Guidelines for histopathological specimen examination and diagnostic reporting of primary bone tumours

This review is intended to provide histopathologists with guidelines for specimen handling and diagnostic reporting of benign and malignant primary bone tumours; the principles of specimen handling required for assessment of secondary bone tumours are similar. As many primary bone tumours are uncommon or rare, experience in diagnostic orthopaedic pathology is required to maintain a high standard of histological reporting of bone tumours; participation in an external quality assessment (EQA) scheme which includes bone tumour pathology is recommended.

Close cooperation is needed between the histopathologist and radiology, surgical, oncology and other clinical colleagues in the diagnosis and treatment of bone tumours; consensus clinical practice guidelines for managing bone sarcomas have been recently published [1, 2]. All primary malignant bone tumour cases should be discussed at a properly constituted sarcoma multidisciplinary team (MDT) meeting.

Relevant clinical information should be provided on the pathology request form and its content should be recorded in the final pathology report. The age of the patient is crucial for bone tumour diagnosis as a number of bone tumours, both benign and malignant, tend to develop most commonly within a given age range. Some tumours and tumour-like lesions have a predilection to arise in certain bones (eg simple bone cyst occurs most often in the proximal humerus of a child or adolescent). Most bone tumours present with bone pain and swelling. Bone pain is dull, aching and characteristically worse at night. Rapid growth is characteristic of some malignant tumours but is also seen in some benign tumours and tumour-like lesions, such as aneurysmal bone cyst, eosinophilic granuloma and osteomyelitis. A history of trauma may be notable in cases where a post-traumatic lesion (eg haematoma) is a possible diagnosis. Local and systemic signs of infection need to be distinguished from those associated with the growth of a bone tumour, such as Ewing's sarcoma. Information regarding a pre-existing skeletal condition should be provided, including developmental conditions where there are multiple skeletal lesions (eg fibrous dysplasia, osteochondromatosis). It is also important to receive information on any relevant extraskeletal disease (eg history of carcinoma) elsewhere in the body. Racial occupational and treatment factors may also be relevant in the assessment of a bone tumour [16, 17].

The results of laboratory investigations which may help in evaluating a bone lesion should be communicated to the reporting pathologist [18]. Details of the white blood cell count and erythrocyte sedimentation rate should be noted if there is a possibility that the lesion is a bone infection, eosinophilic granuloma, leukaemia or other haematological malignancy. Ewing's sarcoma and 'toxic' osteoclasts may present with clinical and laboratory features that resemble osteomyelitis. If myeloma is suspected, protein electrophoresis for the identification of monoclonal immunoglobulin components in the serum or urine should be undertaken. Laboratory tests reflecting bone turnover, such as the serum calcium, phosphate and alkaline phosphatase should also be known, particularly if there is a need to exclude a metabolic cause for the development of a bone tumour, such as a "brown tumour" of hyperparathyroidism or Paget's disease. The alkaline phosphatase may also be elevated in osteosarcoma, 'blastic' metastases, fracture, polyostotic fibrous dysplasia and other conditions. The acid phosphatase may be elevated in prostate carcinoma.

Radiological information is essential for bone tumour diagnosis [15, 19] and it is strongly recommended that, wherever possible, the pathologist should personally view the radiological images of a bone tumour before issuing a diagnostic report. Where this is not possible, it should be recorded in the pathology report.

The precise anatomical location of a lesion in bone is important because tumours have a tendency not only to develop more commonly in certain bones but also more frequently to involve the particular anatomical region of an affected bone. It should also be evident from the radiology whether a lesion has originated in bone or extended into it from surrounding soft tissues.

The matrix composition of the lesion may point to specific diagnostic possibilities (eg calcification within cartilage tumour or ossification within a bone-forming tumour). The interface between the lesion and surrounding bone, particularly whether the lesion is well or poorly defined, should be noted as this may favour a particular benign or malignant diagnosis. A sclerotic rim is commonly present around slow growing lesions and usually points to a benign diagnosis. A non-sclerotic margin is usually found around a more rapidly growing bone lesion; malignant lesions are commonly poorly defined and have a broad zone of transition.

The pattern of bone destruction should be identified as it indicates the rate of growth of a bone lesion. A geographic pattern of bone destruction is characterised by the presence of well-circumscribed lytic areas (maximum dimension more than 1 cm) with a well-defined margin; this reflects the slow growth rate of these lesions, which are usually benign tumours (eg non-ossifying fibroma) or locally aggressive/low-grade malignant tumours (eg giant cell tumour of bone, low-grade chondrosarcoma). A rim of sclerosis between normal host bone and the lytic area may or may not be present. A moth-eaten pattern of bone destruction is characterised by the presence of multiple small lytic areas (usually 2-5 mm) separated by identifiable bone; this indicates an aggressive pattern of growth and is most often seen in malignant neoplasms, although it can be seen in some forms of osteomyelitis and eosinophilic granuloma. A permeative pattern of bone destruction is characterised by diffuse marrow involvement in which there are multiple tiny lytic areas (< 1 mm maximum dimension). This is usually accompanied by a broad zone of transition and reflects rapid growth of a bone lesion. A permeative pattern occurs in malignant tumours such as Ewing's sarcoma and osteosarcoma, but can also be seen in some benign entities such as osteomyelitis and eosinophilic granuloma.

Radiological evidence of extension of the tumour through the bone cortex and involvement of surrounding soft tissue should be noted as this provides evidence of a locally aggressive or malignant tumour. The nature of the periosteal reaction associated with a bone lesion oftens reflects the growth rate of the tumour. When the tumour grows slowly, the periosteum forms a thick layer of bone. Multiple layers of periosteal new bone are formed when there is a succession of fast and slow growth phases associated with the enlargement of the underlying lesion. The presence of tumour on both sides of the cortex (which is not yet destroyed) often indicates a very aggressive lesion.

The presence of multiple lesions within bone should be determined as this may suggest particular conditions such as multiple cartilage tumours (eg enchondromatosis, multiple osteochondromas) or Langerhans cell histocytosis (LCH). This feature is also useful in assessing whether a malignant tumour is more likely to be primary or secondary. With regard to primary malignant bone tumours, it may also point to a diagnosis of multifocal osteosarcoma or metastatic Ewing's sarcoma.

The following information should be recorded for all bone tumour specimens:-

1. Patient and specimen identification details including patient name, age (date of birth), sex, hospital and surgical pathology identification numbers.

2. Identification of the type of specimen received eg fine or core needle (closed) biopsy, surgical (open) biopsy, curettage, excision (segmental resection/en bloc), limb salvage, amputation or specific type of complex resection (eg hemipelvectomy).

It is often useful to receive specimens fresh (unfixed) in the laboratory. This permits the use of a number of specialised investigations where appropriate. These include the provision of material for frozen section diagnosis, the use of specific fixatives for histochemistry and snap freezing of tissue for molecular genetic studies [1, 7, 20, 21]. Fresh tissue can also be sent for microbiological culture or cytogenetic studies. Where the above studies are not anticipated or where there is likely to be a delay in the processing the specimen should be immediately fixed in 10% neutral buffered formalin. It is important that specimens are not placed in a freezer as this may result in formation of ice crystal artefacts.

It may be useful to specify whether the specimen was received fresh or in fixative and to record details of specimen transport to the laboratory; it should be noted if this has led to a delay or problems in processing. 10% neutral buffered formalin fixation is routinely used in most laboratories and is generally suitable for bone specimens. Fixation in absolute alcohol is useful for identifying glycogen in Ewing sarcoma cells. Small biopsy fragments containing only cancellous bone fragments or tumour tissue containing small amounts of bone can be decalcified and fixed in one overnight step through the use of 5% trichloracetic acid or ethylene diaminetetracetic acid (EDTA) dissolved in 10% buffered formalin.

Decalcification with strong (eg nitric) or weak (eg formic) acid solutions is required for the histological processing of most bone tumour specimens. To shorten the period required for decalcification, samples of a tumour biopsy that are not heavily mineralised should be selected; 2-4 mm thick slices should be submitted for decalcification from large specimens. Where there is a need for rapid diagnosis, 5% nitric or 20% formic acid can be used for rapid decalcification. Immersion of bone biopsy specimens in strong acids should not exceed 24 hours. The mineralisation status of the specimens should be constantly monitored. This is usually carried out by specimen radiography but chemical (eg calcium oxalate) tests are also available to determine the end point of decalcification. EDTA made neutral with sodium hydroxide solution removes calcium more slowly but provides better preservation of tissue and cytological details as well as antigenicity. It should be noted that most ancillary stains can be employed without modification to tissues that are decalcified even in strong acids; the antigenicity of many common epitopes is not abrogated by decalcification in strong acids [22]. However, strong acids can interfere with molecular genetic analysis [23].

In general, decalcified tissue is processed in the same way as undecalcified tissue. After paraffin embedding, blocks should be trimmed, placed in the freezer for 30 minutes and then on an ice tray before 3-5 μm sections are cut. A standard rotary microtome is suitable for routine use; larger sections can be cut on a sledge microtome. Haematoxylin-eosin stains are used routinely for morphological diagnosis. Ehrlich's or Cole's formula for haematoxylin is recommended as it gives the best differential staining of calcified bone, osteoid and cement lines. Other useful ancillary stains include PAS (+/- diastase), reticulin, toluidine blue, Alcian blue, Perls, Congo Red and Masson-Fontana.

Frozen section examination of bone tumours should only be carried out by pathologists who have some experience of osteoarticular pathology and who have knowledge of the clinical background and radiological appearances of the lesion; the usefulness of frozen section histological examination is predicated on close cooperation between the surgeon, radiologist and pathologist [1, 2, 20, 21]. Frozen section histology provides information on:-

• Adequacy of the biopsy specimen.

• The nature of the lesion.

• Ancillary investigations which may be required for diagnosis.

• Adequacy of resection margins.

Frozen section analysis is particularly useful in determining whether the sampled tissue is adequate and representative of the biopsied lesion. It is also used intraoperatively for the examination of resection margins to determine the level of excision or amputation of a bone tumour. Frozen section also has a diagnostic role, often indicating to an experienced pathologist the nature of the lesion. It is particularly useful in this regard in indicating whether a lesion is likely to be inflammatory or neoplastic; this may be helpful in directing the surgeon to take further samples for microbiological culture or carrying out cytogenetic and molecular genetic analysis. In some cases, the appearances of the lesion are sufficiently characteristic to permit a definitive diagnosis; in the appropriate clinical and radiological context, this may permit immediate surgical treatment of the lesion. A definitive diagnosis of bone tumour, however, should not be based on the examination of frozen sections alone. Gross specimens submitted for frozen section should be carefully evaluated for the presence of heavily mineralised tissues, such as fragments of cortical bone, which should be removed from the samples submitted for frozen section. Stained touch or imprint preparations can be used to provide supplementary information to a frozen section diagnosis and are particularly useful if the tissue is heavily mineralised. Imprint preparations are particularly useful in the diagnosis of round cell tumours of bone, such Ewing's sarcoma, lymphoma, and osteosarcoma, but may also occasionally suggest a diagnosis of osteomyelitis, eosinophilic granuloma or carcinoma.

Aspiration cytology can also provide useful information on bone tumours [24–27]. However, it has a relatively limited role in the diagnosis of these neoplasms. As with imprint preparations, when combined with frozen section histology, it can provide important cytological information that may point to a specific diagnosis. It may have a particular role in the diagnosis of possible sarcoma recurrence or metastasis of previously well-documented neoplasms. Under these circumstances, when combined with adequate radiological and clinical information, a diagnosis can often be suggested.

Biopsy specimens for histological examination are usually obtained by either open (surgical) biopsy, or closed (percutaneous) needle biopsy [28–30].

Closed needle biopsy is an effective and safe technique and is often used in the initial diagnosis of a bone tumour. Most lesions can be biopsied in this way, yielding adequate material to permit an experienced pathologist to make a diagnosis. With this technique there is little risk of dissemination of the tumour in the course of the procedure. The principal disadvantage of needle biopsy is that it provides less material for histological examination than an open biopsy; this may limit the amount of diagnostic information that can be obtained from the biopsy. In addition, as much of the biopsy tissue is required for histological diagnosis, this can limit or preclude the use of other investigations ( eg molecular genetics), which may provide supplementary diagnostic information. An open biopsy specimen generally provides more material for histological examination than a closed biopsy. Open biopsy is commonly employed for the diagnosis of bone tumours in children from whom it may be difficult to obtain a closed biopsy specimen. It is recommended that an open biopsy is carried out at the centre where the tumour is to be treated as this ensures that the biopsy tract is removed at the time of definitive surgery. When combined with frozen section examination of the biopsy sample, this technique ensures that the biopsy tissue is adequate and representative of the lesion.

Most biopsy specimens require at least three hours fixation. Core needle biopsy specimens, if properly fixed, maybe decalcified overnight in acid or a chelating agent. If the core is 5 mm or more in thickness, it should be divided. In general, if ancillary studies are anticipated then a minimum of three cores may be needed. A general gross description of the biopsy specimen should be given including dimensions, consistency, colour [5, 7, 19]. The presence of necrosis, thrombus, fibrin clot, myxoid change, bone cartilage and fibrous tissue should be noted. It may be possible to separate soft areas of the tumour from calcified areas of the tumour; the former may be submitted for frozen section or rapid histological diagnosis Handling of curettage specimens is essentially similar to that for core needle biopsies in terms of gross description and specimen handling. If a large amount of curetted material is received, then this should be sampled extensively (about 1 section per cm. Specimens should be submitted for overnight formalin fixation and decalcification.

The surgical pathology report on a biopsy or curettage specimen should record the morphological and cytological features of the tumour, including degree of pleomorphism, mitotic count and the presence of tumour necrosis. The report should contain the histological diagnosis (or differential diagnosis), indicating where possible details of the specific tumour type (+/- subtype) and tumour grade. Even when a specific diagnosis appears to have been established by means of a biopsy, careful histological study of the resection and amputation specimen needs to be carried out as the same histological features may not be present throughout a tumour and the appearances of the tumour tissue can change over time or as a result of treatment.

Orthopaedic amputation and segmental resection specimens are often large and will not fix adequately if left un-dissected in formalin. They should be dealt with promptly after arrival in order to expose the key tissues and tumour for subsequent fixation [5–7, 19]. The volume ratio of fixative to specimen large specimens should be at least 3:1.

Before dissecting an amputation or large segmental resection for bone tumour, the pathologist should review the pre-surgical clinical history, relevant radiology and biopsy pathology report(s).

The type of amputation (e.g. left hindquarter amputation, right forequarter amputation, right below nee amputation), or resection (eg left distal femoral resection left second toe amputation) should be noted and a digital photograph of the intact specimen taken from at least two aspects. The following are guidelines for large specimen dissection:

1. Measure and record the length of the amputation or resection specimen in three dimensions. For amputations, the length between the major joints and the distance between the tumour and the osteotomy site and the closest soft tissue amputation site should be noted; for resections, the distance between the palpable tumour and the osteotomy site should be recorded. It may be useful to include a measure of the circumference at the level of the tumour. Specimen radiographs may aid in determining the extent of bone, joint and soft tissue involvement at this stage.

2. Record the size and position of any previous surgical scars. Determine the presence, position, and dimensions of previous biopsy sites in an amputation specimen. For resection specimens, record the size and position of the excised skin ellipse (which should be included in the biopsy tract).

3. Search for the major lymph node groups and identify and place them in separate containers of fixative.

4. Cut a cross section of the proximal bone margin with a bandsaw.

5. Take samples of the major vessels at the amputation site and place in a separate container of fixative.

6. Dissect all the soft tissues (down to the periosteum) around the involved bone. If there is any indication (from the radiographs or at the time of dissection) of soft tissue tumour extension, dissect around this area and keep it in continuity with the bone. Unless the tumour involves an adjacent joint, the entire bone containing the tumour should be excised. If, from the radiographs, the tumour involves an adjacent joint, cut through the adjacent, non-involved bone transversely with the band saw approximately 5-10 cms from the joint, and then excise the involved bone, joint and attached uninvolved length of bone.

7. Once the key part of the amputation specimen has been dissected out, a plain radiograph of the intact tumour and bone can be taken using a laboratory X-ray machine. This will aid the subsequent decision on the preferred initial slice through the specimen.

8. Take tissue samples for histology of any previous surgical sites or biopsy tracts in order to determine the presence/absence of tumour implantation.

9. Long bones containing the tumour (and the excised adjacent joint, if applicable), should be cut longitudinally with a band saw. In most cases, the cut should be in the coronal plane, dividing the specimen into anterior and posterior halves. When the tumour is a surface tumour involving the anterior or posterior aspect of the bone (eg parosteal osteosarcoma, surface chondrosarcoma), a sagittal section may be preferable for tumours of unusual shape/site of origin. It is nearly always sufficient to take an initial coronal slice and then subsequent additional slices to complement the coronal slice. For hindquarter and forequarter amputations, and for pelvic and scapular bone tumours, it may be necessary to cut the bone obliquely; in these bones the ideal cut usually involves the joint (hip and shoulder, respectively) and includes the area of tumour origin/main areas of tumour with subsequent slices used to sample any extra-osseous tumour extension.

10. After fixing the sliced bone specimen for a further 24 hours, a cut is made using a band saw parallel to the initial cut in order to provide a slice of the bone tumour. The thickness of this slice should be approximately 5 mm. This slab should be radiographed to provide a specimen radiograph. For bone resection specimens, the surgical tissue margins should be taken after fixation. Medial, lateral, anterior and posterior (as well as the osteotomy transverse slice) margins should be taken as minimum. Further margins can be taken if there is concern that there is tumour spread; the surgeon should mark with a suture or ink any areas of particular concern alternatively, the surgical margins can be sampled by the pathologist when the surgeon is present. If the tumour has destroyed or breached the cortex, the margin can be sampled with a scalpel or knife.

11. The slab specimen should be photographed.

12. The soft tissues including major vascular structures around the dissected bone of the amputation or resection specimen should be examined. Other apparently uninvolved bones should be sliced sagitally and/or coronally to look for foci of tumour or other lesions and the major joints should be examined.

13. Representative tissue blocks taken for histology from an amputation or resection specimen of a bone tumour should include:-

• Blocks of tumour: a slab of all of the tumour in bone should be blocked out and a photographic record kept of where the tissue blocks for histology have been taken (Figure 1).

• Additional blocks should be taken from areas of tumour or bone not included in the slab if the macroscopic appearance is unusual.

• Blocks of the previous incision site and biopsy tract (if present).

• Blocks of the proximal bone resection margin at the site of amputation or excision.

• Any abnormal-looking areas elsewhere in bone, soft tissues, or skin.

• Lymph nodes: if grossly normal, only representative ones; if grossly abnormal or if there is clinical suspicion of metastasis, all of them.

• Major vessels at the soft tissue amputation site.

14. The macroscopic description should note the following features:-

• Type and side (left/right) of amputation or bone resection.

• The dimensions of the resection specimen (see above).

• The gross presence or absence of exposed tumour in bone or soft tissue of the specimen.

• The presence, position and dimensions of attached skin and the presence of biopsy sites and surgical scars.

• The anatomical site and location of the tumour within bone (ie whether the lesion is in the epiphysis, metaphysis or diaphysis of a long bone and whether located in the medulla, the cortex or on the surface of the bone).

• Dimensions of the tumour (in centimetres).

• Extent of bone, and soft tissue (including joint) involvement.

• The presence or absence of necrosis (approximate percentage) and other descriptive characteristics (eg colour, calcification, hardness, gritty, haemorrhagic, cystic) should be noted.

• The presence or absence or chemotherapy or radiotherapy effect.

• Involvement or invasion of major structures (eg nerve, major blood vessels).

• Presence of satellite lesions of tumour away from the main tumour mass.

• Presence of lymph nodes and description of cut surface of the nodes.

• Relation of the tumour to the resection margins; the distance (in centimetres) should be measured.

In addition, a record should be kept of whether tissue has been submitted for frozen section, cytogenetics, molecular genetic analysis and other investigations.

The following histological information should be included in the surgical pathology report:-

• Tumour type (and subtype) according to WHO histological classification.

• Tumour grade (if relevant)

• Morphological and cytological description of the tumour; this may include details of the mitotic count and degree of pleomorphism.

• Tumour necrosis (presence or absence and percentage necrosis in the resection specimen).

• Extent of local tumour spread, in particular whether the tumour involves specific anatomical components or compartments (eg medullary cavity, cortex, joint, extra-osseous soft tissues).

• Status of resection margins with regard to involvement by tumour.

• Results of cytogenetic/molecular genetic investigations (if available).

• SNOMED coding of bone tumour (Table 3).

Preoperative chemotherapy is commonly used with limb salvage procedures for the treatment of high-grade sarcomas, particularly osteosarcoma and Ewing's sarcoma. To determine the effect of chemotherapy it is generally required to quantify the extent of tumour necrosis as a percentage of the total tumour area [58]. For osteosarcomas, chemotherapy-induced necrosis of 90% or more has a greater than 90% disease-free survival, compared with less than 50% in patients with less than 90% tumour necrosis [58–61]. For Ewing's sarcoma, significant necrosis, according to most series, appears to be roughly defined as greater than 90% of the microscopic tumour mass [62–67]. Tumours demonstrating such massive necrosis are associated with a favourable prognosis, whereas those with less necrosis are associated with poor survival.

To determine the extent of necrosis in an osteosarcoma or Ewing's sarcoma specimen, the slab specimen of the resected bone containing the tumour provides the template for histological analysis [5–7, 66]. A photograph or radiograph of the slice is taken and the site of each numbered block marked on a grid pattern diagram. Additional blocks are also taken in a plane at right angles to this set of blocks to determine the full extent of the tumour. As indicated earlier, blocks should also be taken from other representative areas of the specimen including areas of unusual appearance in bone and soft tissue surrounding the tumour and possible satellite lesions in order to get a clear picture of the volume and extent of the tumour. Treated osteosarcomas may contain large atypical cells with hyperchromatic nuclei, smudged or clumped chromatin and vacuolated cytoplasm in areas of necrosis, calcification or fibrosis [66, 67]. The nature of these cells is not certain but they are currently considered to represent viable tumour cells in assessing the extent of tumour necrosis. Other effects of chemotherapy on tumour histology include ghost-like cells with loss of nuclear and cytoplasmic detail, granulation tissue formation, fibrosis, haemosiderin deposition, mucinous change and inflammation.