp
overexpression in primary, diagnostics biopsies to be predictive of poor
prognosis [12, 50, 53, 59], which may be of relevance in planning the
chemotherapy [60]. Other authors have not been able to detect
prognostic value of the Pgp expression, as determined by immunohistochemistry [40, 61].
Some of the confusion may be related to differences in documenting and scoring
immunohistochemical Pgp expression. We have employed the international
recommendation of using cocktails of two or more vendor-standardized anti-Pgp
antibody reagents that recognize different epitopes [20]. This may
improve the reliability of the immunohistochemical, overall detection of Pgp,
but the maybe unique clinical or prognostic impact of the individual antibody
clones used remains
undetected by our approach.The immunohistochemical
overexpression of Pgp may not, however, reflect true genetic amplification of
the MDR-gene [62], and quantitative RT-PCR analysis of osteosarcomas
from 123 patients was unable to demonstrate any relationship between the
genetic MDR expression and prognosis [63]. So far it seems that most
investigations, using immunohistochemical study design, favor prognostic
significance of Pgp overexpression, which also is the conclusion of the
meta-analyses mentioned above [56].Investigations of angiogenesis in human
osteosarcomas are rather sparse, but some studies have shown prognostic impact
of estimates of angiogenesis, either directly [15–17] or indirectly [64, 65],
whereas others have not been able to prove any prognostic value by angioenesis [18].
Some research based on animal models suggests, however, a role for angiogenesis
in the evaluation of progression of osteosarcoma [66], or in the
treatment of such tumors with antiangiogenetic drugs [67, 68]. We could
not demonstrate any prognostic impact by angiogenesis, and were unable to find
any significant differences in the degree of vascularity in pre- and
post-chemotherapy tumor specimens. Moreover, there seems to be no obvious relationship
between the grade of angiogenesis and the expression of Pgp in individual osteosarcomas,
pointing to the conclusion that angiogenesis is not associated with the accessibility
of chemotherapeutics to the vascular bed of the tumor cells. Only a
nonsignificant tendency to lower estimates of angiogenesis in nonconventional
osteosarcomas was revealed.We have used the Chalkley technique [22], as
recommended by Vermeulen et al. [23],
whereas other studies [14–18, 64] on human osteosarcomas have
used the microvessel density score for estimating the grade of vascularity within
tumors investigated. Although estimates of angiogenesis obtained by these two
different approaches may be correlated, a direct comparison is not possible.
The sampling approach for both techniques is based on estimation within preselected hot spots. Dealing with the highly
heterogeneous vascular morphology in osteosarcomas, this sampling method seems
rational. Optimal sampling schemes, like random systematic sampling, as used
for scoring of proliferative activity and p53-expression, seemed in our hands
unsuited for angiogenesis estimation. Necrosis, condroblastic, and bony-differentiated
areas within the often small diagnostic biopsies from osteosarcomas, as related
to the focality of neovascularisation (see Figure 2(a)), made such
stereological approach for estimating architectural structures like vessels futile,
in contrast to the suitability of this methodology in quantifying cellular
events like p53-expression and proliferation.Furthermore, in our investigation,
we have experienced great difficulties in the immunohistological staining by
antibodies to CD34, reducing the number of cases available for statistical
analysis regarding patient survival and recurrence of osteosarcoma. In our
preliminary investigations, we also tested out the usability of CD31 for
immunohistochemical detection of neoangiogenesis. However, CD31 showed an
immense, nonspecific background staining in the decalcified tissue sections,
making it impossible to obtain reliably quantitative estimates of angiogenesis.Also, the end points for various analyses regarding
prognostic impact of angiogenesis are varying, in that some studies focus on
survival and recurrences, whereas others primarily are addressing the response
to preoperative chemotherapy, another
point disabling bona fide comparisons
between various investigations. Moreover, the number of cases in the studies,
published so far, is rather sparse. Adding together all these problems
associated with estimation of neovascularity in osteosarcomas, we believe it is
too early to dismiss angiogenesis as a possible parameter for prognostic
evaluation, and for purposes of planning treatment in the event
antiangiogenetic drugs are introduced in treatment trials of patients with
osteosarcomas.In searching for new morphologic and
molecular predictors of prognosis and treatment outcome, the oncopathologist is
faced with the fight against tumor heterogeneity [69]. Indeed, this battle
seems insurmountable if one considers the sparseness of the primary, diagnostic
biopsy from the mostly bulky osteosarcomas. The intratumoral heterogeneity may
be attributed to intrinsic, tumor cell-specific characteristics or caused by
the environment of the tumor growth. Moreover, the tissue sampling within the
tumors and the technical approach used makes heterogeneity one of the most
difficult obstacles to manage in both a scientific and clinical contexts, even in the
case of monomorphic, small cell malignancies like, for example,Ewing sarcoma [70]. The diagnostic biopsy from an
osteosarcoma will always represent a keyhole of the neoplastic reality. Dealing
with cellular events like p53 expression and proliferative activity, systematic
random sampling can combat some of the problems related to heterogeneity.
Angiogenesis is more difficult in this regard, and a semiquantitative approach
seems most feasible for quantifying such architectural aspects. Monitoring the
expression of MDR/Pgp is also highly sensitive to tumor heterogeneity, but one
molecular study, aimed at this particular issue, has revealed very little variation
of intratumoral MDR expression [71]. The overall impact of all these
problems suggests humbleness in interpreting, and in taking clinical
consequence of studies of prognostic variables in all kinds of human neoplasms.


## 7. Conclusion

This retrospective, immunohistochemical study
of patients treated for osteosarcoma does not disclose prognostic impact of
quantitative estimates regarding angiogenesis, tumor cell proliferation, p53
status, or P-glycoprotein expression. Likewise, the use of preoperative
chemotherapy and the extent of induced tumor cell necrosis were without
prognostic value. Although the study has a retrospective design which may
inflict data confounding and bias, these findings may be associated to the fact
that the overall survival was around 50% even before the introduction of
preoperative chemotherapy. Conflicting evidence in the literature regarding the
true, prognostic impact of the dogmatized, predictive value of the
histopathologic response to preoperative chemotherapy in patients suffering
from osteosarcoma may ask for intensified research regarding the pretreatment,
predictive value of, for example, patient-specific qualities like MDR status.



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*Source: 874075-2009-02-25.xml*