Algorithm of diagnosis in bone metastasis of unknown primary origin – experience of a single clinical center over a five-year period

Introduction

Metastatic bone cancer (secondary bone cancer)*, is the term used to describe tumors that originate in other tissues and spread (metastasize) to the bone⁽¹⁾. Bone is the third most frequent site of metastasis, besides lung and liver⁽²⁾. Breast, prostate, lung, kidney, gastrointestinal tract and thyroid carcinomas are the most common tumors to develop bone metastasis⁽³⁾. Prostate and breast cancer are responsible for the majority of the skeletal metastases (up to 70%). The overall incidence of bone metastasis is not known⁽⁴⁾. We reviewed and evaluated the existing cases of assessed bone metastasis in a given period of time in the specialized Foişor Orthopedics-Traumatology and Osteoarticular TB Clinical Hospital, Bucharest, Romania.

Our objective was to analyze the histopathological features and, based on the already existing clinical and imaging data, to establish the minimal immunohistochemical panel necessary to rend a histopathological diagnosis compatible with the origin of the tumor on a small tissue sample of needle biopsy specimen.

Materials, method and acquired data

We reviewed the medical files of patients who were admitted and treated in our institution, and we selected the cases that presented bone metastases confirmed on the histopathological examination and completed with immunohistochemical necessary panel. This review outlines the experience of the Pathology Department of the Foişor Orthopedics-Traumatology and Osteoarticular TB Clinical Hospital, Bucharest, Romania. In a 5-year period, from January 2019 to December 2023, a number of 59 cases of bone metastases with unknown primary origin were recorded. In the total 59 cases of metastatic tumors involving the bone, the age of the patients ranged from 44 to 82 years old (mean age: 63 years old), with a majority of male patients – 23 female patients (39%) and 36 male patients (61%). The most affected skeletal sites were the femur and the pelvis. The distribution of the affected skeletal sites is summarized in Table 1.

In our statistics, the most common primary sites were the lung (25.4%), the gastrointestinal tract (22%), the breast (15.2%) and renal (10.2%). The rest of the primary sites were thyroid (1.7%), prostate (1.7%), gynecological (1.7%), skin (1.7%), urothelial (1.7%), rhinopharyngeal (1.7%) and hepatic (1.7%). Immunohistochemistry (IHC) confirmed the diagnosis in most cases, and the remaining cases had compatible clinical history supporting the diagnoses. There were some cases (11.9%) of poorly differentiated malignant neoplasms classified as metastatic carcinoma of unknown origin, and some (3.4%) with the diagnostic of mucinous type carcinoma with unknown origin. Furthermore, additional workup utilizing immunohistochemistry to classify the type of carcinoma in these cases were inconclusive. The distribution of primary sites of metastatic carcinomas is summarized in Table 2.

Method

Tumor tissues came from a needle biopsy of the bone lesion, collected in our clinic in accordance with the imaging data. The fresh material was then fixed for 1-3 days in neutral 10% buffered formalin and then processed for decalcification protocol if needed, followed by paraffin embedding procedures. Fixed specimens were decalcified when needed in 5% formic acid solution, usually for less than 24 hours. Slides obtained from the paraffin blocks, with 4-µm thick sections, were stained with Hematoxylin & Eosin (HE) after a standard procedure. In each case, the immunohistochemistry panel was established after histopathological aspects correlated with clinical and imaging data. The immunohistochemistry (IHC) tests were performed using LEICA BOND III automatic machine using Leica Bond antibodies for the following: CK7 (clone RN7), CK20 (clone Ks20.8), TTF1 (clone SPT24), Napsin A (clone IP64), CK8/18 (clone 5D3), Vimentin (clone V9), AMACR (clone EPMU1), PSA (clone 35H9), ER (clone 6F11), PR (clone 16), SOX10, Ki-67 (clone MM1), MCK (AE1/AE3), Mammaglobin (EP249), CK5 (XM26), S100 (EP32), Beta-catenin (17C2), p63 (7JUL). Some IHC were performed manually: Thyroglobulin (cocktail, mouse monoclonal 2H11 + 6E1), CDX2 (clone EP25), SATB2 (rabbit monoclonal ABCAM – EPNCIR130A), SOX10 (rabbit monoclonal ABCAM – SP267), CD10 (mouse monoclonal NOVOCASTRA – Cl.56C6), PAX8 (rabbit polyclonal – BIOCARE), Arginase1 (rabbit monoclonal BIOCARE – EPR667b).

Clinical characteristics and symptoms

The patients with bone metastasis usually have no specific symptoms. Patients with metastatic disease often present with progressive pain or other skeletal-related events (SRE). It is not uncommon for a patient to associate an unrelated or nonspecific low energy injury with SRE. Often, a patient is treated with pain medication, and the symptoms do not completely resolve. A detailed history and physical exam can identify the red flags, such as night pain, unintentional weight loss, or an enlarging mass in the area of concern⁽¹⁾. Most of our cases in the Foişor Clinical Hospital presented with weight loss, enlarging bone mass, or pathological bone fracture.

Radiological and imaging types of bone metastatic lesions

Usually, the imaging findings are not characteristics of a metastatic lesion in bones. These are mainly classified by the number, as unique or multiple, and by the type of interference with normal bone remodeling, as osteolytic, osteoblastic or mixed:

• Osteolytic – characterized by the destruction of normal bone, generally seen in renal cell carcinoma, breast carcinoma, melanoma, non-small cell lung carcinoma, thyroid carcinoma. These tumors are usually highly vascularized, and they have an important osteoclastic reactive component which facilitates extension of the tumor by bone resorption (Figure 1).

• Osteoblastic (or sclerotic) – characterized by deposi­tion of new bone, generally present in prostate cancer, carcinoid, or small cell lung cancer.
• Mixed – if a lesion presented in bones of a patient has both osteolytic and osteoblastic lesions. This type of bone metastasis may be present in breast carcinoma, gastrointestinal cancers, and squamous cell carcinomas. Although breast carcinoma gives origin predominantly to osteolytic lesions, 15-20% of women have osteoblastic lesions or a mixed type of lesion⁽⁴⁾.

Patients with bone metastases included in our study had an imaging workup (plain radiographs, CT scans, MRI scans, bone scans) before the biopsy was performed. The nature of the bone lesions after imaging and clinical criteria was suspected to be secondary, even though the primary site was unknown, except for the cases with multiple bone lesions which were more compatible with a metastatic origin.

Most of our cases presented with a pathological bone fracture or with unique osteolytic bone lesion without any other known tumor localization, a fact that was making it also a possible primary lesion.

Histopathological features
and characteristics. Primary site origin assessment on biopsy specimens using H&E and immunohistochemistry

The histologic diagnosis of bone metastasis on needle bone biopsy samples can be sometimes challenging on Hematoxylin & Eosin (HE) slides. The determination of the primary tumor is facilitated by ancillary techniques, such as histochemical staining (PAS, Alcian Blue) and immunohistochemistry; it is possible, in about 65% of cases, with clinical and imaging correlation. Additional immunohistochemistry or molecular analyses are becoming more useful in various tumors (breast, lung, melanoma etc.) for targeted therapy⁽⁵⁾.

To begin with the HE cytomorphological characteristics, they may vary a lot, depending on the primary cancer origine and the cancer type.

We present a general line of some histopathological characteristics, the ones that are useful to correlate with the cases from our study presented in this article. Meta­stasis in bone can be from epithelial origin (carcinoma) or nonepithelial origin. The carcinoma can have various grades of differentiation, depending on the resemblance of the tissue of origin, ranging from well differentiated to poorly differentiated. Some of the carcinomas’ meta­stases are so poorly differentiated that the histological features may take the aspects of a sarcoma. These cases benefit most from the immunohistochemical tests. In all cases, the IHC panel that we chose consisted of 4-8 antibodies depending on the histological features, clinical and imaging data. We summarize in Table 3 the IHC marker – primary cancer site correlation⁽⁶⁾. Some of these poorly differentiated carcinomas remain with unknown primary source even after effectuating IHC markers.

It is also very important to note that histopathological exams need to be correlated with the clinical and imaging data.

As we discussed before, we firstly analyzed the HE samples, and after that we proceeded to IHC markers to find the primary site of bone metastasis, taking into account the clinical and imaging data. Adenocarcinomas (ADK) constitute the predominant histological type among the metastatic bone carcinomas⁽⁷⁾. Similar was noted in the present study. We present some examples of cases in Figure 2.

After a thorough examination of the HE slides of each case, which confirmed the presence of a metastasis in the bone, we established a minimal IHC panel of markers in order to assess the origin of the tumor. We were guided by the histopathological aspects of the tumor on HE slides to evaluate the possible primary site source of the meta­stases, and we chose the corresponding IHC markers that would confirm or not our first probable diagnosis on the HE slides. If needed, we then proceed to do more IHC markers, depending on each case (Figure 3).

The distribution by origin, histologic pattern and IHC of metastatic carcinomas with unknown primary source in the Foişor Clinical Hospital, in a five-year period, is summarized in Table 4.

Discussion

The gastric carcinomas are most frequently adenocarcinomas (ADK). The WHO classification includes different subtypes of gastric ADK, like tubular, papillary and poorly cohesive carcinomas, including signet-ring cell carcinomas and other subtypes⁽⁸⁾. The Laurén classification separates gastric adenocarcinomas into two primary subtypes, intestinal and diffuse, and tumors exhibiting features of both the intestinal and diffuse types (>25% of either component) are designated as mixed-type adenocarcinoma. The intestinal type is characterized by the formation of glands exhibiting various degrees of differentiation, either with or without extracellular mucin production. The diffuse type is composed of poorly cohesive cells without gland formation. This type of tumor often may contain cells with or without intracytoplasmic mucin, known as “signet ring cells”⁽⁹⁾. In our clinic, most of the bone metastasis of gastric origin presented with pathological bone fracture or as osteolytic bone lesions. Most of bone metastasis from gastric carcinoma were adenocarcinomas and poorly differentiated carcinomas.

The colorectal carcinomas are in the majority of cases adenocarcinomas. Adenocarcinomas may be further differentiated histologically by the predominant cell morphology. Variants include cribriform comedo-type, medullary, micropapillary, serrated, mucinous, and signet-ring cells, which are usually replicated in the metastatic tumor. Adenocarcinomas are graded, by the degree of gland formation, into well, moderately, and poorly differentiated tumors. Undifferentiated tumors show no evidence of any discernible gland formation or squamous differentiation. Cytokeratin 20 (CK20) and caudal-type homeobox 2 (CDX2) immunohistochemistry can accurately identify cells of colonic origin⁽¹⁰⁾. In our clinic, these bone metastases presented as osteolytic, osteoblastic, or mixed bone lesions in radiological findings. SATB2, originally defined for the epithelial colonic origin, is a very reliable marker. It is also positive in osteoblasts which can be useful as positive tissue control in bone metastases (Figure 4).

The lung carcinomas are generally adenocarcinomas, squamous carcinomas and small cell carcinomas^(11,12). Lung adenocarcinomas can be of mucinous type, and the IHC panel of these type of carcinomas is the same as in gastrointestinal carcinomas. On the other hand, squamous carcinomas do not have an origin site specific panel. In our clinic, bone metastases of lung carcinoma were mostly adenocarcinoma type, but there were also cases of squamous carcinomas, small cell type carcinoma and mucinous carcinomas (Figures 5 and 6).

The small cell lung carcinoma presented as a pathological bone fracture (humeral). The HE slides showed bone tissue infiltrated by a tumoral proliferation consisting of small undifferentiated cells, disposed in discohesive groups. The IHC markers showed AE1/AE3 intense positivity in tumoral cells, CK7 positive in tumoral cells, CK20 negative in tumoral cells, Vimentin negative in tumoral cells, CD20 negative, CD45 negative, CD138 negative in tumoral cells, TTF1 intense nuclear positivity in tumoral cells, CD56 intense positive in tumoral cells, and Ki67 index positive 70% in tumoral cells. In poorly differentiated tumors, as the one presented before, the markers CD45 and CD138, both negative, excluded a lymphoid proliferation (CD45) and a plasmocytic myeloma (CD138). On the other hand, CD56 intense positivity correlates with a high proliferation index Ki67 (70%) to sustain the diagnostic of small cell neuroendocrine carcinoma.

Prostate carcinomas are the main adenocarcinomas (acinar/ductal)⁽¹³⁾. In our clinic, the case with prostatic bone metastasis presented as a pathological bone fracture (peritrochanteric left femoral fracture). The biopsy showed a paucicellular tumoral proliferation with a microacinar arrangement on the HE slides. IHC was very useful in this case, as it showed positivity for CK8/18, PSA and AMACR markers, which confirmed the prostatic origin of the metastasis (Figure 7).

Renal carcinomas. The most frequent histological subtypes of renal carcinomas include clear cell renal cell carcinomas, papillary renal cell carcinomas, and chromophobe renal cell carcinomas. These three subtypes together represent more than 90% of all renal cell carcinomas⁽¹⁴⁾. In our clinic, metastasis to the bone were primarily clear cell renal carcinomas. They presented as osteolytic lesions on bone radiography. In one case encountered on our clinic, the HE slides showed areas of bone infiltrated by a proliferation consisting of clear cells arranged in tubulo-papillary configurations with high-grade nuclei. The IHC test revealed AE1/AE3 positive in tumoral cells, CD10 positive cytoplasmatic, CK7 negative in tumoral cells, CK 8/18 positive in tumoral cells, AMACR positive in tumoral cells, Ki67 index 30%, and Vimentin positive in tumoral cells. This case was concluded as a renal cell carcinoma meta­stasis in bone. Taking into consideration the IHC profile with AMACR positive, CD10 positive and CK7 negative, the histological subtype of renal primary carcinoma would be a papillary renal cell carcinoma. The high proliferation index Ki67 in this case (more than 30%) correlates with the high-grade nuclei of the tumor (Figure 8).

Breast carcinomas. The two most prevalent histologic types of invasive breast carcinomas are ductal carcinoma and lobular carcinoma. Invasive ductal carcinoma constitutes around 55% of all breast cancers. Invasive lobular carcinoma is the second most common histological subtype of breast cancer, that accounts for 5-15%⁽¹⁵⁾ of cases. In our clinic, bone metastasis of breast carcinoma presented as pathological fractures (humerus) and osteolytic bone lesions in the femur and spine. All our cases of bone metastasis of breast carcinoma were positive for ER and PR markers and for the mammaglobin marker (Figure 9). The cases were sent for further molecular tests, useful for targeted therapy. Some of our cases of bone metastasis of breast carcinoma appeared many years after the primary tumor (10 to 15 years), and there was a suspicion of another primary tumor, other than breast. Nevertheless, it is well known that breast carcinoma can appear many years later after the primary tumor was surgically removed and treated. In these cases, the molecular profile of the metastatic tumor is necessary for the diagnosis, but also for the treatment of the new metastatic tumor.

Thyroid carcinomas are histopathologically classified as well-differentiated thyroid carcinomas, which include papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC), and Hürthle cell thyroid carcinomas, and the less differentiated forms – i.e., poorly differentiated thyroid carcinomas and anaplastic, or undifferentiated, thyroid carcinomas. PTC is the most common endocrine malignancy, accounting for approximately 85% of all follicular-derived thyroid cancers, while FTC occurs in less than 10% of all thyroid⁽¹⁶⁾. In our clinic, the case of thyroid carcinoma was of follicular type, and it presented as an osteolytic bone lesion in the right humerus, suspected of osteosarcoma. Anamnestic data showed thyroid ablation, but without known histopathological diagnosis. The HE slides revealed a bone infiltrating proliferation consisting of well-formed follicles resembling normal thyroid tissue (Figure 10). Taking into consideration the anamnestic data, we suggested the reevaluation of the thyroid ablation specimen.

Differential diagnosis of multiple osteolytic lesions – case presentation

One case that presented with multiple osteolytic lesions (in femoral bones, costal arches and pelvis) was suspec­ted as a secondary metastatic bone lesion on clinical and imagistic grounds (the patient had a history of prostatic enlargement). The HE slides showed areas of trabecular bone infiltrated by a proliferation consisting of sheets of plasma-like cells, all of these cells having a monotonous appearance. IHC test showed CD138 positivity in tumor cells, CD56 positivity in reactive osteoblasts, S-100 positive in isolated stromal cells, CD34 positive in vascular walls, CD68 positive in osteoclasts, and Ki67 index positive in approximately 40% of tumoral cells (Figure 11). Finally, after examining the resulting HE slides and performing IHC tests, the lesions revealed to be of multiple myeloma diagnosis, and the case was sent to a specialized hematopathological center to further refine the diagnostic.

Discussion on the limitations and benefits of histopathological diagnosis on biopsy samples

1. Needle biopsy: advantages and limitations

Several studies have shown that needle biopsies are useful methods for diagnosing bone metastatic tumors, since they are safe, accurate, minimally invasive, and have high diagnostic significance. Unfortunately, sample volumes obtained from needle biopsies are sometimes scant^(17,18). Likewise, some of the specific characteristics of the lesions, like extended areas of necrosis, can make the diagnosis on biopsy difficult. In fact, some proven basic strategies for a successful biopsy include establishing the apparently more aggressive portion of the lesion as the main target and avoiding areas of necrosis⁽¹⁸⁾. The remaining areas of true tumoral cells may be very limited, consequently making the precise diagnosis on HE slides and IHC markers challenging. Sometimes, the collected biopsies can incorporate normal tissue – for example, skin sweat glands artificially introduced by the transcutaneous needle biopsy technique. Careful examination and eventually IHC markers will rule out the metastatic nature of the lesion.

Other limitations of biopsy samples are the availability and disposal of IHC markers in the pathological laboratory of the hospital. Small biopsy samples can be representative if they incorporate sufficient tumoral material even for processing a sufficient number of IHC markers. On the other hand, generous biopsy samples sometimes may not contain the representative lesion of the bone or only small groups of tumor cells. This gives limited sections and choices of IHC markers. This is why it is important to guide the histopathological diagnosis from the HE examination and give valuable IHC markers that can help with the histopathological diagnosis, in the limits of the available paraffin block.

Generally speaking, needle biopsy is an effective me­thod, as it is minimal invasive, more comfortable for the patient, and can give precious information to the histopathologist, as it is examined, leading to a high probability diagnosis.

2. Cost-effective diagnosis

The IHC panel for the identification of the primary site and of the type of tumor on a biopsy material can be much wider.

IHC provides diagnostic guidance in approximately 90% of undifferentiated malignant tumors, but usually at the end of a fastidious and expensive algorithm based on both morphology and IHC. The expansion of diagnostic IHC is also associated with an increased cost and burden of work for laboratory staff⁽¹⁹⁾. However, the identification of the primary site of origin may represent a difficult challenge for the pathologist when dealing with a small sample size, along with increased generation of tumor-specific primary antibodies and the need for complementary molecular analysis.

No single antibody is fully sensitive and specific for a particular tumor; however, some antibodies are especially useful when used within small panels. However, in approximately 5% of cases, IHC may not provide any definitive information, and the final diagnosis of an undifferentiated malignant neoplasm is given without any reference to the tumor origin, despite all the investigations⁽²⁰⁾.

Conclusions

Needle biopsies are generally an effective method for the histopathological diagnosis of bone metastasis, but having its own limitations, such as the quality of the biopsy material and the limited availability of the paraffin block. A careful histopathological and cytopathological assessment in biopsy sample from metastasis to the bone is essential for an accurate diagnosis which is further elaborated by sending the cases to the oncology department for other investigations and appropriate treatment for each case in part. Immunohistochemical markers are needed and necessary most of the times in identifying the possible primary site in cases with unknown primaries, especially in biopsy samples of the bone lesion. Immunohistochemical markers must be chosen attentively and effectively in order to give a correct diagnosis, taking into consideration the capacity of the laboratory, their availability, the limited small biopsy sample, and the cost-effectiveness of the immunohistology examinations. Radiological and clinical information represents useful resources for the needed medical correlations and the final histopathological diagnosis.   

Note: All the images used and presented in this article belong to the database of the Foişor Orthopedics-Traumatology and Osteoarticular TB Clinical Hospital, Bucharest, Romania.

 

Corresponding author: Florinel Pop E-mail: florinel.pop@spcaroldavila.ro

Conflict of interest: none declared.

Financial support: none declared.

This work is permanently accessible online free of charge and published under the CC-BY licence.