P ediatric and D e v e lo p m e n ta l P a th o lo g y 17, 278-285, 2014 DOI: 10.2350/14-02-1436-OA.1 © 2014 Society fo r P ediatric P a th o lo g y
S ig n ific a n t Im m u n o h is to c h e m ic a l E xp ressio n o f H u m a n C h o rio n ic G o n a d o tr o p in in H ig h G ra d e O s te o s a rc o m a is R are, B u t M a y Be A s s o c ia te d w it h C lin ic a lly E le v a te d S e ru m Levels A n na F. L ee, 1,2 B ruce R. Paw el , 1 a n d L isa M . Su lliv a n 1* ’ Department of Anatomical Pathology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA d e p a rtm e n t of Laboratory Medicine and Pathology, Children's and Women's Health Centre o f British Columbia, Vancouver, BC, Canada
Received February 4, 2014; accepted May 23, 2014; published online May 23, 2014.
ABSTRACT
Survival rates have plateaued at 70% for osteosarcoma. Proteins ectopically produced by malignant tumors may provide insight into new therapeutic targets. Osteosarco mas secreting human chorionic gonadotropin (hCG) have been suggested to have a worse prognosis. We examined the frequency of expression of P-subunit of hCG (P-hCG) in pretreatment osteosarcoma biopsies, and asked if it was associated with various clinical prognostic parameters, and the development of metastases. We subjected 51 pretreatment biopsies of high-grade osteosarcoma, from 51 patients, to P-hCG immunohistochemistry. In 19 of these patients, postchemotherapy metastatic biopsies also were examined for P-hCG expression. Clinical informa tion (patient age, sex, survival status, and serum hCG in females only), and tumor characteristics (site, size, and presence of metastases) were recorded. The p-hCG positive and negative biopsies were separated and compared. Of 49 interpretable pretreatment biopsies, 28 (57%) showed positive cytoplasmic P-hCG expression: 27 with sparse positivity (1% of tumor cells) and 1 with frequent positivity (10% of tumor cells). The patient with frequent P-hCG positivity in her pretreatment biopsy had elevated serum hCG (88.2 mlU/mL) at diagnosis, decreasing to undetectable following chemotherapy and definitive resection. There was no difference in clinical parameters or rate of metastasis between P-hCG positive versus negative groups. Expression of P-hCG may be seen in high-grade osteosarcoma, but frequent P-hCG immu nohistochemical expression by tumor cells, associated with clinically elevated serum P-hCG, is rare. Recogni tion that some nongenn cell tumors may produce P-hCG *Corresponding author, e-mail: [email protected]
can prevent confusion with malignancies containing neoplastic syncytiotrophoblast cells, including germ cell and trophoblastic tumors. Key words: ectopic, human chorionic gonadotropin,
metastasis, osteosarcoma, paraneoplastic, primary
INTRODUCTION
Osteosarcoma, a malignant neoplasm of bone-forming cells, is the most common primary sarcoma of bone [1]. Biologic behavior is predicted in part by histologic tumor grade, stratified into low and high grade. The treatment of choice for low-grade osteosarcoma is surgical resection. High-grade osteosarcomas, in contrast, usually require neoadjuvant chemotherapy followed by surgical resec tion. In addition, postresection chemotherapy also may be offered based on specific histologic findings in the resection specimen. Before the institution of systemic combination chemotherapy regimens starting in the late 1980s [2], survival was less than 20%, but the survival rate has risen to just over 70% for nonmetastatic patients [3], No new treatment strategies have substantially improved the survival over this figure. New therapeutic targets may be identified by studying ectopic production of proteins by malignant cells that could confer a survival advantage. Thus, characterization of substances not produced by normal host cells may prove helpful for the diagnosis or treatment of osteosarcoma, as well as determination of prognosis. One such substance is human chorionic gonadotropin (hCG), a heterodimeric protein composed of noncovalently-linked a and P subunits, which normally is secreted by placental syncytiotrophoblast and is required for
maintenance of the corpus luteum in early pregnancy [4,5]. Human chorionic gonadotropin may be found in neoplasms, secreted by the neoplastic counterpart of the syncytiotrophoblast cell. Such neoplasms include gesta tional trophoblastic disease, choriocarcinoma, and other germ cell tumors that contain neoplastic syncytiotropho blast cells. Malignancies derived from tissues unrelated to placental tissue also may secrete hCG. For instance, ectopic expression of hCG, has been observed in a wide variety of epithelial cancers, including those of the bladder, lung, breast, colon, and head/neck [6,7]. Case reports of sarcomas, including osteosarcoma [8-13], synovial sarcoma [14], and rhabdomyosarcoma [15], also have described hCG expression. Tumoral production of hCG may be clinically silent, or it may manifest as a paraneoplastic syndrome, such as amenorrhea in repro ductive age females. To date, the function of hCG in tumor biology is unclear and its role as a prognostic factor varies depending on the tumor. Human chorionic gonadotropin shows regions of homology with the tumor growth factor (TGF) superfamily of proteins, leading to speculation that hCG may crosstalk with the TGF signaling pathway, which is prosurvival in some conditions [6,7]. Another potential effect of tumoral hCG production is angiogen esis; recent in vitro experiments suggest that P-hCG may promote or enhance this phenomenon, which is necessary for tumor invasion and metastasis [16]. Some reports suggest that elevated serum and/or tissue P-hCG is an independent prognostic factor for adverse outcome in multiple tumor types [17-21], although one study on patients with colon carcinoma found this only to be the case for females [22]. However, negative studies also exist, in which no relationship between tissue P-hCG and survival was found [23]. To explore the role of hCG in osteosarcoma, Masrouha and colleagues [11] recently examined the immunohistochemical expression of the P-subunit of human chorionic gonadotropin (P-hCG) in a series of 32 osteosarcoma pretreatment biopsies and definitive resections. They reported P-hCG positivity in 5 cases. All 5 patients had suboptimal response to treatment (which was defined as greater than 10% viable tumor cells in the postchemotherapy specimen), and 4/5 developed metastases. The sample size was too small to draw definitive conclusions, but the investigators suggested that a potential link between tumor-associated hCG and poor outcome might be demonstrated by a larger study [11], In this present study, the relationship between P-hCG immunohistochemistry staining and urine/serum P-hCG was explored in pretreatment osteosarcoma biopsies and postchemotherapy metastatic lesions. To detennine whether the degree of P-hCG expression may be related to tumor aggressiveness and metastatic potential, P-hCG immunohistochemistry was compared between primary tumors and metastatic foci.
METHODS Ethics approval, case selection, and acquisition o f clinical in fo rm atio n
Approval to conduct this retrospective study was granted by the Institutional Review Board of the Children’s Hospital of Philadelphia (CHOP, protocol #12-009417). The pathology files at CHOP were searched for diagnostic biopsies, postneoadjuvant therapy resections, and biopsies of metastatic osteosarcoma (and malignant bone tumors consistent with osteosarcoma), accessioned between 2006 and 2012. We retrieved 51 diagnostic biopsies (taken before giving chemotherapy) from 51 patients, all showing high-grade osteosarcoma. All 51 patients received neoad juvant chemotherapy before definitive resection. Of these patients 19 suffered metastatic disease, which was surgi cally biopsied or sampled at autopsy. The retrieved cases were reviewed by two authors (A.F.L. and L.M.S.) for confirmation of the diagnosis. Data on age, sex, deaths, primary tumor site, primary tumor size, histologic response to neoadjuvant treatment at resection, and metastases (if any), were collected from pathology reports and the electronic health record. Information on whether the metastatic lesions were synchronous with the primary tumor, or developed subsequently, also was collected. Urine and/or serum [3-hCG levels were recorded where available. Only female patients were tested for phCG. The end of the follow-up period was April 2013. Preparation o f fo rm alin -fixed , p a raffin -em b ed d ed tissues
Fresh biopsy tissues were fixed in formalin for a minimum of 2 hours to overnight depending on the size of the biopsy. If required, formalin-fixed tissues were decalcified with Decalcifying Solution B, a hydrochloric acid-based decalcification solution (catalog #245-683; Thermo Fisher Scientific, Waltham, MA, USA), until soft enough to cut. Tissues then were dehydrated and embedded in paraffin wax as per standard operating procedure. Sections (4 p) were cut and mounted onto glass slides for routine hematoxylin and eosin (H&E) staining or for immunohistochemistry. Im m unohistochem istry and scoring o f staining
A representative block from each case with the largest amount of viable tumor was selected for immunohisto chemical staining. On the Leica Bond-Ill automated immunostaining machine (Leica Biosystems, Buffalo Grove, IL, USA), the sections were subjected to epitope retrieval (ER1-20 epitope retrieval solution; Leica Biosystems), and then incubated with 1:5000 dilution of rabbit polyclonal antibody directed against the P subunit of hCG (catalog #A0231; Dako, Carpinteria, CA, USA). Staining was visualized with a polymer-based secondary antibody. Placenta served as external positive control. Three immunohistochemistry staining patterns were observed: sparse positivity (approximately 1% of tumor hCG in H igh-G rade O steosarcoma
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Table 1.
Clinicopathologic characteristics of the study population separated by females and males
N o f patients N o f evaluable pretreatment biopsies N o f metastases N o f deaths Age (years)a Tumor size post resection (cm)» Response to neoadjuvant therapy (%)a
O verall
Fem ales
M ales
51 49 19 10 13.7 ± 3.0 10.8 ± 5.0 79.8 ± 21.8
26 24 7 6 13.7 ± 3.1 9.8 ± 4.1 75.6 ± 22.4
25 25 12 4 13.7 ± 3.0 11.6 ± 5.7 81.7 ± 21.9
aAverage ± SD. All comparisons, P > 0.05.
cells showing cytoplasmic [3-hCG positivity), frequent positivity (approximately 10% of tumor cells showing cytoplasmic P-hCG positivity), and negative staining. Two diagnostic biopsies, in which excessive hemosiderin confounded IHC interpretation, were excluded from scoring. Three metastatic tumor samples had too few tumor cells, either due to small sample size or block exhaustion, for meaningful assessment. One metastatic tumor sample was not available for staining. In total, 49/ 51 diagnostic biopsies and 15/19 biopsies of metastases had interpretable immunohistochemistry. Nondecalcified tissues were preferentially selected for immunohisto chemistry, but decalcified tissues were used if no other choice was available.
ostesoarcomas were captured in our retrospective study. The histologic subtypes included 44 conventional, 3 epithelioid, 1 periosteal, 1 periosteal chondroblastic, 1 small cell, and 1 telangiectatic. For the 19 patients with metastases, 8 patients had synchronous metastatic disease at the time of initial diagnostic biopsy (defined as metastatic disease clearly visible on imaging studies at the time of initial diagnostic biopsy). Ten patients had nonsynchronous metastases. Information was not available for one patient. Focal im m unohistochem ical p-hCG expression m ay be seen in high-grade osteosarcoma, bu t clinically elevated serum levels are rare
Statistical analysis
The 2-tailed Student’s /-test was performed to evaluate any difference in mean clinical parameters between groups. The 2-tailed Fisher’s exact test was performed to examine the significance of the association between two groups. Significance level (a) was set at 0.05. RESULTS Clinical characteristics o f th e study population
Table 1 shows the major clinical characteristics and demographics of the study population, and Supplemental Table SI (http://dx.doi.org/14-02-1436-OA.Sl) is an expanded table showing clinical characteristics as well as staining results obtained in all of the pretreatment biopsies and biopsies of metastases. The study population consisted of 51 patients (26 females and 25 males), with an average age of 13.7 ± 3.0 years (range, 4.5-18.9 years). No cases of secondary osteosarcoma were identified. There was no significant difference in sex distribution with respect to number of metastases (P = 0.24), number of deaths (P = 0.72), patient age (P = 0.71), tumor size (P = 0.34), and response to neoadjuvant therapy {P = 0.39) in these two groups. Consistent with previous large-scale epidemiologic studies of osteosarcoma [1], the most common tumor sites in our study population involved long bones of the limbs, with femur, tibia, and humerus being the most frequently affected. Less common sites involved fibula, sacrum, scapula, and sphenoid. All of the biopsies showed highgrade osteosarcoma; no low or intermediate grade
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Of the 49 interpretable biopsies of pretreatment osteosar comas, 28 (57%) showed some degree of cytoplasmic staining of (3-hCG in tumor cells (13 females and 15 males, see Supplemental Table SI; http://dx.doi.org/14-02-1436OA.S1). O f the 28 positive biopsies 27 showed only sparse p-hCG staining, involving approximately 1% of tumor cells, identified either as scattered single cells or small clusters (examples shown in Fig. 1). One biopsy of epithelioid osteosarcoma showed frequent p-hCG staining, which involved approximately 10% of tumor cells (Fig. 2A-C). The effects of decalcification were consid ered when interpreting the biopsy samples. For the cases with interpretable immunohistochemistry, 14/49 (28.6%) of diagnostic biopsies and 3/15 (20.0%) of metastatic biopsies had prior decalcification (Supplemental Table SI; http://dx.doi.org/14-02-1436-OA.Sl). Because there was P-hCG positivity in 7 of 17 decalcified samples (Supple mental Table SI; http://dx.doi.org/14-02-1436-OA.Sl), it was concluded that decalcification did not lead to total loss of P-hCG antigenicity or immunoreactivity. We next asked whether patients whose osteosarco mas showed P-hCG expression by immunohistochemistry had clinically detectable levels of P-hCG in the urine or serum. At our institution, before invasive testing or imaging, a routine screening urine pregnancy test and/or a serum P-hCG test are administered to females of reproductive age. In contrast, male patients undergoing osteosarcoma workup are not screened routinely for PhCG. In our series, 24/26 females underwent P-hCG screening, 23 of whom tested negative.
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Figure 1. Sparse expression o f p-hCG in h ig h -g ra d e osteosarcoma. A. H igh -g ra de c o n ve n tio n a l osteosarcoma. H em atoxylin and eosin, o rig in a l m a g n ific a tio n x 2 0 . B. Same case as A, s h o w in g sparse c y to p la s m ic p-hCG p o s itiv ity by im m u n o h isto ch e m istry (in v o lv in g 1% o r fe w e r tu m o r cells). C,D. T w o d iffe re n t cases o f h ig h -g ra d e co n ve n tio n a l osteosarcoma, also sh ow ing sparse p-hCG p o s itivity. A ll im m u n o h isto ch e m istry images, x 4 0 o rig in a l m a g n ific a tio n . A co lo r version o f th is Figure is availab le o nline .
One patient in the series had a positive P-hCG screen. This female patient was 14 years old when she presented with a distal femur mass. Her routine screening urine pregnancy test, performed before invasive investigations, was positive. However, intrauterine or ectopic pregnancy was ruled out by imaging studies and history. She then underwent a diagnostic biopsy of the distal femur mass, which showed epithelioid osteosarcoma with frequent (1hCG immunohistochemical staining (Fig. 2A-C). Before commencement of neoadjuvant chemotherapy, her quan titative serum P-hCG test peaked at 88.2 mlU/mL (negative, less than 5 mlU/mL). Following one cycle of chemotherapy, the serum P-hCG decreased to 1.2 mlU/mL, and following resection her serum P-hCG was virtually undetectable (Fig. 3). Although the pretreatment biopsy showed malignant epithelioid cells with no definitive osteoid matrix production, the post-treatment resection specimen showed abundant osteoid matrix production diagnostic of osteosarcoma, with widespread tumor cell necrosis, and a prominent acute inflammatory
cell infiltrate (Fig. 2D). Immunohistochemistry for PhCG was attempted on the resection specimen, but due to a paucity of viable tumor cells, the staining was not interpretable. There was no evidence of recurrent or metastatic disease at 1 year of follow-up.
C lin ic a l c h a ra c te ris tic s o f |5-hCG e x p re s s in g a n d p-h C G n o n e x p r e s s in g o s te o s a rc o m a s a re s im ila r
To determine whether immunohistochemical expression of P-hCG is associated with any prognostic clinical characteristics, the immunohistochemistry staining results were separated into P-hCG-positive and P-hCG—negative groups. No significant difference between the two groups was observed with respect to the number of deaths (P = 0.15), tumor size post resection (P = 0.06), and response to neoadjuvant therapy (P = 0.79) in each group (Table 2). Because tumoral hCG production has been associated with worse prognosis in some studies [17-22], we hypothesized that primary osteosarcomas with immunohCG in
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Figure 3. Serial se ru m (3-hCG m e a s u re m e n ts o f th e p a tie n t w h o s e b io p s y s h o w e d fr e q u e n t p-hCG e xp re ssio n (b io p s y a n d re s e c tio n im a g e s s h o w n in F ig u re 2). T h e g ra p h d e m o n s tra te s m a rk e d decreases in s e ru m p-hCG a ft e r n e o a d ju v a n t c h e m o th e ra p y a n d d e fin itiv e re s e c tio n . T he da sh e d lin e re p re se n ts th e m in im u m le ve l f o r c lin ic a lly p o s itiv e re su lts (less th a n 5 m lU /m L ).
histochemical evidence of hCG production would be associated with more metastases than osteosarcomas that were negative. We found that there was no significant difference between the two groups with respect to the number of patients that had metastases (9 metastases in the (3-hCG-positive group versus 10 metastases in the PhCG-negative group, Table 2, P = 0.38). The frequency o f p-hCG positivity is not increased in m etastatic biopsies versus p re tre atm e n t biopsies
To determine whether there is a difference in P-hCG expression in pretreatment and metastatic biopsies, we obtained 19 biopsies of metastases (from 19 patients within our overall sample of 51 patients) and subjected them to P-hCG immunohistochemistry. Of the biopsies 15 had interpretable immunohistochemistry, with 5 biopsies (2 females, 3 males) showing sparse staining and 10 biopsies showing no staining. No frequent p-hCG staining was seen in any of the metastatic biopsies. The 6 patients with sparse-positive P-hCG on the pretreatment biopsy had 2 P-hCG-sparse-positive and 4 p-hCG-negative metastases. The 9 patients with negative P-hCG on the pretreatment biopsy had 3 P-hCG-sparse-positive and 6
Figure 2. F re q u e n t p-hCG expression in a case o f e p ith e lio id osteosarcom a. A. H e m a to xylin a nd eosin (H&E). B,C. T w o fie ld s d e m o n s tra tin g cytoplasm ic p o s itiv ity f o r p-hCG in a p p ro x i m a te ly 10% o f tu m o r cells. D. Resection specim en fr o m th e same p a tie n t, d e m o n s tra tin g a b u n d a n t o ste o id m a trix p ro d u c tio n a n d tu m o r cell necrosis w ith a p ro m in e n t acute in fla m m a to ry cell in filtra te . H&E, all im ages X 40 o rig in a l m a g n ific a tio n . A c o lo r version o f th is F igure is a va ila b le o n lin e .
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Table 2. Clinical and tumor characteristics of the 49 patients with evaluable pretreatment biopsies, separated by P-hCG immunohistochemistry status
N o f patients Age at initial diagnosis (years)3 Sex distribution
N o f patients with metastases N o f deaths Tumor size post-resection (cm)3 Response to neoadjuvant therapy (%)3
P-hCG IH C negative biopsy
p-hCG IH C positive biopsy
21 13.6 ± 3.0 11 females 10 males 10 6 12.6 ± 5.9 75.9 ± 25.6
28 13.5 ± 3.0 13 females 15 males 9 3 9.8 ± 4.2 77.9 ± 24.8
IHC indicates immunohistochemistry. aAverage ± standard deviation. All comparisons: P > 0.05.
(3-hCG-negative metastases. We found no statistically significant association between pretreatment biopsy [3hCG status and metastatic biopsy P-hCG status (Fisher’s exact test, P = 1.000). DISCUSSION To our knowledge, the present study is the largest series to date to examine the P-hCG immunohistochemical staining pattern in pediatric osteosarcomas, and was performed to elucidate the incidence of P-hCG expression in pretreatment tumors, and determine any relationship between tumoral P-hCG production and prognosis. In our series, frequent P-hCG expression by immunohistochem istry accompanied by detection of the protein in urine and serum was rarely identified, in 1/49 cases (2%). In contrast, we found that the majority of cases (27/49 cases, 55%) showed sparse expression, suggesting that immunohisto chemistry may be more sensitive at identifying tumoral (3hCG production than either serum or urine P-hCG tests. In our study, we did not find a relationship between immunohistochemical p-hCG expression in pretreatment biopsies and clinical prognostic factors, such as deaths, tumor size, and response to neoadjuvant treatment. Our findings contrasted with other published studies that show an association between increased P-hCG levels and worse prognosis [17-22], From our study, we were unable to draw conclusions regarding the relationship between elevated serum P-hCG levels and clinical outcomes, likely because our sample did not include enough cases demonstrating frequent P-hCG expression. However, multiple case reports demonstrate high serum P-hCG in osteosarcoma patients [9,10,12,13,24,25], so it may be of value to study a larger sample of osteosarcoma patients with clinically elevated P-hCG levels to confidently determine the relationship between serum P-hCG and clinical outcomes in osteosarcoma patients. Our findings differed from the conclusion of Masrouha and colleagues [11], which suggested that immunohistochemical expression of p-hCG in tumor cells was associated with suboptimal tumor response, metas tases, and/or recurrence. In that case series, the frequency
of P-hCG-positive staining (5/32 patients) was less than encountered in our study (28/49 patients). It may be difficult to compare our raw data directly to that reported by Masrouha and colleagues [11] as they do not clearly outline their criteria for calling a biopsy positive for |3hCG immunostaining. They reported weak versus strong staining intensity, which may be subjective. Our study used a different antibody against [3-hCG (polyclonal rabbit) than Masrouha and colleagues [11] (monoclonal mouse), and these antibodies may have different perfor mance characteristics, like sensitivity, specificity, and staining intensity. We chose to use a semi-quantitative system and defined 1% of staining as positive. Since this is a low threshold, it is possible that we called more biopsies positive than Masrouha and colleagues [11], However, it is clear from one of their illustrative cases that they considered 1% of positive tumor cells as positive. This is what we defined in our study as “ sparse” staining, the most common staining pattern we observed. It should be noted that a large proportion of cases in the study of Masrouha and colleagues [11] consisted of resection specimens. When we attempted to perform [3hCG immunohistochemistry on resection specimens, we found the results difficult to interpret due the low density of viable tumor cells and confounding hemosiderin deposits. Therefore, we did not include resection specimens in our study. Our cases, which comprised a more homogeneous population of pediatric pretreatment biopsies, and demonstrated a low level of artifact, may not be directly comparable to the more heterogeneous mix of pretreatment biopsies and resections from a wide age range, which was studied by Masrouha and colleagues [11]. Furthermore, because we evaluated P-hCG expres sion on biopsy material (not resections), the material may not have been representative of the entire tumor and it is possible that we underestimated the overall frequency of P-hCG immunohistochemical positivity in osteosarcoma patients. Another potential explanation for underestima tion of P-hCG immunohistochemical positivity may be decalcification. Approximately 1/3 of our biopsies (14/49 pretreatment biopsies and 3/15 metastatic biopsies) underwent decalcification (Supplemental Table SI). We h CG in
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found that our decalcification protocol did not lead to a total loss of antigenicity or immunoreactivity, as demonstrated by the observation of (i-hCG positivity in 7/17 decalcified samples. The resection specimens, which often contained cortical bone and, therefore, were decalcified longer than the biopsies, had uninterpretable staining. Therefore, we cannot dismiss the effects of decalcification on the pretreatment and metastatic biopsies. Based on the findings of previously published studies suggesting a worse outcome for P-hCG expressing tumors [17-22], we hypothesized that metastases would be more common in patients whose primary osteosarcomas expressed (3-hCG. Our results showed no relationship between expression of P-hCG in pretreatment biopsies and the number of patients in whom metastases developed. We also saw no statistical difference between P-hCG expression frequency in pretreatment and meta static biopsies. These results suggested that immunohistochemical evidence of P-hCG production does not predict the biological behavior of osteosarcoma, including the likelihood for metastasis. Taking this notion a step further, our results did not point toward a pathogenic role for P-hCG in osteosarcomas; the expression of this protein may be an epiphenomenon. The possibility remains that we did not detect differences because of a limited follow-up period, during which only a small number of metastases developed, leading to reduced sample size for metastases. It could be argued that the sparse staining was nonspecific staining unrelated to |3hCG protein production. However, in these cases, the background was clean, and the positive-staining cells were histologically similar to the other adjacent tumor cells. Future studies focusing on osteosarcoma patients who have clinically elevated levels of serum P-hCG may be useful in revealing a threshold of P-hCG production above which it may affect the tumor’s biological behavior. To enrich for this population, it may be necessary to evaluate serum hCG levels of male and female osteosarcoma patients. Osteosarcoma with positive P-hCG immunohistochemistry is a rare, but well reported phenomenon in the literature. These reports [9,10,12,13,24—26] have been well-summarized by Masrouha and colleagues [11], and we refer the reader to this manuscript for further details. The meta-analysis by Masrouha and colleagues [11] revealed 13 cases positive for P-hCG expression of a total of 54 cases reported. Adding our pretreatment biopsy results to these totals, there now are 41 cases positive for P-hCG expression of a total of 103 reported. Production of P-hCG by tumor cells unrelated in lineage to trophoblast (“ ectopic” production) is well documented in multiple tumor types [6,7]. This present study confirmed the frequent occurrence of this phenom enon in osteosarcoma. Increased [3-hCG may cause amenorrhea, which could lead to the initial erroneous diagnosis of pregnancy or gestational trophoblastic disease, before subsequent investigations reveal that the etiology is a malignant tumor. This scenario has been 28 4
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reported in a case of synovial sarcoma [14], Other (female) patients may initially present with a malignant neoplasm, but are found only to have elevated (3-hCG when screened for pregnancy before invasive testing, which is the situation that happened to the patient described in this present report. Either scenario presents a major clinical pitfall (a false diagnosis of pregnancy), which could result in delay of appropriate treatment. Moreover, a positive urine or serum P-hCG test may influence the pathologic interpretation of the subsequent diagnostic biopsy, and could lead to confusion with other P-hCG positive nongerm cell tumors or tumors that contain scattered syncytiotrophoblasts, including chorio carcinoma, trophoblastic tumors, dysgerminoma/seminoma, and yolk sac tumor. Recognition that numerous types of malignancies of nongerm cell origin (ie, carcinomas and sarcomas) may infrequently express P-hCG can help in making the correct diagnosis, clinically and pathologically. The identification of novel classes of treatments for malignancies is aided, in part, by making new observa tions about tumor behavior and the microenvironment that affects it. For example, data from animal studies and in vitro experiments suggest that hCG produced by tumors may be involved in angiogenesis, metastasis, and immune tolerance of malignant neoplasms [6,7,27]. From these findings came the hypothesis that using vaccines against hCG may have antineoplastic effects. A number of such vaccines currently are under development, with some having undergone Phase 1 and II trials [28-32]. It is important to note that the target patients of these vaccines all have been those diagnosed with epithelial cancers. Before considering similar therapies in patients with osteosarcoma, it is important to elucidate the function, if any, of ectopic P-hCG production in osteosarcoma. Nevertheless, the possibility of new treatment modalities for a subset of osteosarcoma patients is promising, since the survival rate has plateaued in recent years. In summary, we find that significant positivity for (3hCG is an infrequent immunohistochemical finding in pediatric osteosarcoma biopsies. Nonetheless, awareness of this fact may help avoid diagnostic pitfalls. In contrast to previous studies on the topic, our data suggested that there is no relationship between low-level immunohisto chemical demonstration of [3-hCG expression in osteo sarcoma biopsies, various clinical prognostic parameters, or the development of metastases. Future studies focusing on osteosarcoma patients who have clinically elevated levels of serum [3-hCG may be useful in revealing a threshold of P-hCG production above which it may affect the tumor’s biological behavior. REFERENCES 1. Fletcher CDM, World Health Organization, International Agency for Research on Cancer. WHO Classification of Tumours of Soft Tissue and Bone. Lyon: 1ARC Press, 2013. 2. Fuchs N, Bielack SS, Epler D, et al. Long-term results of the co-operative German-Austrian-Swiss osteosarcoma study group’s protocol COSS-86 of intensive multidrug chemotherapy and surgery for osteosarcoma of the limbs. Amt Oncol 1998;9:893-899.
3. Federman N, Bernthal N, Eilber FC, et al. The multidisciplinary management of osteosarcoma. Curr Treat Options Oncol 2009; 10: 82-93. 4. LeMaire WJ, Conly PW, Moffett A, et al. Function of the human corpus luteum during the puerperium: its maintenance by exogenous human chorionic gonadotropin. Am J Obstet Gynecol 1971; 110: 612-618. 5. Midgley AR Jr, Pierce GB Jr. Immunohistochemical localization of human chorionic gonadotropin. J Exp Med 1962;115:289-294. 6. lies RK. Ectopic hCGbeta expression by epithelial cancer: malignant behaviour, metastasis and inhibition of tumor cell apoptosis. Mol Cell Endocrinol 2007:260-262:264—270. 7. lies RK, Delves PJ, Butler SA. Does hCG or hCGbeta play a role in cancer cell biology? Mol Cell Endocrinol 2010;329:62-70. 8. Boss DS, Glen FI, Beijnen JH, et al. Serum beta-FICG and CA-125 as tumor markers in a patient with osteosarcoma: case report. Tumori 2011;97:109-114. 9. Demirtas E, Krishnamurthy S, Tulandi T. Elevated serum betahuman chorionic gonadotropin in nonpregnant conditions. Obstet Gynecol Surv 2007;62:675-679, quiz 691. 10. Leidinger B, Bielack S, Koehler G, et al. High level of beta-hCG simulating pregnancy in recurrent osteosarcoma: case report and review of literature. J Cancer Res Clin Oncol 2004;130:357-361. 11. Masrouha KZ, Khattab R, Tawil A, et al. A preliminary investigation of Beta-hCG expression in patients with osteosarcoma. J Bone Joint Surg Br 2012;94:419^124. 12. Ordonez NG, Ayala AG, Raymond AK, et al. Ectopic production of the beta-subunit of human chorionic gonadotropin in osteosarcoma. Arch Pathol Lab Med 1989;113:416^119. 13. Tuy BE, Obafemi AA, Beebe KS, et al. Case report: elevated serum beta human chorionic gonadotropin in a woman with osteosarcoma. Clin Orthop Relat Res 2008;466:997-1001. 14. Stevens EE, Aquino J, Barrow N, et al. Ectopic production of human chorionic gonadotropin by synovial sarcoma of the hip. Obstet Gynecol 2013; 121:468^471. 15. Inoue N, Watanabe H, Takehara H, et al. Refractory pediatric nonrhabdomyosarcoma soft tissue sarcoma associated with ectopic production of beta hCG and hypercalcemia induced by PTHrP. Pediatr Blood Cancer 2011;57:1244-1246. 16. Bourdiec A, Shao R, Rao CV, et al. Human chorionic gonadotropin triggers angiogenesis via the modulation of endometrial stromal cell responsiveness to interleukin 1: a new possible mechanism underlying embryo implantation. Biol Reprod 2012;87:66. 17. Birgisson H, Jirstrom K, Stenman UH. Serum concentrations of human chorionic gonadotropin beta and its association with survival in patients with colorectal cancer. Cancer Biomark 2012; 11:173— 181. 18. Louhimo J, Carpelan-Holmstrom M, Alfthan H, et al. Serum HCG beta, CA 72-4 and CEA are independent prognostic factors in colorectal cancer. Int J Cancer 2002;101:545-548. 19. Lundin M, Nordling S, Lundin J, et al. Tissue expression of human chorionic gonadotropin beta predicts outcome in colorectal cancer:
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S ig n ific a n t Im m u n o h is to c h e m ic a l E xp ressio n o f H u m a n C h o rio n ic G o n a d o tr o p in in H ig h G ra d e O s te o s a rc o m a is R are, B u t M a y Be A s s o c ia te d w it h C lin ic a lly E le v a te d S e ru m Levels A n na F. L ee, 1,2 B ruce R. Paw el , 1 a n d L isa M . Su lliv a n 1* ’ Department of Anatomical Pathology, The Children's Hospital of Philadelphia, Philadelphia, PA, USA d e p a rtm e n t of Laboratory Medicine and Pathology, Children's and Women's Health Centre o f British Columbia, Vancouver, BC, Canada
Received February 4, 2014; accepted May 23, 2014; published online May 23, 2014.
ABSTRACT
Survival rates have plateaued at 70% for osteosarcoma. Proteins ectopically produced by malignant tumors may provide insight into new therapeutic targets. Osteosarco mas secreting human chorionic gonadotropin (hCG) have been suggested to have a worse prognosis. We examined the frequency of expression of P-subunit of hCG (P-hCG) in pretreatment osteosarcoma biopsies, and asked if it was associated with various clinical prognostic parameters, and the development of metastases. We subjected 51 pretreatment biopsies of high-grade osteosarcoma, from 51 patients, to P-hCG immunohistochemistry. In 19 of these patients, postchemotherapy metastatic biopsies also were examined for P-hCG expression. Clinical informa tion (patient age, sex, survival status, and serum hCG in females only), and tumor characteristics (site, size, and presence of metastases) were recorded. The p-hCG positive and negative biopsies were separated and compared. Of 49 interpretable pretreatment biopsies, 28 (57%) showed positive cytoplasmic P-hCG expression: 27 with sparse positivity (1% of tumor cells) and 1 with frequent positivity (10% of tumor cells). The patient with frequent P-hCG positivity in her pretreatment biopsy had elevated serum hCG (88.2 mlU/mL) at diagnosis, decreasing to undetectable following chemotherapy and definitive resection. There was no difference in clinical parameters or rate of metastasis between P-hCG positive versus negative groups. Expression of P-hCG may be seen in high-grade osteosarcoma, but frequent P-hCG immu nohistochemical expression by tumor cells, associated with clinically elevated serum P-hCG, is rare. Recogni tion that some nongenn cell tumors may produce P-hCG *Corresponding author, e-mail: [email protected]
can prevent confusion with malignancies containing neoplastic syncytiotrophoblast cells, including germ cell and trophoblastic tumors. Key words: ectopic, human chorionic gonadotropin,
metastasis, osteosarcoma, paraneoplastic, primary
INTRODUCTION
Osteosarcoma, a malignant neoplasm of bone-forming cells, is the most common primary sarcoma of bone [1]. Biologic behavior is predicted in part by histologic tumor grade, stratified into low and high grade. The treatment of choice for low-grade osteosarcoma is surgical resection. High-grade osteosarcomas, in contrast, usually require neoadjuvant chemotherapy followed by surgical resec tion. In addition, postresection chemotherapy also may be offered based on specific histologic findings in the resection specimen. Before the institution of systemic combination chemotherapy regimens starting in the late 1980s [2], survival was less than 20%, but the survival rate has risen to just over 70% for nonmetastatic patients [3], No new treatment strategies have substantially improved the survival over this figure. New therapeutic targets may be identified by studying ectopic production of proteins by malignant cells that could confer a survival advantage. Thus, characterization of substances not produced by normal host cells may prove helpful for the diagnosis or treatment of osteosarcoma, as well as determination of prognosis. One such substance is human chorionic gonadotropin (hCG), a heterodimeric protein composed of noncovalently-linked a and P subunits, which normally is secreted by placental syncytiotrophoblast and is required for
maintenance of the corpus luteum in early pregnancy [4,5]. Human chorionic gonadotropin may be found in neoplasms, secreted by the neoplastic counterpart of the syncytiotrophoblast cell. Such neoplasms include gesta tional trophoblastic disease, choriocarcinoma, and other germ cell tumors that contain neoplastic syncytiotropho blast cells. Malignancies derived from tissues unrelated to placental tissue also may secrete hCG. For instance, ectopic expression of hCG, has been observed in a wide variety of epithelial cancers, including those of the bladder, lung, breast, colon, and head/neck [6,7]. Case reports of sarcomas, including osteosarcoma [8-13], synovial sarcoma [14], and rhabdomyosarcoma [15], also have described hCG expression. Tumoral production of hCG may be clinically silent, or it may manifest as a paraneoplastic syndrome, such as amenorrhea in repro ductive age females. To date, the function of hCG in tumor biology is unclear and its role as a prognostic factor varies depending on the tumor. Human chorionic gonadotropin shows regions of homology with the tumor growth factor (TGF) superfamily of proteins, leading to speculation that hCG may crosstalk with the TGF signaling pathway, which is prosurvival in some conditions [6,7]. Another potential effect of tumoral hCG production is angiogen esis; recent in vitro experiments suggest that P-hCG may promote or enhance this phenomenon, which is necessary for tumor invasion and metastasis [16]. Some reports suggest that elevated serum and/or tissue P-hCG is an independent prognostic factor for adverse outcome in multiple tumor types [17-21], although one study on patients with colon carcinoma found this only to be the case for females [22]. However, negative studies also exist, in which no relationship between tissue P-hCG and survival was found [23]. To explore the role of hCG in osteosarcoma, Masrouha and colleagues [11] recently examined the immunohistochemical expression of the P-subunit of human chorionic gonadotropin (P-hCG) in a series of 32 osteosarcoma pretreatment biopsies and definitive resections. They reported P-hCG positivity in 5 cases. All 5 patients had suboptimal response to treatment (which was defined as greater than 10% viable tumor cells in the postchemotherapy specimen), and 4/5 developed metastases. The sample size was too small to draw definitive conclusions, but the investigators suggested that a potential link between tumor-associated hCG and poor outcome might be demonstrated by a larger study [11], In this present study, the relationship between P-hCG immunohistochemistry staining and urine/serum P-hCG was explored in pretreatment osteosarcoma biopsies and postchemotherapy metastatic lesions. To detennine whether the degree of P-hCG expression may be related to tumor aggressiveness and metastatic potential, P-hCG immunohistochemistry was compared between primary tumors and metastatic foci.
METHODS Ethics approval, case selection, and acquisition o f clinical in fo rm atio n
Approval to conduct this retrospective study was granted by the Institutional Review Board of the Children’s Hospital of Philadelphia (CHOP, protocol #12-009417). The pathology files at CHOP were searched for diagnostic biopsies, postneoadjuvant therapy resections, and biopsies of metastatic osteosarcoma (and malignant bone tumors consistent with osteosarcoma), accessioned between 2006 and 2012. We retrieved 51 diagnostic biopsies (taken before giving chemotherapy) from 51 patients, all showing high-grade osteosarcoma. All 51 patients received neoad juvant chemotherapy before definitive resection. Of these patients 19 suffered metastatic disease, which was surgi cally biopsied or sampled at autopsy. The retrieved cases were reviewed by two authors (A.F.L. and L.M.S.) for confirmation of the diagnosis. Data on age, sex, deaths, primary tumor site, primary tumor size, histologic response to neoadjuvant treatment at resection, and metastases (if any), were collected from pathology reports and the electronic health record. Information on whether the metastatic lesions were synchronous with the primary tumor, or developed subsequently, also was collected. Urine and/or serum [3-hCG levels were recorded where available. Only female patients were tested for phCG. The end of the follow-up period was April 2013. Preparation o f fo rm alin -fixed , p a raffin -em b ed d ed tissues
Fresh biopsy tissues were fixed in formalin for a minimum of 2 hours to overnight depending on the size of the biopsy. If required, formalin-fixed tissues were decalcified with Decalcifying Solution B, a hydrochloric acid-based decalcification solution (catalog #245-683; Thermo Fisher Scientific, Waltham, MA, USA), until soft enough to cut. Tissues then were dehydrated and embedded in paraffin wax as per standard operating procedure. Sections (4 p) were cut and mounted onto glass slides for routine hematoxylin and eosin (H&E) staining or for immunohistochemistry. Im m unohistochem istry and scoring o f staining
A representative block from each case with the largest amount of viable tumor was selected for immunohisto chemical staining. On the Leica Bond-Ill automated immunostaining machine (Leica Biosystems, Buffalo Grove, IL, USA), the sections were subjected to epitope retrieval (ER1-20 epitope retrieval solution; Leica Biosystems), and then incubated with 1:5000 dilution of rabbit polyclonal antibody directed against the P subunit of hCG (catalog #A0231; Dako, Carpinteria, CA, USA). Staining was visualized with a polymer-based secondary antibody. Placenta served as external positive control. Three immunohistochemistry staining patterns were observed: sparse positivity (approximately 1% of tumor hCG in H igh-G rade O steosarcoma
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Table 1.
Clinicopathologic characteristics of the study population separated by females and males
N o f patients N o f evaluable pretreatment biopsies N o f metastases N o f deaths Age (years)a Tumor size post resection (cm)» Response to neoadjuvant therapy (%)a
O verall
Fem ales
M ales
51 49 19 10 13.7 ± 3.0 10.8 ± 5.0 79.8 ± 21.8
26 24 7 6 13.7 ± 3.1 9.8 ± 4.1 75.6 ± 22.4
25 25 12 4 13.7 ± 3.0 11.6 ± 5.7 81.7 ± 21.9
aAverage ± SD. All comparisons, P > 0.05.
cells showing cytoplasmic [3-hCG positivity), frequent positivity (approximately 10% of tumor cells showing cytoplasmic P-hCG positivity), and negative staining. Two diagnostic biopsies, in which excessive hemosiderin confounded IHC interpretation, were excluded from scoring. Three metastatic tumor samples had too few tumor cells, either due to small sample size or block exhaustion, for meaningful assessment. One metastatic tumor sample was not available for staining. In total, 49/ 51 diagnostic biopsies and 15/19 biopsies of metastases had interpretable immunohistochemistry. Nondecalcified tissues were preferentially selected for immunohisto chemistry, but decalcified tissues were used if no other choice was available.
ostesoarcomas were captured in our retrospective study. The histologic subtypes included 44 conventional, 3 epithelioid, 1 periosteal, 1 periosteal chondroblastic, 1 small cell, and 1 telangiectatic. For the 19 patients with metastases, 8 patients had synchronous metastatic disease at the time of initial diagnostic biopsy (defined as metastatic disease clearly visible on imaging studies at the time of initial diagnostic biopsy). Ten patients had nonsynchronous metastases. Information was not available for one patient. Focal im m unohistochem ical p-hCG expression m ay be seen in high-grade osteosarcoma, bu t clinically elevated serum levels are rare
Statistical analysis
The 2-tailed Student’s /-test was performed to evaluate any difference in mean clinical parameters between groups. The 2-tailed Fisher’s exact test was performed to examine the significance of the association between two groups. Significance level (a) was set at 0.05. RESULTS Clinical characteristics o f th e study population
Table 1 shows the major clinical characteristics and demographics of the study population, and Supplemental Table SI (http://dx.doi.org/14-02-1436-OA.Sl) is an expanded table showing clinical characteristics as well as staining results obtained in all of the pretreatment biopsies and biopsies of metastases. The study population consisted of 51 patients (26 females and 25 males), with an average age of 13.7 ± 3.0 years (range, 4.5-18.9 years). No cases of secondary osteosarcoma were identified. There was no significant difference in sex distribution with respect to number of metastases (P = 0.24), number of deaths (P = 0.72), patient age (P = 0.71), tumor size (P = 0.34), and response to neoadjuvant therapy {P = 0.39) in these two groups. Consistent with previous large-scale epidemiologic studies of osteosarcoma [1], the most common tumor sites in our study population involved long bones of the limbs, with femur, tibia, and humerus being the most frequently affected. Less common sites involved fibula, sacrum, scapula, and sphenoid. All of the biopsies showed highgrade osteosarcoma; no low or intermediate grade
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Of the 49 interpretable biopsies of pretreatment osteosar comas, 28 (57%) showed some degree of cytoplasmic staining of (3-hCG in tumor cells (13 females and 15 males, see Supplemental Table SI; http://dx.doi.org/14-02-1436OA.S1). O f the 28 positive biopsies 27 showed only sparse p-hCG staining, involving approximately 1% of tumor cells, identified either as scattered single cells or small clusters (examples shown in Fig. 1). One biopsy of epithelioid osteosarcoma showed frequent p-hCG staining, which involved approximately 10% of tumor cells (Fig. 2A-C). The effects of decalcification were consid ered when interpreting the biopsy samples. For the cases with interpretable immunohistochemistry, 14/49 (28.6%) of diagnostic biopsies and 3/15 (20.0%) of metastatic biopsies had prior decalcification (Supplemental Table SI; http://dx.doi.org/14-02-1436-OA.Sl). Because there was P-hCG positivity in 7 of 17 decalcified samples (Supple mental Table SI; http://dx.doi.org/14-02-1436-OA.Sl), it was concluded that decalcification did not lead to total loss of P-hCG antigenicity or immunoreactivity. We next asked whether patients whose osteosarco mas showed P-hCG expression by immunohistochemistry had clinically detectable levels of P-hCG in the urine or serum. At our institution, before invasive testing or imaging, a routine screening urine pregnancy test and/or a serum P-hCG test are administered to females of reproductive age. In contrast, male patients undergoing osteosarcoma workup are not screened routinely for PhCG. In our series, 24/26 females underwent P-hCG screening, 23 of whom tested negative.
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Figure 1. Sparse expression o f p-hCG in h ig h -g ra d e osteosarcoma. A. H igh -g ra de c o n ve n tio n a l osteosarcoma. H em atoxylin and eosin, o rig in a l m a g n ific a tio n x 2 0 . B. Same case as A, s h o w in g sparse c y to p la s m ic p-hCG p o s itiv ity by im m u n o h isto ch e m istry (in v o lv in g 1% o r fe w e r tu m o r cells). C,D. T w o d iffe re n t cases o f h ig h -g ra d e co n ve n tio n a l osteosarcoma, also sh ow ing sparse p-hCG p o s itivity. A ll im m u n o h isto ch e m istry images, x 4 0 o rig in a l m a g n ific a tio n . A co lo r version o f th is Figure is availab le o nline .
One patient in the series had a positive P-hCG screen. This female patient was 14 years old when she presented with a distal femur mass. Her routine screening urine pregnancy test, performed before invasive investigations, was positive. However, intrauterine or ectopic pregnancy was ruled out by imaging studies and history. She then underwent a diagnostic biopsy of the distal femur mass, which showed epithelioid osteosarcoma with frequent (1hCG immunohistochemical staining (Fig. 2A-C). Before commencement of neoadjuvant chemotherapy, her quan titative serum P-hCG test peaked at 88.2 mlU/mL (negative, less than 5 mlU/mL). Following one cycle of chemotherapy, the serum P-hCG decreased to 1.2 mlU/mL, and following resection her serum P-hCG was virtually undetectable (Fig. 3). Although the pretreatment biopsy showed malignant epithelioid cells with no definitive osteoid matrix production, the post-treatment resection specimen showed abundant osteoid matrix production diagnostic of osteosarcoma, with widespread tumor cell necrosis, and a prominent acute inflammatory
cell infiltrate (Fig. 2D). Immunohistochemistry for PhCG was attempted on the resection specimen, but due to a paucity of viable tumor cells, the staining was not interpretable. There was no evidence of recurrent or metastatic disease at 1 year of follow-up.
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To determine whether immunohistochemical expression of P-hCG is associated with any prognostic clinical characteristics, the immunohistochemistry staining results were separated into P-hCG-positive and P-hCG—negative groups. No significant difference between the two groups was observed with respect to the number of deaths (P = 0.15), tumor size post resection (P = 0.06), and response to neoadjuvant therapy (P = 0.79) in each group (Table 2). Because tumoral hCG production has been associated with worse prognosis in some studies [17-22], we hypothesized that primary osteosarcomas with immunohCG in
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Figure 3. Serial se ru m (3-hCG m e a s u re m e n ts o f th e p a tie n t w h o s e b io p s y s h o w e d fr e q u e n t p-hCG e xp re ssio n (b io p s y a n d re s e c tio n im a g e s s h o w n in F ig u re 2). T h e g ra p h d e m o n s tra te s m a rk e d decreases in s e ru m p-hCG a ft e r n e o a d ju v a n t c h e m o th e ra p y a n d d e fin itiv e re s e c tio n . T he da sh e d lin e re p re se n ts th e m in im u m le ve l f o r c lin ic a lly p o s itiv e re su lts (less th a n 5 m lU /m L ).
histochemical evidence of hCG production would be associated with more metastases than osteosarcomas that were negative. We found that there was no significant difference between the two groups with respect to the number of patients that had metastases (9 metastases in the (3-hCG-positive group versus 10 metastases in the PhCG-negative group, Table 2, P = 0.38). The frequency o f p-hCG positivity is not increased in m etastatic biopsies versus p re tre atm e n t biopsies
To determine whether there is a difference in P-hCG expression in pretreatment and metastatic biopsies, we obtained 19 biopsies of metastases (from 19 patients within our overall sample of 51 patients) and subjected them to P-hCG immunohistochemistry. Of the biopsies 15 had interpretable immunohistochemistry, with 5 biopsies (2 females, 3 males) showing sparse staining and 10 biopsies showing no staining. No frequent p-hCG staining was seen in any of the metastatic biopsies. The 6 patients with sparse-positive P-hCG on the pretreatment biopsy had 2 P-hCG-sparse-positive and 4 p-hCG-negative metastases. The 9 patients with negative P-hCG on the pretreatment biopsy had 3 P-hCG-sparse-positive and 6
Figure 2. F re q u e n t p-hCG expression in a case o f e p ith e lio id osteosarcom a. A. H e m a to xylin a nd eosin (H&E). B,C. T w o fie ld s d e m o n s tra tin g cytoplasm ic p o s itiv ity f o r p-hCG in a p p ro x i m a te ly 10% o f tu m o r cells. D. Resection specim en fr o m th e same p a tie n t, d e m o n s tra tin g a b u n d a n t o ste o id m a trix p ro d u c tio n a n d tu m o r cell necrosis w ith a p ro m in e n t acute in fla m m a to ry cell in filtra te . H&E, all im ages X 40 o rig in a l m a g n ific a tio n . A c o lo r version o f th is F igure is a va ila b le o n lin e .
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Table 2. Clinical and tumor characteristics of the 49 patients with evaluable pretreatment biopsies, separated by P-hCG immunohistochemistry status
N o f patients Age at initial diagnosis (years)3 Sex distribution
N o f patients with metastases N o f deaths Tumor size post-resection (cm)3 Response to neoadjuvant therapy (%)3
P-hCG IH C negative biopsy
p-hCG IH C positive biopsy
21 13.6 ± 3.0 11 females 10 males 10 6 12.6 ± 5.9 75.9 ± 25.6
28 13.5 ± 3.0 13 females 15 males 9 3 9.8 ± 4.2 77.9 ± 24.8
IHC indicates immunohistochemistry. aAverage ± standard deviation. All comparisons: P > 0.05.
(3-hCG-negative metastases. We found no statistically significant association between pretreatment biopsy [3hCG status and metastatic biopsy P-hCG status (Fisher’s exact test, P = 1.000). DISCUSSION To our knowledge, the present study is the largest series to date to examine the P-hCG immunohistochemical staining pattern in pediatric osteosarcomas, and was performed to elucidate the incidence of P-hCG expression in pretreatment tumors, and determine any relationship between tumoral P-hCG production and prognosis. In our series, frequent P-hCG expression by immunohistochem istry accompanied by detection of the protein in urine and serum was rarely identified, in 1/49 cases (2%). In contrast, we found that the majority of cases (27/49 cases, 55%) showed sparse expression, suggesting that immunohisto chemistry may be more sensitive at identifying tumoral (3hCG production than either serum or urine P-hCG tests. In our study, we did not find a relationship between immunohistochemical p-hCG expression in pretreatment biopsies and clinical prognostic factors, such as deaths, tumor size, and response to neoadjuvant treatment. Our findings contrasted with other published studies that show an association between increased P-hCG levels and worse prognosis [17-22], From our study, we were unable to draw conclusions regarding the relationship between elevated serum P-hCG levels and clinical outcomes, likely because our sample did not include enough cases demonstrating frequent P-hCG expression. However, multiple case reports demonstrate high serum P-hCG in osteosarcoma patients [9,10,12,13,24,25], so it may be of value to study a larger sample of osteosarcoma patients with clinically elevated P-hCG levels to confidently determine the relationship between serum P-hCG and clinical outcomes in osteosarcoma patients. Our findings differed from the conclusion of Masrouha and colleagues [11], which suggested that immunohistochemical expression of p-hCG in tumor cells was associated with suboptimal tumor response, metas tases, and/or recurrence. In that case series, the frequency
of P-hCG-positive staining (5/32 patients) was less than encountered in our study (28/49 patients). It may be difficult to compare our raw data directly to that reported by Masrouha and colleagues [11] as they do not clearly outline their criteria for calling a biopsy positive for |3hCG immunostaining. They reported weak versus strong staining intensity, which may be subjective. Our study used a different antibody against [3-hCG (polyclonal rabbit) than Masrouha and colleagues [11] (monoclonal mouse), and these antibodies may have different perfor mance characteristics, like sensitivity, specificity, and staining intensity. We chose to use a semi-quantitative system and defined 1% of staining as positive. Since this is a low threshold, it is possible that we called more biopsies positive than Masrouha and colleagues [11], However, it is clear from one of their illustrative cases that they considered 1% of positive tumor cells as positive. This is what we defined in our study as “ sparse” staining, the most common staining pattern we observed. It should be noted that a large proportion of cases in the study of Masrouha and colleagues [11] consisted of resection specimens. When we attempted to perform [3hCG immunohistochemistry on resection specimens, we found the results difficult to interpret due the low density of viable tumor cells and confounding hemosiderin deposits. Therefore, we did not include resection specimens in our study. Our cases, which comprised a more homogeneous population of pediatric pretreatment biopsies, and demonstrated a low level of artifact, may not be directly comparable to the more heterogeneous mix of pretreatment biopsies and resections from a wide age range, which was studied by Masrouha and colleagues [11]. Furthermore, because we evaluated P-hCG expres sion on biopsy material (not resections), the material may not have been representative of the entire tumor and it is possible that we underestimated the overall frequency of P-hCG immunohistochemical positivity in osteosarcoma patients. Another potential explanation for underestima tion of P-hCG immunohistochemical positivity may be decalcification. Approximately 1/3 of our biopsies (14/49 pretreatment biopsies and 3/15 metastatic biopsies) underwent decalcification (Supplemental Table SI). We h CG in
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found that our decalcification protocol did not lead to a total loss of antigenicity or immunoreactivity, as demonstrated by the observation of (i-hCG positivity in 7/17 decalcified samples. The resection specimens, which often contained cortical bone and, therefore, were decalcified longer than the biopsies, had uninterpretable staining. Therefore, we cannot dismiss the effects of decalcification on the pretreatment and metastatic biopsies. Based on the findings of previously published studies suggesting a worse outcome for P-hCG expressing tumors [17-22], we hypothesized that metastases would be more common in patients whose primary osteosarcomas expressed (3-hCG. Our results showed no relationship between expression of P-hCG in pretreatment biopsies and the number of patients in whom metastases developed. We also saw no statistical difference between P-hCG expression frequency in pretreatment and meta static biopsies. These results suggested that immunohistochemical evidence of P-hCG production does not predict the biological behavior of osteosarcoma, including the likelihood for metastasis. Taking this notion a step further, our results did not point toward a pathogenic role for P-hCG in osteosarcomas; the expression of this protein may be an epiphenomenon. The possibility remains that we did not detect differences because of a limited follow-up period, during which only a small number of metastases developed, leading to reduced sample size for metastases. It could be argued that the sparse staining was nonspecific staining unrelated to |3hCG protein production. However, in these cases, the background was clean, and the positive-staining cells were histologically similar to the other adjacent tumor cells. Future studies focusing on osteosarcoma patients who have clinically elevated levels of serum P-hCG may be useful in revealing a threshold of P-hCG production above which it may affect the tumor’s biological behavior. To enrich for this population, it may be necessary to evaluate serum hCG levels of male and female osteosarcoma patients. Osteosarcoma with positive P-hCG immunohistochemistry is a rare, but well reported phenomenon in the literature. These reports [9,10,12,13,24—26] have been well-summarized by Masrouha and colleagues [11], and we refer the reader to this manuscript for further details. The meta-analysis by Masrouha and colleagues [11] revealed 13 cases positive for P-hCG expression of a total of 54 cases reported. Adding our pretreatment biopsy results to these totals, there now are 41 cases positive for P-hCG expression of a total of 103 reported. Production of P-hCG by tumor cells unrelated in lineage to trophoblast (“ ectopic” production) is well documented in multiple tumor types [6,7]. This present study confirmed the frequent occurrence of this phenom enon in osteosarcoma. Increased [3-hCG may cause amenorrhea, which could lead to the initial erroneous diagnosis of pregnancy or gestational trophoblastic disease, before subsequent investigations reveal that the etiology is a malignant tumor. This scenario has been 28 4
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reported in a case of synovial sarcoma [14], Other (female) patients may initially present with a malignant neoplasm, but are found only to have elevated (3-hCG when screened for pregnancy before invasive testing, which is the situation that happened to the patient described in this present report. Either scenario presents a major clinical pitfall (a false diagnosis of pregnancy), which could result in delay of appropriate treatment. Moreover, a positive urine or serum P-hCG test may influence the pathologic interpretation of the subsequent diagnostic biopsy, and could lead to confusion with other P-hCG positive nongerm cell tumors or tumors that contain scattered syncytiotrophoblasts, including chorio carcinoma, trophoblastic tumors, dysgerminoma/seminoma, and yolk sac tumor. Recognition that numerous types of malignancies of nongerm cell origin (ie, carcinomas and sarcomas) may infrequently express P-hCG can help in making the correct diagnosis, clinically and pathologically. The identification of novel classes of treatments for malignancies is aided, in part, by making new observa tions about tumor behavior and the microenvironment that affects it. For example, data from animal studies and in vitro experiments suggest that hCG produced by tumors may be involved in angiogenesis, metastasis, and immune tolerance of malignant neoplasms [6,7,27]. From these findings came the hypothesis that using vaccines against hCG may have antineoplastic effects. A number of such vaccines currently are under development, with some having undergone Phase 1 and II trials [28-32]. It is important to note that the target patients of these vaccines all have been those diagnosed with epithelial cancers. Before considering similar therapies in patients with osteosarcoma, it is important to elucidate the function, if any, of ectopic P-hCG production in osteosarcoma. Nevertheless, the possibility of new treatment modalities for a subset of osteosarcoma patients is promising, since the survival rate has plateaued in recent years. In summary, we find that significant positivity for (3hCG is an infrequent immunohistochemical finding in pediatric osteosarcoma biopsies. Nonetheless, awareness of this fact may help avoid diagnostic pitfalls. In contrast to previous studies on the topic, our data suggested that there is no relationship between low-level immunohisto chemical demonstration of [3-hCG expression in osteo sarcoma biopsies, various clinical prognostic parameters, or the development of metastases. Future studies focusing on osteosarcoma patients who have clinically elevated levels of serum [3-hCG may be useful in revealing a threshold of P-hCG production above which it may affect the tumor’s biological behavior. REFERENCES 1. Fletcher CDM, World Health Organization, International Agency for Research on Cancer. WHO Classification of Tumours of Soft Tissue and Bone. Lyon: 1ARC Press, 2013. 2. Fuchs N, Bielack SS, Epler D, et al. Long-term results of the co-operative German-Austrian-Swiss osteosarcoma study group’s protocol COSS-86 of intensive multidrug chemotherapy and surgery for osteosarcoma of the limbs. Amt Oncol 1998;9:893-899.
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