—  SYMPOSIUM #03  —

Fine Needle Aspiration Cytopathology: Bone and Soft Tissue
Moderator: William J. Frable

Section 3 - Fine Needle Aspiration Biopsy of Bone Lesions

William J. Frable


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In the United States the first significant recording of aspiration biopsy of bone was by Coley et al, 1931, 35 consecutive cases, at Memorial Sloan Kettering Cancer Center. An 18-gauge needle was used. Three errors were reported in that series [1]. By 1945, in a follow-up study of Snyder and Coley, the series had expanded to 567 aspirations performed on 474 patients. The authors found this biopsy method was most valuable in primary bone tumors and that there was no evidence of dissemination of tumor or needle tract implantation [2]. As the fine needle aspiration biopsy developed and expanded in Europe following World War II, both bone and soft tissue tumors became targets. Today large series have been compiled and biopsy methods have been aided by modern imaging techniques [3, 4, 5, 6, 7, 8, 9].

Indications for FNA of bone are: an obvious malignant tumor on clinical and radiologic evidence. Rapid confirmation of the type of tumor to begin treatment; a tumor that appears to be benign on clinical and radiologic information but confirmation by morphology is needed; tumor identity is in doubt and cytologic study may determine whether the tumor is benign or malignant, primary or metastatic. Large palpable tumors can be aspirated directly while smaller tumors will require image guidance. Imaging can also determine the optimal approach. A malignant tumor in the medullary cavity that has penetrated the cortex and elevated the periosteum can be targeted to that area where tumor cells lie between the outer cortex and elevated periosteum. Fine needles will not penetrate an intact cortex but a small hole can be drilled through the cortex with image guidance and the aspirate performed thru that area. Anesthesia, at least local, is generally required for FNA of bone. With improved imaging, FNA is being combined with thin core needle biopsies. Onsite evaluations of smears and or touch preparations from the core biopsies can insure that an adequate sample is available for study and ancillary techniques when required. Optimally, the cytopathologist should be directly involved in reviewing the imaging studies, clinical history and the taking of the biopsies. All elements need to be correlated to make a reliable interpretation.

Conventional osteogenic sarcoma is the most common primary malignant tumor of bone. It is most common in the second decade while there is a second peak incidence in patients over 50 years of age. Usually more than half of all the cases occur around the knee, either the distal femur or proximal tibia. Pain in the most common presenting symptom and swelling and warmth of skin around the site may suggest an infection. While radiographic features are variable, most commonly there is a combination of a lytic and sclerotic process that is infiltrative. Tumor may elevate the periosteum stimulating new reactive bone formation, the classic Codman's triangle. Codman's triangle can result from both benign and malignant conditions that elevate the periosteum but it is never a normal radiographic finding. [10] Conventional radiographs and or other imaging studies along with physical examination should be correlated to determine the best approach for aspiration biopsy. Use of small 21 gauge needles, particularly of the Franzen design are recommended to provide a micro core sample that can be touched to a slide to provide an initial interpretation or at least to confirm that diagnostic tissue is present. Fine needles, 22 gauge or higher, should be used to make separate aspirates for smear preparation. This author does not recommend any of the liquid based cytology methods for handling aspirations of bone or soft tissue tumors.

Aspirates of conventional osteosarcoma are typically quite cellular. Cells are large with enlarged nuclei with irregular nuclear membranes and overall hyperchromasia. There is moderate to marked pleomorphism of the tumor cells and the pattern of the cells has been noted to be similar to an aspirate of a poorly differentiated squamous cell carcinoma. Mitotic figures may be found. Tumor cells have relatively abundant cytoplasm that may be amphophilic and or metachromatic with use of the Romanowsky type stains, the most popular currently being Diff-Quik. Though it may be sometimes difficult to identify, there can be a background of fibrillary metachromatically staining material that represents osteoid and in some parts of a given smear this osteoid material may intermix haphazardly with the tumor cells simulating the tissue pattern that has been described as malignant osteoid. Conventional osteosarcomas may be dominated by giant cells, fibrosarcomatous or a chondrosarcomatous pattern and this may be reflected in the aspiration smear with either many osteoclastic type giant cells, atypical or malignant appearing spindle cells or moderately to poorly differentiated chondroblasts. These cytologic and histologic patterns do not impact significantly on the radiographic presentations. The rare small cell osteosarcoma may mimic an epithelial neoplasm and be confused with metastatic carcinoma. Conventional osteosarcoma may also occur primarily in a periosteal location. However the smear patterns are the same as the predominantly intramedullary tumor as is the clinical behavior. The telangiectatic form of osteosarcoma may also be a challenge to interpret on aspiration biopsy because only scattered tumor cells are seen in a background of blood. Sampling based on image guidance to the more solid areas can overcome this difficulty. In contrast, intramedullary well differentiated osteosarcoma and parosteal osteosarcoma are not likely to be diagnosed by aspiration biopsy. Both can be difficult to interpret correctly even with open surgical biopsy.

Correctly interpreting osteosarcoma from aspiration or micro core biopsy is of value since preoperative chemotherapy and post excision assessment for degree of necrosis significantly impacts the prognosis, which has improved substantially over surgical excision, often amputation alone. More limited resections are achievable with the use of preoperative chemotherapy and the ability to replace the distal part of long bones that interface with joints. [10]

Conventional chondrosarcoma is a tumor of the 5th-7th decade with a predominant location in the proximal extremities and pelvic bones. [10] Chondrosarcoma that is grade I is slow growing and is as cytologically bland as it is in tissue sections. The diagnosis may be made on FNA with good clinical correlation, typically a large lesion of the extremity or pelvic bone with heavy calcification noted radiographically. The aspirate is of low cellularity but has abundant myxochondroid background substance that stains brightly metachromatic with Diff-Quik stain. Cytologic atypia is minimal and only a rare binucleated chondrocyte may be found. Such a tumor has no metastatic potential but can recur if incompletely excised, a major surgical consideration for tumors in the pelvic bones. The same degree or even more cytologic atypia in an aspirate of a chondroid lesion in the phalanges is a benign enchondroma. Grade 2 and 3 conventional chondrosarcoma have much more cellular aspirates with more cytologic atypia of the tumor cells but still retaining some features of chondroid cells in a myxoid metachromatically staining background. A plump spindle cell pattern may predominate but the background remains myxoid. Radiographically these higher-grade chondrosarcomas have an infiltrative and destructive pattern. Dedifferentiated chondrosarcomas take on a spindle cell pattern and my become pleomorphic to the point of both histologically and cytologically being interpreted as malignant fibrous histiocytoma. Such tumors are aggressive. Clear cell chondrosarcoma may resemble metastatic renal cell carcinoma histologically and in aspiration biopsy smears but the age for this tumor is between 25-35 years, an uncommon age range for renal cell carcinoma. Mesenchymal chondrosarcoma has highly cellular aspirates with small round to plump spindle cells that cytologically place it in the small round blue cell tumor category generically. Evidence of cartilaginous differentiation may be limited and positive S-100 staining may be present in tumor cells or micro core biopsies that correspond to cartilaginous differentiation.

Two important medullary tumors, Ewing's sarcoma and myeloma occur at opposite ends of the age spectrum, Ewing's sarcoma being seen predominantly in the first and second decades, myeloma usually beyond the 5th decade. Ewing's sarcoma is the prototype of a number of related tumors that include primitive neuroectodermal tumor (PNET), Askin tumor (ES/PNET of the thoracopulmonary region), and questionably small cell osteosarcoma, mesenchymal chondrosarcoma and a primitive pluripotential sarcoma. Ewing's sarcoma and PNET are distinguished by the 11:22 translocation (t(11;22)(q24;q12), that has also been identified in some of the latter three entities. Ewing's sarcoma is typically found in long tubular bones, favors the lower extremities and is generally a lesion of the midshaft. It presents with pain and often fever, swelling of the soft tissue over the lesion, all signs of an infection in bone, which is the immediate differential diagnosis clinically. This tumor permeates the bone quickly with elevation of the periosteum the classic radiologic picture of a permeative lytic pattern and onionskin appearance of the periosteum. [10] The cortex may appear relatively intact and therefore a good site for aspiration biopsy is the area between the cortex and the elevated periosteum. Large numbers of small primitive and monotonous round tumor cells may be obtained by targeting that area. There may be a fine fibrillary background to the smears of Ewing's sarcoma and some suggestion of rosette formation, a pattern that dominates with neuroblastoma, one of the differentiatials among the small round blue cell tumors of childhood. Malignant lymphoma and embryonal rhabodmyosarcoma are the other two important differential interpretations. Lymphoma is quite uncommon in children, particularly as a primary bone tumor, is usually of the large B-cell type with lack of uniformity of the nuclei in comparison to Ewing's sarcoma, and abundant lymphoglandular bodies that identify the tumor cells as lymphoid. Embryonal rhabdomyosarcoma is very rare as a primary tumor of bone and typically has nuclei with small cytoplasmic tags and the presence of some tumor cells with large nuclei well beyond the range seen in Ewing's sarcoma. A variety of immunohistochemical markers can be used to distinguish these tumors and are applicable to aspiration biopsy smears and small core biopsies as well as cell blocks. Identification of the 11;22 translocation can be accomplished by fluorescent in situ hybridization (FISH) performed on aspiration samples as confirmation of the interpretation. [11]

The presence of myeloma is identified on aspirates as sheets of plasma cells in the presence of clinically highly lytic bone lesions that usually affect multiple bones particularly vertebra, skull and pelvic bones. The markedly lytic nature of myeloma makes the use of aspiration biopsy relatively easy to confirm the diagnosis. Hyperglobulinemia is present with elevated urine proteins and the presence of globulins can be detected in the tumor cells using immunohistochemical methods on aspiration smears, cellblocks or core biopsies. A variety of other immunohematopoetic tumors involving bone primarily may be diagnosed on aspiration biopsy, the only limiting factor being adequate samples to perform confirming immunohistochemical studies. One of the more common lesions, seen principally in children and young adults is eosinophilic granuloma, part of the Langerhan's histiocytosis group of lesions that have different clinical manifestations. The predominant cell and the really diagnostic feature in aspirations are the Langerhan's histiocytes with the vesicular footprint shaped nucleus usually having a prominent nuclear groove. In typical solitary lesion of eosinophilic granuloma eosinophils may predominate but not in every case.

There are several bone lesions where osteoclastic giant cells are or can be a prominent feature, both histologically and on aspiration biopsy. They are giant cell tumor, benign chondroblastoma, chondromyxoid fibroma, aneurysmal bone cyst, and fibrous cortical defect. The first four entities have radiologic features that may be quite similar, a lytic but circumscribed lesion with a thin rim of calcification and a location predominantly at the end of long bones. [10] Aneurysmal bone cyst may also be the result of degenerative changes within one of these neoplasms, particularly giant cell tumor and it is generally not possible to make that distinction specifically on aspiration biopsy. FNA of classic giant cell tumor will have cellular smears with a dominance of plump uniform stromal cells and multiple osteoclastic giant cells interspersed within the stromal cells. It is actually easier to see this arrangement in well-prepared aspiration smears than on frozen sections of a biopsy where the giant cells may blend imperceptively into the background stromal cells. The rarity of benign chondroblastoma and chondromyxoid fibroma make definitive recognition on aspiration biopsy difficult. Only a few cases have been reported. However, there is an over all benign appearance to the aspirate and micro core biopsies may provide histology that is adequate with the radiographic/clinical correlation to make a correct interpretation.

In an adult, a solitary lytic bone lesion immediately raises the question of metastatic carcinoma. This is the most common problem encountered in adults with a solitary bone lesion. FNA is the simplest method to obtain material for the interpretation and provide some information based on both morphology and immunostaining as to possible primary sites. Much more often there is a prior history of carcinoma and FNA merely confirms that the bone lesion is metastatic. Both the prior clinical information and comparison of the aspirate with the prior histology, if available, is very important. While any primary site of carcinoma can metastasize to bone, the common ones are breast, lung and prostate. While most metastases to bone are lytic, some, particularly breast and prostate are blastic. These are difficult if not impossible to aspirate and will require either a cutting core needle type biopsy or an open biopsy. Both breast cancer and lung cancer can also metastasize to the area between the cortex and the periosteum with elevation of the latter producing an onionskin pattern radiologically. These are usually painful metastases, which bring them to clinical attention. With good imaging for localization this is an easy aspirate to obtain material similar to the approach in Ewing's sarcoma.

For the unknown primary and undifferentiated malignancy found on FNA of a bone lesion a basic screening panel may consist of Cytokeratin Cam 5.2. Epithelial membrane antigen (EMA), S-100, Leukocyte common antigen, and Placenta-like Alkaline Phosphatase. Having established that a carcinoma is present from this panel there are quite a variety of additional immunomarkers, particularly the cytokeratin family that may help to pinpoint a potential primary site. [12] Given modern chemotherapy this may be of some value and in the future specifically tailored therapy based on protein profiles may be utilized to improve treatment and survival.

References
  1. Coley BL, Sharp GS, Ellis EB:Diagnosis of bone tumors by aspiration. Am J Surg 1931;13:215-224.

  2. Snyder RE, Coley BL:/Further studies on the diagnosis of bone tumors by aspiration biopsy. Surg Gynecol Obstet 1945;80:517-522.

  3. Handa U, Bal A, Hohan H, Bhardwaj S:Fine needle aspiration cytology in the diagnosis of bone lesions. Cytopathology 2005;16:59-64

  4. lLerma E, Tani E, Brosjo O, Bauer H, Oderlung V, Skoog L:Diagnosis and grading of chondrosarcomas on FNA biopsy material. Diagn Cytopathol 2003;28:13-17.

  5. Kabukcuoglu R, Kabukcuoglu Y, Kuzgun U, Evren I:Fine needle aspiration of malignant bone lesions. Acta Cytol 1998;43:875-882.

  6. Koscick RL, Petersilge CA, Makley JT, Abdul-Karim FW:CT-guided fine needle aspiration and needle core biopsy of skeletal lesions. Complementary diagnostic techniques. Acta Cytol 1998;42:697-702.

  7. Bommer KK, Ramzy I, Mody D:Fine-needle aspiration biopsy in the diagnosis and management of bone lesions: a study of 450 cases. Cancer 1997;81:148-156.

  8. Willen H:Fine needle aspiration in the diagnosis of bone tumors. Acta Orthop Scand Suppl 1997;273:47-53.

  9. Ayala AG, Ro JY, Fanning CV, Flores JP, Yasko AW:Core needle biopsy and fine-needle aspiration in the diagnosis of bone and soft-tissue lesions. Hematol Oncol Clin North Am 1995;9:633-651.

  10. Dorfman H, Czerniak B:Bone Tumors. Mosby St. Louis 1998.

  11. Bridge RS, Rajaram V, Dehner LP, Pfeifer JD, Perry A:Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT_PCR in malignant round cell tumors. Mod Pathol 2006;19:1-8.

  12. Dabbs DJ:Diagnostic Immunohistochemistry. Churchill Livingstone, New York 2002 pp.163-196.