Recently, using transcriptional gene profiling comple- mented with, e.g. tissue microarrays, new potential ther- apeutic markers of histiotype-specific soft tissue tu- mours have emerged, e.g. CD117 (c-kit) and DOG1 for gastrointestinal stromal tumours, PDGFRb in dermatofi- brosarcoma protuberans and EGFR1 for synovial sarco- mas, which in some cases might even be able to predict therapy response and prognosis [1–3]. These advances have led to an increased appreciation of the importance that histological typing might have in predicting natural history and treatment sensitivity of soft tissue tumours.
Tumour tissue procurement and exact tissue han- dling are critical issues to provide the pathologist with adequate and well preserved tissue, in order to perform the necessary ancillary studies for making an exact di- agnosis. Cross-talk between pathologists, radiologists and clinicians is crucial in obtaining this goal.
8.2 When Pathology Meets Radiology
The importance for the pathologist of both clinical and radiological information cannot be overemphasised.
Some tumours merely occur in children, e.g. rhab- domyosarcomas, while others are rare in children and are mostly found in adults, e.g. pleiomorphic liposarco- ma [4].
Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) give the pathologist information about the size, consistency, homogeneity, location, pres- ence or absence of necrosis, infiltration pattern, inter- face with adjacent structures, etc.
Anatomic pathologists rendering a diagnosis on mostly limited material without having the advantages of clinical and radiological information might be in a disadvantageous position in many cases. It is imperative that pathologists with a special interest in soft tissue tu- mours do at least have a rudimentary knowledge of the radiological and clinical features of the tumours, in analogy with pathologists interested in bone tumours.
On the other hand, it is equally important to have a ra- diologist and clinicians with a keen interest in soft tis- sue tumour pathology.
8.1 Introduction
The diagnosis of soft tissue tumours has always been difficult and controversial. This was mainly due to the rarity of these lesions, comprising <1% of all malignant tumours. Nevertheless, according to the Surveillance, Epidemiology, and End Results (SEER) database, about 15,000 cases of both bone and soft tissue tumours are diagnosed annually in the US. This puts soft tissue and bone tumours in the same order of magnitude as myeloma, gliomas and cervical carcinoma.
Previously, the clinical importance of the exact histo- logical typing of soft tissue tumours by the pathologist was limited, knowing that grade and stage, and not his- tological type determined treatment options. The pathologist merely served to grade the lesion and to ex- clude malignant non-sarcomatous, benign or pseudotu- moral lesions. The small number of lesions – in particu- lar of single histological types – and the poorly defined pathological criteria are the major sources of discor- dances in diagnostic soft tissue pathology. Even in spe- cialist centres about 5–10% of soft tissue lesions remain unclassified.
Soft Tissue Tumours:
the Surgical Pathologist’s Perspective
Roberto Salgado, Eric Van Marck
8
8.1 Introduction . . . 107
8.2 When Pathology Meets Radiology . . . 107
8.3 When Pathology Meets the Clinics . . . 108
8.4 When Morphology is not Enough . . . 109
8.5 World Health Classification of Soft Tissue Tumours 2002 . . . 111
8.6 To Grade or not to Grade? . . . 112
8.7 Reporting Soft Tissue Tumours . . . 114
8.8 Gene Expression Profiling and Tissue Microarrays in Soft Tissue Tumours . . . . . 114
8.9 Conclusions and Future Perspectives . . . 115
References . . . 115 Contents
Some soft tissue tumours such as liposarcomas are quite heterogeneous. Therefore, the pathologist might aid in choosing which area of the tumour is best suited for sampling, considering that sampling in areas of overt necrosis is best avoided. Sampling near necrotic areas, however, can be meaningful since the presence or absence of necrosis is considered an integral part of grading schemes [5]. Meanwhile, in analogy with bone tumours, e.g. osteosarcoma and chondrosarcoma, imaging features might provide the pathologist with spatial features of soft tissue lesions that are highly sug- gestive of being the high grade areas of the tumour. For example, chondrosarcomatous tumours having myxoid areas juxtaposed to cartilaginous areas are highly sug- gestive of being of high grade malignancy [6]. Sampling in these areas might be necessary for arriving at an ex- act diagnosis, meanwhile trying to avoid underestima- tion of the malignant potential of the tumour when sampling of non-high grade areas occurs. Sampling is also imperative for performing additional studies such as cytogenetic analysis, as will be explained later.
Estimating the percentage of necrosis preoperatively by the radiologist might also be important, since some tumours, e.g. Ewing’s sarcoma and extra-renal rhabdoid tumours, are treated preoperatively, and the pathologist is asked to estimate the percentage of viable tumour tis- sue left after treatment, as a measure of tumour re- sponse. This is quite elaborate and subjective since it is quite impossible, histologically, to distinguish between spontaneous or therapy-induced necrosis.
Close collaboration between radiologists and pathol- ogists is important, not only to decide where to sample but also in determining how to sample.
8.3 When Pathology Meets the Clinics
Once the lesion is localised, obtaining a biopsy in the appropriate tumour area is the next logical step. Ideally, the biopsy should occur in the centre where the patient will be treated. It is not surprising that a pathologist of- ten requests larger tumour samples or asks for a second opinion when being confronted with a soft tissue tu- mour. This is mainly due to the rarity of the lesions, the lack of adequately defined diagnostic criteria and the
“unearthing” of new entities. There are some 50 differ- ent subtypes of soft tissue tumours. Considering the rarity of the lesions and the difficulties associated in ob- taining an adequate biopsy from representative tumour areas, which can only occur once the tumour has been investigated thoroughly by adequate imaging means (MRI and CT), biopsy procedures in the primary care settings should be discouraged. Biopsy procedures without having adequate radiological information giv- en by CT or preferable by MRI, should be avoided, un- less the lesion is small and superficially located. Exci-
sion biopsies should only be performed on superficial or subcutaneous tumours which are usually smaller than 2–3 cm in size. Excision biopsies of larger and/or deep seated masses are best avoided.
Open, incisional biopsies are performed in most cen- tres. Open biopsies enable the pathologist to have enough tissue, not only with a preserved architecture of the tumour which is crucial for histological subtyping, but also for performing ancillary studies, e.g. immuno- histochemistry, cytogenetic and gene expression profil- ing-studies, when deemed necessary.
Recently, more emphasis is being placed on the abili- ty of pathologists to make a reliable diagnosis on rela- tively smaller amounts of tissues. This is mainly due to economic reasoning and patient comfort. Core Needle Biopsies (CNB) and Fine Needle Aspiration Biopsies (FNAB) are temporarily quite popular [7, 8]. The main advantages of both these methods include the feasibili- ty in performing these kinds of biopsies in outpatient clinical settings, the minimal risk of complications, the cost-effectiveness and the acceptability of the patient.
Moreover, the reduced risk of spilling tumour cells dur- ing the biopsy procedure and the lack of compromising the subsequent surgical management are considered additional features in favour of FNAC or CNB vs open biopsy. Spilling of tumour cells and compromising here- with the surgical bed and subsequent therapeutic mea- sures are often the case when open, incisional biopsies are performed by inexperienced surgeons. This urges that for soft tissue lesions suspicious of being malig- nant, biopsy procedures should be decided on after in- tense cross-talk between pathologists, radiologists and clinicians, and should preferable be performed in refer- ence centres.
Nevertheless, despite the above-mentioned obvious advantages of sampling limited material, this does not imply that pathologists are willing or able to make a specific diagnosis on small amounts of material. Limit- ed sampling might make grading and exact subtyping quite difficult. Moreover, preoperative chemo- and/or radiotherapy for some tumours, e.g. Ewing sarcoma, might make adequate and reliable histological typing and grading evaluation of resected specimens extreme- ly difficult, if not impossible.
Preoperatively, with limited material, especially after FNAC, the pathologist is in some cases only able to pro- vide diagnoses such as “malignant soft tissue tumour, not otherwise specified” or “spindle” or “round cell” tu- mour. This would imply in some cases that it would even not be possible to know exactly which tumour was treated, since preoperative therapy might distort mor- phology considerably.
Deep seated lesions, however, are amenable for CNB, avoiding the discomfort of open, incisional biopsy in these specific circumstances. FNAC is indicated mostly for assessing local recurrence or metastasis or in cases
ples stored in culture media for subsequent cytogenetic analysis and fixing tissue in glutaraldehyde for electron microscopic investigation are necessary when morpho- logic features on haematoxylin and eosin slides are deemed insufficient. Tumour tissue stored in this man- ner is never wasted and can be consulted later on for di- agnostic and research purposes, when appropriate.
Yet, in most cases the pathologist does not have the luxury of having enough material to be stored in a way as mentioned before.
Immunohistochemistry might be beneficial in these circumstances. Immunohistochemistry is the use of an- tibodies specific to epitopes located on the cells of inter- est. A range of immunohistochemical markers are avail- able nowadays. These antibodies should be applied ac- cording to the morphology of the tumour (Table 8.1), which should enable the pathologist to arrive at an ac- curate diagnosis. The rationale of the use of these anti- bodies lies with the assumption that antigens expressed by the tumour cells are the same as those expressed by their normal counterparts. For example, myogenin and MyoD-1 seem to be specific for muscle cells, since foetal muscle cells do express these proteins. Similarly, blood vessels are CD31 and CD34 immunoreactive (Figs. 8.1 and 8.2), and consequently their tumoral counterparts, the angiosarcomas, are immunoreactive for both these proteins. In a number of cases, however, this simplifica- tion does not hold. Some soft tissue tumours, e.g. an- giosarcomas might have an anomalous expression of proteins pointing to another lineage, e.g. cytokeratin.
For example, only two soft tissue tumours do have
“true” cytokeratin expression, namely epitheloid sarco- ma and synovial sarcoma. These two tumours have so far an undetermined origin. The example of the syn- ovial sarcoma is interesting in another respect. Al- though the name might imply a synovial origin, most synovial tumours do not arise in or near joints. Using transcriptional gene profiling experiments it has been shown that these tumours might have a neuroectoder- mal (stem cell?) origin [9]. Another example illustrative of the incongruent relation between a normal cell and its tumoral counterpart are the rhabdomyosarcomas.
Some rhabdomyosarcomas are located in bladder and gall bladder, yet in these organs no striated muscle cells are found. Expression of a single marker should there- fore not be considered in all cases as indicative of the origin of a specific tumour. The example mentioned be- fore of the synovial sarcoma might also be applied to the gastrointestinal soft tissue tumours (GIST), of which a plethora of publications have arisen during the past years. One of the criteria to diagnose GIST-tu- mours is the presence of the membrane protein c-kit (Figs. 8.3 and 8.4). Since this protein is detected on the interstitial cells of Cajal in the intestine, it has been speculated that these cells might be the origin of these tumours. However, some tumours are c-kit negative and where incisional biopsy is difficult to perform. Never-
theless, in some specialised centres, with extensive ex- pertise in soft tissue tumour pathology, reliable subtyp- ing might be possible in most of the (untreated) cases using FNAC or CNB. Similarly, distinguishing between benign and malignant lesions can be done in these spe- cialist centres, which, as mentioned before, is currently considered sufficient to initiate treatment. Failure of ex- act subtyping does not in all instances withhold initiat- ing therapy, since quite limited information is needed by the clinician. This information might be rendered by limited samples, with FNAB yielding less material than CNB. Obviously, when both FNAB and CNB are inade- quate, an open and incisional biopsy is mandatory. Nev- ertheless, an open, incisional biopsy is recommended in centres without explicit experience in FNAC or CNB.
As already mentioned, in this era of targeted therapy, histological subtyping is crucial. Since imaging modali- ties cannot reliably distinguish between benign and definitely malignant lesions, morphology has the last word. This can only be accomplished in a reliable man- ner with substantial material.
8.4 When Morphology is not Enough
Considering the increased importance of histological typing and the presence/absence of molecular markers for diagnostic, predictive or prognostic purposes, ade- quate handling of biopsy material is mandatory.
It is imperative that radiologists and clinicians need to be aware of not only the total amount of material they (need to) sample, but also on the handling of soft tissue tumour tissue by the pathologist. The tissue needs to be transported fresh in an unfixed state to the pathology department. This allows an accurate and exact orienta- tion of the specimen by, ideally, both the surgeon and the pathologist who is going to diagnose the lesion. This cross-talk is also important for assessing the often com- plex resection margins of soft tissue tumour specimens.
Resection margins, be it other than fascia or perios- teum, less than 1 cm are associated with a substantial risk of recurrence, prompting additional treatment such as. e.g. radiotherapy or wider surgical excision.
When the tissue arrives at the pathology department, critical decisions are made with respect to how the tis- sue is handled. With limited amounts of material, as ob- tained by FNAC and CNB, the pathologist is deemed to include all tissue for, e.g. formalin fixation and subse- quent paraffin embedding. Yet, as mentioned before, histological typing of the tumour should be the main objective of the pathologist. With small amounts of tis- sue, it is sometimes quite difficult, even impossible to asses the histology of the tumour. Moreover, differenti- ating between benign and low grade malignant lesions is not always possible. Snap-frozen tissue, tissue sam-
some do not at all arise in the gastrointestinal tract, e.g.
the extra-gastrointestinal GIST. C-kit expression in most of these cases is related to an acquired gain of function mutation of the c-kit gene. C-kit expression in GIST-tumours is also related to the therapeutic effec- tiveness of imatinib mesylate (Glivec). Intriguingly, GIST-tumour cells expressing the protein without hav- ing the corresponding mutation have less response to imatinib mesylate (Glivec) therapy.
Considering that GIST-tumours were previously cat- egorised as being of smooth muscle cell or neural ori- gin, the comments above illustrate that identification of genetic alterations in histiotype-specific tumours is able to change the morphological classification of soft tissue tumours.
If sufficient tissue is available, additional investiga- tions can be performed such as cytogenetic analysis and electron microscopic studies.
Table 8.1. Immunohistochemical panel according to the morphology of the tumour Immunohistochemical panels
Spindle cell tumours Cytokeratin, epithelial membrane antigen (EMA), desmin, smooth muscle actin (SMA), h-caldesmon, S-100 Round-cell tumours CD99, myogenin, desmin, cytokeratin, EMA, synaptophysin, FLI-1
Epitheloid tumours Cytokeratin, EMA, CD34, CD31, S-100, FLI-1
Fig. 8.1. Angiosarcoma (magnification:¥200) Fig. 8.2. Angiosarcoma tumour cells staining for CD31 (magnifi- cation:¥200)
Fig. 8.3. Gastrointestinal stromal tumour (GIST) (magnification:
¥200) Fig. 8.4. GIST-tumour cells staining for CD117 (c-kit) (magnifica-
tion:¥200)
8.5 World Health Classification of Soft Tissue Tumours 2002 [10]
Once a specific diagnosis is reached the lesion should be categorized accordingly. Recent advances in cytogenetic and molecular genetic information, the recognition of new entities and the enhanced understanding of the bi- ological behaviour have prompted a new morphological classification of soft tissue tumours.
It is recommended to categorise the lesions in a be- nign, intermediate (locally aggressive), intermediate (rarely metastasizing) or a malignant category:
1. Benign lesions: e.g. lipoma. These tumours mostly do not recur and if recurrence occurs it is most of the time in a non-destructive fashion. In exceptional cir- cumstances metastasis might occur, as in e.g. benign fibrous histiocytoma.
2. Intermediate (locally aggressive): e.g. kaposiform haemangioendothelioma and desmoid fibromatosis.
These tumours are infiltrative and might recur local- ly, sometimes in a destructive fashion when incom- pletely excised.
3. Intermediate (rarely metastasizing): e.g. retiform haemangioendothelioma. These tumours are locally aggressive, recur if incompletely excised and may give rise to metastasis. Histological features are not, in most cases, predictive for metastasis.
4. Malignant: e.g. round cell liposarcoma. These tu- mours are infiltrative, recur locally if incompletely excised and have a significant risk of metastasis.
Significant changes have occurred in the new WHO- classification compared with the previous classification published in 1994. Major adaptations include:
1. Adipocytic tumours: new entities include myolipoma and chondroid lipoma. Four types of liposarcoma oc- cur:
a) Atypical lipomatous tumour/ well differentiated li- posarcoma: these terms are synonyms, although the term well differentiated liposarcoma is prefer- able in retroperitoneal and paratesticular areas, due to its higher morbidity and mortality in these specific areas.
b) Dedifferentiated liposarcoma: previously were these tumours categorised as being definitively high grade. In the current classification a low- grade variant is included. These tumours, although being of the dedifferentiated type, do have a more protracted clinical course compared with other dedifferentiated soft tissue tumours.
c) Myxoid liposarcoma: both round-cell liposarcoma and myxoid liposarcoma are considered to be one entity, with the round-cell liposarcoma type being at the less dedifferentiated spectrum of this tu- mour.
Cytogenetic analysis is the identification of specific molecular alterations thought to be specific for certain tumours. This concept will be discussed elsewhere by A.
Sandberg.
Performing electron microscopic investigations for soft tissue tumours is not routine in most pathology de- partments. This might be due to the pathologists them- selves who are unwilling to embrace and further devel- op or invest in “old” techniques such as electron mi- croscopy. In any branch of medicine any technique which might offer additional clinical information is dif- ficult to be discarded, in contrast to electron microscopy for soft tissue tumour diagnostics.
It is argued that electron microscopy does not add much to the diagnosis of soft tissue tumours.
Specific examples highlight the importance electron microscopy might have in soft tissue tumours:
1. Distinguishing between malignant peripheral nerve sheath tumours and monophasic synovial tumours.
Monophasic synovial sarcomas are much more chemosensitive than malignant peripheral nerve sheath tumours.
2. Distinguishing between mimics of soft tissue tu- mours such as sarcomatous carcinoma and metastat- ic melanoma.
3. Diagnosing alveolar soft tissue sarcoma in an aspe- cific clinical or metastatic setting.
All the above-mentioned features highlight a few issues:
1. The pathologist needs to have sufficient material to perform the ancillary studies which he seems neces- sary to arrive at a diagnosis as accurate as possible.
2. Without sufficient material it is difficult to distin- guish between benign, low grade and sometimes even high grade malignant lesions.
3. Ancillary immunohistochemical techniques might help the pathologist in arriving at a diagnosis.
4. The expression of molecules on soft tissue tumour cells does not always imply a corresponding normal counterpart.
5. The expression of molecules on tumour cells, is sometimes not merely diagnostic, but might in some cases also have prognostic and predictive informa- tion.
6. It might be argued that the classification of soft tissue tumours by the corresponding normal counterpart of the tumours should be abandoned, considering the anomalous expression of proteins by some tumour cells. Some (all?) soft tissue tumours seem to arise from somatic stem cells, and not from its normal counterpart.
d) Pleiomorphic liposarcoma: these are the rarest lipomatous tumours and are considered to have a worse prognosis.
2. Fibroblastic/myofibroblastic tumours: keloid is not longer regarded as a soft tissue tumour. New benign entities include, e.g. giant cell fibroblastoma, Gardner fibroma and angiomyofibroblastoma. New malignant entities include, e.g. sclerosing epitheloid fibrosarco- ma and low-grade fibromyxoid sarcoma. Important- ly, the term “hemangiopericytoma” is included in the chapter of the solitary fibrous tumours considering the lack of morphological dissimilarities between these two tumour types.
3. Fibriohistiocytic tumours: pleiomorphic malignant fibriohistiocytic tumour (MFH) is considered to be a high grade undifferentiated tumour.
4. Smooth muscle tumours: inclusion of GIST-tumours in different organs, e.g. in the gastrointestinal tract is considered in the respective WHO-volumes.
5. Pericytic/perivascular tumours: only glomus and myopericytoma are included in this category.
6. Skeletal muscle tumours: the embryonal, alveolar and pleiomorphic subtypes are included.
7. Vascular tumours: in this category is only the epith- eloid haemangioendothelioma included as a malig- nant subtype of the haemangioendotheliomas.
8. Chondro-osseous: myositis ossificans and fibro-os- seous pseudotumours are considered to be variants
of nodular fasciitis. Since extra-skeletal myxoid chondrosarcoma does not seem to have chondroid differentiation, this lesion is placed in the category
“tumours of uncertain differentiation”.
8.6 To Grade or not to Grade?
The purpose of grading is to segregate tumours in those with a favourable prognosis, amenable to surgical exci- sion only, and in those with an unfavourable prognosis, amenable to surgical treatment and additional chemo- therapeutic and/or radiotherapeutic treatment. More- over, adequate grading is considered to be more repro- ducible than histological typing, might even circumvent diagnostic disagreement between pathologists and might triage unclassified sarcoma in a low or high risk category. As a matter of fact, most contemporary oncol- ogists triage their patients for subsequent treatment by merely relying on the tumour grade provided by the pathologist. Relying in a way that grading is in all cir- cumstances able to distinguish between benign, low grade and high grade lesions, irrespective of histiotype.
Currently, two grading classifications exist: the National Cancer Institute (NCI)-grading system (Table 8.2) and the Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC)-grading system, the so called “Trojani system” (Table 8.3). The FNCLCC-
Table 8.2. National Institute of Cancer grading system
Common histological types
Grade 1 Grade 2 Grade 3
Well-differentiated liposarcoma Pleiomorphic liposarcoma Alveolar rhabdomyosarcoma
Myxoid liposarcoma Fibrosarcoma Soft tissue osteosarcoma
Deep-seated dermatofibrosarcoma protuberans Malignant fibrous histiocytoma Primitive neuroectodermal tumour
Some leioymyosarcomas Alveolar soft part sarcoma
Epitheloid haemangioendothelioma Synovial sarcoma Mesenchymal chondrosarcoma
Spindle cell haemangioendothelioma Leiomyosarcoma
Infantile fibrosarcoma Neurofibrosarcoma
Subcutaneous myxofibrosarcoma or or
0–15% necrosis >15% necrosis
Table 8.3. Fédération Nationale des Centres de Lutte Contre le Cancer grading system
Differentiation score Mitosis score (per 10 HPF) Necrosis
Score 1 Sarcomas resembling adult mesenchymal tissue 0–9 No necrosis
Score 2 Sarcomas of certain histiotype >9; <20 <50% necrosis
Score 3 Embryonal/undifferentiated sarcomas 20 or more >50% necrosis
and sarcomas of uncertain histiotype
HPF: high power field.
The total scores for each grade are as follows: grade 1, 2–3; grade 2, 4–5; grade 3; 6–8
giosarcomas. Some tumours are definitively of low ma- lignant potential, e.g. infantile fibrosarcoma and well differentiated liposarcoma. Some tumours are not yet amenable for grading, e.g. malignant giant cell tumour and hemangiopericytoma. Conversely, some tumours are amenable for grading, e.g. leiomyosarcoma, myxoid liposarcoma and fibrosarcoma.
When looking at both grading systems it is obvious that some subjectivity in applying these parameters is unavoidable. Although, e.g. mitotic figure counting should be performed rigidly in soft tissue tumours, the inter-observer reproducibility, equally as e.g. in breast carcinoma, is in the range of 60–70%, even among soft tissue specialists. Cellularity, mitotic figure counting and, e.g. extent of necrosis are all influenced by sam- pling, fixation, section thickness, the number of blocks taken and microscopic field size. Also, as previously mentioned, having small amounts of material precludes grading in most cases. Considering the intra-tumoral heterogeneity of soft tissue tumours, sampling error is likely to occur, especially when trying to avoid highly necrotic areas. It is also difficult to asses macroscopical- ly the extent of necrosis since myxoid and hyalinized ar- eas might mimic necrosis. There is also no consensus as whether the mere presence or the extent of necrosis de- termines prognosis. Similarly, how to measure necrosis, micro- or macroscopically, is not at all defined.
Counting mitoses is, as previously mentioned, inher- ently subjective. Distinguishing mitotic figures from py- knosis is sometimes difficult. There is also no universal consensus on which cut-off and microscopic field size to use.
Some have argued that intra-tumoral (lympho)vas- cular invasion should also be searched for. In analogy with epithelial tumours, there is considerable difficulty in distinguishing artefactual retraction from “true”
lymphatic or blood vessels. Immunohistochemistry might be helpful in this respect.
In the FNCLCC-system differentiation is one the pa- rameters to asses. As previously mentioned, no normal counterpart exists for some tumours, e.g. epitheloid sarcoma.
Also, some tumours, e.g. GIST-tumours do have their own grading system including tumour size, mitotic fig- ure counting and location [12]. Here also, estimating mitotic figure counting is subjected to the same inher- ent subjectivity as mentioned before. Size should be as- sessed by the pathologist on a fresh resected specimen.
It is argued that the FNCLCC- and the NCI-grading system should be restricted to adult tumours, consider- ing the unique features of childhood soft tissue tu- mours. For example, childhood rhabdomyosarcoma- tous tumours are graded according to the system pro- vided by the Intergroup Rhabdomyosarcoma Study Group where risk assignment, rather than grading is performed by a combination of both exact histology grading system is considered to be more reproducible
and more apt to classify patients in either grade 1 or grade 3, avoiding more frequently the inclusion of pa- tients in the “indeterminate” grade 2 category, com- pared with the NCI-system. Both systems are included in the latest WHO Soft Tissue Tumour Classification.
Both are three-tiered systems, assigning different weights to histiotype, necrosis, mitotic figures, cellulari- ty, pleiomorphism and differentiation.
Grading should be performed on untreated tumours on representative and well preserved tissue. This should be the case for all tumours, be it sarcomatous or non- sarcomatous. Previous therapy, whether be it chemo- therapy or radiotherapy, precludes grading in all cir- cumstances since both mitotic figures and the extent of necrosis can be affected. It is also well known that soft tissue tumours acquire a higher grade in local recur- rence or metastatic lesions. Grading of visceral sarco- mas should also be avoided.
It is a genuine fact that some soft tissue tumours are not amenable at all to exact histological grading. Histo- logical typing does not in all cases provide the neces- sary prognostic and/ or predictive information. Some soft tissue tumours should not at all be graded, especial- ly considering the inherent malignant potential of these tumours. According to the WHO 2002 Classification of Soft Tissue Tumours grading should not be performed on malignant peripheral nerve sheath tumours, an- giosarcomas, extraskeletal myxoid chondrosarcoma, alveolar soft part sarcoma, clear cell sarcoma and epith- eloid sarcoma.
Considering both grading systems it is quite clear that some histological typing is crucial for grading soft tissue tumours. As previously mentioned, exact histo- logical typing should be the main aim of the patholo- gist, yet, it is not always possible, as is the case when hav- ing only small amounts of tissue. On the other hand, if a pathologist wants to use one of the grading systems, an attempt at histological typing is necessary, despite the inherent difficulties of soft tissue diagnostics. It is even elaborated that both tumour type and tumour size are able to adequately predict survival, withholding grading in these cases [11].
Mitotic counts are inherently associated with the FNLCC-grading system. However, as mentioned before, histological typing is mandatory because some tumours behave in a benign fashion despite having many mi- toses, e.g. cellular schwannoma and atypical fibroxan- thoma. Some tumours are considered low grade despite the presence of mitotic figures, e.g. infantile fibrosarco- ma. Conversely, some tumours are inherently malignant despite the lack of mitotic figures and/ or necrosis, e.g.
the round-cell liposarcoma.
Grading systems can thus not be meaningfully ap- plied to all soft tissue tumours. Some tumours are defin- itively malignant, e.g. Ewing sarcoma/MPNET and an-
and the location of the tumour. A modified TNM stag- ing system has even developed for these tumours, with sites being defined as unfavourable (e.g. bladder, prostate) and favourable (e.g. orbit, head and neck) [13].
Importantly, currently no method, e.g. DNA flow cy- tometry, other than histological subtyping and grading provides more prognostic information in soft tissue tu- mours. It is clear that there is no single universal para- meter applicable to all tumours. In ideal circumstances should grading systems be developed and applied for each histological subtype.
8.7 Reporting Soft Tissue Tumours (Table 8.4) A template for reporting soft tissue tumours should in- clude the following:
1. Histological type.
2. Grade (1–3) or risk assessment, when applicable.
3. Size, assessed by the pathologists on fresh, unfixed tissue.
4. Location, cross-talk with radiologist and surgeon is crucial, especially when trying to identify infiltration in critical structures, e.g. blood vessels and nerves.
5. Margins, especially mentioning those margins <1 cm and whether there is a surrounding fascia or perios- teum.
6. Necrosis, if assessed macroscopically it should ideal- ly be on fresh, unfixed and untreated tissue.
7. Additional investigations, e.g. karyotyping.
8.8 Gene Expression Profiling
and Tissue Microarrays in Soft Tissue Tumours Soft tissue tumours are heterogeneous, both with re- spect to the morphology and the clinical behaviour. Al- though grade and histiotype currently determine treat- ment options and prognosis, prediction of clinical be- haviour remains elusive in most circumstances, especially in individual cases. Tumour growth, even within the same subtype, ranges from slow growing with low risk of metastasis, to fast growing lesions with considerable morbidity and mortality. This precludes adequate morphological classification.
Furthermore, progress in the adequate identification of molecules that might be useful for therapeutic target- ing is hampered by the examination of only a small set of genes by the more conventional methods such as, e.g.
Northern and Southern blotting.
Gene expression profiling might be useful in this re- spect. The basis of microarray studies is the labelling of cDNA of the tumour with a fluorescent dye. This la- belled cDNA is hybridised together with a reference fluorescent labelled cDNA onto a microarray chip. The slide is scanned after hybridisation on a gene specific probe and the ratio values of the intensity of the sample cDNA vs the reference cDNA is measured and calculat- ed. Thousands of mRNA transcripts can be analysed in this manner.
A number of different array technologies are avail- able, each with its advantages and disadvantages. A main review of all aspects of gene expression profiling is beyond the scope of this review. These are covered more extensively in excellent reviews ([14] and references therein).
Sample preparation is crucial for these kinds of ex- periments. This method measures the abundance of gene transcripts (mRNA). One caveat is the fragility of mRNA by its rapid degradation by RNAses present in cells and tissues. Thus, it is crucial that the tissue is stored as rapid as possible at –80 °C when sampling the tissue. Although formalin fixed and paraffin embedded tissue might also be useful for RNA extraction, this is more laborious and the risk of not having substantial RNA is considerable. This is mainly due to the slow in- activation of RNAses by the fixative.
Gene expression profiling has revealed novel sub- types in other tumours, e.g. in breast cancer and malig- nant lymphoma. An illustrative example of the applica- tion of gene expression profiling in soft tissue tumours include a study performed by Nielsen and colleagues.
Unsupervised analysis of a set of 5500 genes demon- strated that synovial sarcomas, GIST-tumours, malig- nant peripheral nerve sheath tumours and some leiomyosarcomas clustered in separate groups, in sup- port of the morphological classification of these tu- mours. When applying a supervised analysis, several
Table 8.4. Template for soft tissue tumour reporting Template
a) Histological type
b) Grade (1–3), or risk assessment c) Size
d) Location e) Margin status f) Necrosis
h) Additional investigations
for ancillary studies, such as karyotyping and electron microscopy. Pathologists should aim at trying to histio- type and grade soft tissue tumours, whenever consid- ered adequate. Clinicians need to be aware that grade does not replace histology. It is mandatory that soft tis- sue tumoral specimens should always be stored in liq- uid nitrogen for future diagnostic and research purpos- es. Gene expression profiling and tissue microarrays re- fine the morphological classification and are currently the best ways to identify new diagnostic, prognostic and predictive molecules in soft tissue tumours. Clinical tri- als with target specific compounds are ongoing. Cen- tralised pathological review is considered critical in these circumstances.
Things to remember:
1. Pathological diagnosis of soft tissue tumours is difficult. Second opinion or even preferable a cen- tralised pathological review should be mandatory for difficult cases.
2. Biopsy and diagnosis should be performed in the centre were the patient will be treated.
3. Cross-talk between radiologists, clinicians and pathologists should occur at all phases of patient management.
4. Clinicians and radiologist need to be aware of the amount of tissue the pathologist needs for an ac- curate diagnosis.
5. Grading does not replace histology.
6. Pathologists should aim at trying to histiotype and grade soft tissue tumours.
7. It is mandatory that soft tissue tumoral tissue should always be stored in liquid nitrogen for future diagnostic and research purposes.
References
1. West RB, Corless CL, Chen X, Rubin BP, Subramanian S, Mon- togomery K, Zhu S, Ball CA, Nielsen TO, Patel R, Goldblum JR, Brown PO, Heinrich MC, van de Rijn M (2004) The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation sta- tus. Am J Pathol 165(1):107–113
2. Blay JY, Ray-Coquard I, Alberti L, Ranchere D (2004) Targeting other abnormal signalling pathways in sarcoma; EGFR in syn- ovial sarcoma, PPAR-gamma in liposarcoma. Cancer Treat Res 120:151–167
3. McArthur G (2004) Molecularly targeted treatment for der- matofibrosarcoma protuberans. Semin Oncol 31(2 Suppl 6):
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4. Enzinger and Weiss’s soft tissue tumors. Fourth Edition (2001) 5. Guillou L, Coindre JM, Bonichon F, Binh Bui N, Terrier P, Collin F, Vilain M, Mandard A, Le Doussal V, Leroux A, Jacquemier J, Duplay H, Sasttre-Garau X, Costa J (1997) Comparative study of the National Cancer Institute and French Federation of Cancer Centers Sarcoma Group Grading Systems in a popula- tion of 410 adult patients with soft tissue sarcoma. J Clin Oncol 15:350–362
6. Dahlin’s Bone Tumors. Sixth Edition (1996)
transcripts were found to be specifically upregulated in the respective subgroups. For GIST-tumours this in- cluded c-kit, for synovial sarcomas EGFR-1 and SSX- transcripts were upregulated [15].
Importantly, the identification of these transcripts in specific tumour histiotypes prompted the development of agents directed specifically at these molecules (Glivec directed at c-kit positive GIST-tumours and Iressa at EGFR-1 positive synovial sarcoma). Clinical trials are in progress.
Moreover, using gene expression profiling it has also been shown that synovial sarcomas clustered on the one hand separately from other tumours, but on the other hand clustered closely to malignant peripheral nerve sheath tumours and expressed genes that are involved in the migration of neural crest cells, such as, e.g.
ephrin-3 and endothelin-3, raising suspicion that syn- ovial sarcomas might be of neuroectodermal origin.
Considering the plethora of data provided by gene expression profiling, many new markers have emerged which need to be tested and validated on large tumour sets.
Tissue microarrays might be useful in this respect.
Tissue microarrays utilises 0.6-mm core needle biopsies of representative areas of the tumour on a paraffin block. This core is subsequently re-embedded in a nov- el paraffin block. With this method a large number of tumour samples, ranging to 1000 samples on a single slide, can be analysed at the RNA, DNA or protein level.
The GIST-marker DOG-1 was evaluated and validated in such a manner, following identification by gene ex- pression profiling [1].
8.9 Conclusions and Future Perspectives
Soft tissue tumours are rare tumours. Pathological diag- nosis is difficult. This is mainly due to the poorly de- fined pathological criteria and the more than 50 differ- ent histological subtypes. Second opinion or, even preferable, a centralised pathological review should be mandatory for difficult cases. The Belgian Soft Tissue Neoplasm Registry (BSTNR) is worth mentioning in this respect. The BSTNR is composed partly of experi- enced soft tissue tumour pathology- and radiology ex- perts confirming or even providing an alternative diag- nosis in submitted cases. Biopsy and diagnosis should be performed in the centre were the patient will be treated. It is imperative that cross-talk between radiolo- gists, clinicians and pathologists occurs at all phases of patient management. Preoperatively, when choosing the biopsy technique and the area to sample and postopera- tively, at the bench side. Clinicians and radiologist need to be aware of the amount of tissue the pathologist needs for an accurate diagnosis. Adequate sampling is mandatory, not only for diagnostic purposes, but also
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mours. A prospective study of malignancy and tumor pheno- type on MRI. Epidemiological studies and evaluation of char- acterization parameters. Thesis, University of Antwerp 9. Allander SV, Illei PB, Chen Y, Antonescu CR, Bittner M,
Ladanyi M, Meltzer PS (2002) Expression profiling of synovial sarcoma by cDNA microarrays: association of ERBB2, IGFBP2, and ELF3 with epithelial differentiation. Am J Pathol 161(5):
1587–1595
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of malignancy and prognosis of gastrointestinal stromal tu- mors: a review. Hum Pathol 33(5):478–483
13. Meyer W, Spunt S (2004) Soft tissue sarcoma of childhood.
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14. Yang YH, Speed T (2002) Design issues for cDNA microarray experiments. Nat Rev Genet 3(8):579–588
15. Nielsen TO, West RB, Linn SC, Knowling MA, O’Connel JX, Zhu S, Fero M, Sherlock G, Pollack JR, Botstein D, van de Rijn M (2002) Molecular characterisation of soft tissue tumours: a gene expression study. Lancet 359(9314):1301–1307
