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 . 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 . 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
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
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.  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
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. 
Conventional chondrosarcoma is a tumor of the 5th-7th decade with a predominant
location in the proximal extremities and pelvic bones.  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
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.  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. 
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.  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
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.  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.
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