Pathologic Assessment

Principles of Biopsy

A biopsy for histological diagnosis is pivotal in the investigation and subsequent management of bone and STS.10This biopsy should be performed by the same surgeon who will be performing the surgical resection.11

A biopsy should be kept as minimally invasive as possible, while still yielding sufficient tissue to obtain a diagnosis, as more immunohistochemistry and genomic tests are being done to classify sarcoma than previously. Fine needle aspiration cytology is, however, not appropriate as the pathologist needssufficient tissue for analysis. A percutaneous trucut core needle biopsy, under ultrasound or computed tomogram guidance if necessary, is preferred. 12, 13Open biopsy may still be indicated if the anatomy precludes percutaneous core needle biopsy or if percutaneous core needle biopsy is unsuccessful.

A biopsy should be performed only after detailed imaging of the lesion has been completed.14This allows for careful planning of the biopsy tract.14A poorly performed biopsy can compromise treatment outcomes, especially those of possible limb salvage surgery.13

The biopsy skin incision should be kept to the minimum required for access.9Planning ahead for possible future surgical resection, the biopsy skin incision should be orientated longitudinally, allowing for an extensile surgical approach to the limb.13,15

In general the most direct route is taken to the lesion, avoiding neurovascular structures and traversing of joints. The biopsy should ideally be kept to a single compartment and should minimise the opening of fascial planes. Meticulous haemostasis throughout the biopsy is critical.16A muscle splitting approach whenever traversing muscle planes is advised.

If a bone window is required to be made for access, attention should be paid to its size and shape. The bone window should only be made as large as necessary for access and sharp edges are to be avoided as they act as stress risers.

Sampling of the lesion should be directed by the pre-biopsy imaging which would identify the part of the lesion with the greatest suspicion for sarcomatous tissue. Intra-operative frozen section analysis to confirm lesional tissue is advised.17It is important to take adequate amounts ofrepresentative tissue for the pathologist.12These samples should ideally be sent to a pathologist with an interest in musculoskeletal oncology. 19A detailed clinical and radiological patient history in the accompanying histology request form would be helpful to the pathologist and should be included.13,14Samples should also be taken for cultures by the microbiologist to exclude infection, such as tuberculosis and fungal cultures.

Principles of Pathologic Assessment

The diagnostic approach of limb sarcomas is highly complex and labour intensive due to the sheer volume of differential diagnoses to consider covering bone, STS, and sometimes carcinomas and melanomas, and therefore requires a multidisciplinary approach. This is especially relevant for bone tumours where input by the clinicians and radiologists are critical to avoid well-known diagnostic pitfalls.

Before interpreting the biopsy, pathologists need to be furnished with relevant clinicoradiological information and radiological images. This information should include at least:

a)site of the tumour (for instance, bone versus soft tissue centred, anatomical region of the bone),

b)characteristics of the tumour (for instance, benign versus aggressive, homogenous versus heterogeneous, bone or cartilage forming) and

c)tumour relationship with other structures such as nerves, bone, muscle, vessels, etc.

Besides influencing the diagnosis, this information may help the pathologist decide whether the biopsies are likely to be diagnostic or representative.

Assessment of Biopsy Specimen

A preliminary investigation of limb sarcomas usually involves obtaining multiple core biopsies (at least four to five cores) or incisional biopsies from the patient. Excision biopsies may be considered for STS that are small, superficial, and easily resectable. However, fine needle aspiration is not recommended as a primary diagnostic modality, except for confirming disease recurrence or obtaining material for ancillary tests. For bone tumours or specimens, it is likely that the tissue will undergo decalcification, potentially affecting immunohistochemical results or genomic analyses. The frozen-section technique for immediate diagnosis should be avoided in bone tumours due to suboptimal histological interpretation, sampling issues, and lack of immediate ancillary support.

Assessment of Resection Specimen

Histological evaluation of limb sarcomas usually involves a description of the tumour in the resected specimens which should at least include its site, size, gross characteristics and relationships with surrounding structures and margins. A detailed description of the margins (such as intra-lesional, marginal, or wide) with close communication between the clinician and pathologist is important since the margin status often determines the tumour recurrence rate.

For post-treated osteosarcoma specimens, there is an additional requirement of detailed tumour mapping and estimation of the amount of tumour necrosis by percentage. Detailed discussions on the gross handling and tumour reporting datasets for resected limb sarcoma specimens are covered comprehensively in the websites of various pathology organisations and colleges18-20.

Histological Diagnosis

Histological diagnosis of limb sarcomas should be made according to the latest (2013) World Health Organisation (WHO) classification for bone and STS. 21For STS, grading should be provided following the Fdration Nationale des Centres de Lutte Contre le Cancer (FNCLCC) grading system, which is based on tumour differentiation, mitotic count, and necrosis. Malignancy grading may be underestimated in small biopsies particularly in heterogeneous tumours.

The histological assessment of both STS and osteosarcoma requires a combination of careful morphological examination complemented by ancillary investigations especially immunohistochemistry and molecular diagnostic tests to determine the subtype. Morphological examination involves a systematic pattern-based approach taking into consideration the architecture, cytomorphological features and matrix of tumours. The diagnosis of osteosarcoma requires the identification of osteoid forming cells or matrix for both low- and high-grade osteosarcoma variants.21However, diagnostic difficulties may arise where the presence of osteoid cannot be confirmed (for instance, sampling problem related to small biopsies, osteoid-poor osteosarcoma variants) or in low-grade tumours.

Ancillary Investigations

A panel of immunohistochemistry stains is often required for the diagnosis of most STS and more rarely in bone sarcoma. Comprehensive discussions of the appropriate immunohistochemistry panels with the corresponding differential diagnoses can be found in most specialised pathology textbooks.

Molecular diagnostic tests play a critical role in determining the final diagnosis of many sarcomas particularly in those with recurrent chromosomal translocations (for instance, Ewing sarcoma, synovial sarcoma, alveolar rhabdomyosarcoma, etc.). Recent studies have also shown that these tests can be utilised to diagnose certain types of low-grade osteosarcoma22and benign bone tumours (for instance, aneurysmal bone cyst).23 Molecular tests are often employed in problematic tumours that demonstrate unusual clinical presentation, atypical morphology, encompass numerous differential diagnoses (for instance, small round cell tumours) and overlapping immune-phenotype.

In summary, the diagnostic algorithm of limb sarcoma is a highly specialised and complex process that often requires a sarcoma pathologist with clinical and morphological expertise and a deep familiarity with modern ancillary techniques.