A CURRENT PERSPECTIVE ON THE ROLE FOR MOLECULARSTUDIES IN SOFT TISSUE TUMOR PATHOLOGY
Special thanks to my resident Dr Babar Yasin for preparing the presentation
ADAPTED FROM THE ABOVE ARTICLE FROM Seminar in diagnostics Pathology 30 (2013) 375-381
Angelo P. Dei Tos, MD Department of Pathology, Treviso General Hospital,
Piazza Ospedale,1 31100 Treviso, Italy
INTRODUCTION
The marriage of Molecular Genetics and soft tissue
Pathology.
IMPACTS
More accurate definition of disease entities and validation of classification schemes.
Improved diagnostic accuracy. Identification of molecular predictive
and prognostic markers. Discovery and validation of therapeutic
molecular targets.
MOLECULAR TECHNIQUES ANALYZE: Nucleic acids (RNA and DNA), either via
hybridization on a slide(i.e., fluorescent in situ hybridization—FISH)
OR On isolated DNA or RNA via polymerase
chain reaction (PCR) techniques (i.e.,reverse transcriptase PCR and quantitative PCR)
It is MANDAOTORY that the results get interpreted in context with
morphology. Genetic assessement is
an important ADJUNCT not a replacement to
conventional morphological tools.
MOLECULAR GENETICS/PATHOLOGY AS A TOOL TO IMPROVEDIAGNOSTIC ACCURACY
Soft tissue tumors are heterogeneous group of neoplasm which can be :
Benign Boderline Malignant
Challenge for Pathologists
Diagnostic inaccuracy affects the treatment and
development of new drugs as clinical trials depend upon
tumor classification.
REASONS: Rare group of diseases (< 2% of all
cancers) Extremely heterogenous. Over 100
subtypes are being described. Usual features of malignancy (?) are not
always applicable to these.Histologically worrisome leisons may actually be benign e.g.
Nodular fascitis
Cytollogically innocent neoplasm may behave aggressively e.g.
Low-Grade Fibromyxoid Sarcoma.
DIAGNOSTIC UTILITY OF MOLECULAR GENETICS TESTING CAN BE FORESEEN IN:
Distinguishing specific subtypes of sarcomas.
Supporting diagnosis in non-canonical clinical presentations.
Distinguishing sarcomas from benign mimickers.
DISTINGUISHING SPECIFIC SUBTYPES OF SARCOMAS
Molecular genetics has proved diagnostically useful in two relatively large groups of mesenchymal malignancies: round cell sarcomas and pleomorphic sarcomas.
Round cell sarcomas include : Ewing sarcoma Desmo-plastic small round cell tumor Alveolar rhabdomyo-sarcoma Poorly differentiated round cell synovial
sarcoma Mesenchymal chondrosarcoma Minority of cases of round cell liposarcoma
Distinction is crucial
because the therapeutic approach differs.
KERATIN + EWING SARCOMA VS POOFRLY DIFF ROUND CELL SYNOVIAL SARCOMA
SYT REARRANGEMENTS IDENTIFIED BY FISH REPRESENT AN EXTREMELY USEFUL DIAGNOSTIC TOOL
The demonstration by FISH of EWSR1,SS18, and FOXO1 rearrangements in EWS, PDSS, and ARMS, respectively, or, alternatively, of specific chimerical transcriptsby PCR-based techniques is of great help for achieving acorrect diagnosis.EWSR1 FISH results need
to be interpreted in context with morphology and IHC
findings.
PLEOMORPHIC SARCOMAS
Sub classification is very important. myogenic differentiation in pleomorphic
sarcomas is associated with a less favorable outcome.
Another important point is the distinction, among retroper-itoneal sarcomas, of dedifferentiated liposarcoma (DDLPS) from other pleomorphic sarcomas, most often pleomorphic leiomyosarcoma.
DEDIFFERENTIATED LIPOSARCOMA (DDLPS)
The recognition of DDLPS is based on the identification of a well-differentiated lipogenic component associated with a high-grade, most often non-lipogenic, sarcoma.
Core biopsies usedfor diagnostic purposes may leave the lipogenic component unsampled.
DDLPS exhibit better out- comes when compared to other pleomorphic sarcomas, and its accurate recognition may lead to adopt a more aggressive surgical strategy. (Locally aggressive)
Detection of MDM2 amplification by FISH or quantitative RT-PCR certainly represents a useful diagnostic adjunct.
The MDM2 gene (as well as CDK4 and HMGA2) maps at the 13q12–15 chromosome region and is amplified in both well-differentiated and dedifferentiated liposarcomas.
MDM2 testing is also potentially useful in distinguishing between myxoid liposar-coma (MDM2 negative) and WD/DDPLS with myxoid change.
The separation of the two conditions again allows adoption of proper treatment in consideration of the high sensitivity of myxoid liposarcoma to the marine-derived alkaloid named trabectedin.
DETECTION OF MDM2 AMPLIFICATION BY FISH
A LIST OF THE MOST COMMON GENETIC ALTERATIONS IN SOFT TISSUE TUMORS.
Tumor Gene Mutation Gene InvolvedAlveolar rhabdomyosarcoma
t(2;13)(q35;q14)t(1;13)(p36;q14)
PAX3–FOXO1APAX7–FOXO1A
Alveolar soft part sarcoma
(X;17)(p11.2;q25) ASPL–TFE3
Angiomatoid fibrous histiocytoma
t(12;16)(q13;p11)t(12;22)(q13;q12)t(2;22)(q34;q12)
FUS–ATF1ATF1–EWSR1CREB1–EWSR1
Aneurysmal bone cyst t16;17)(q22;p13) CDH11–USP6Atypical lipomatous tumour/dedifferentiated liposarcoma
Amplification MDM2, CDK4, and HMGA2
Central/periosteal osteosarcoma
Point mutation IDH1/IDH2
Clear cell sarcoma t(12;22)(q13;q12)t(2;22)(q34;q12)
ATF1–EWSR1CREB1–EWSR1
Dermatofibrosarcoma protuberans
t(17;22)(q22;q13) COL1A1–PDGFB
Desmoid-type fibromatosis
Activating mutation BCTN1
Desmoplastic round cell tumor
t(11;22)(p13;q12) WT1–EWSR1
Endometrial stromal sarcoma
t(7;17)(p15;q21)t(6;7)(p21;p15)t(6;10)(p21;p11)t(10;17)(q22;p13)
JAZF1–JJAZ1PHF1–JAZF1PHF1–EPC1YWHAE–FAM22
Ewing sarcoma/PNET t(11;22)(q24;q12)t(21;22)(q22;q12)t(7;22)(p22;q12)t(17;22)(q12;q12)t(16;21) (q13;q22)t(2;22)(q33;q12)
EWSR1–FLI1EWSR1–ERGETV1–EWSEIAF–EWSFUS–ERGFEV–EWS
Extraskeletal Myxoid Chondrosarcoma
t(9;22)(q22;q12)t(9;15)(q22;q21)
EWSR1–NR4A3TCF12–NR4A3TFG–NR4A3
GIST Activating mutation KIT and PDGFRA
Intramuscular myxoma
Activating mutation GNAS1
Infantile fibrosarcoma t(12; 15)(p13;q25) ETV6–NTRK3
Inflammatory myofibroblastic tumor
t(2;19)(p23;p13.1)t(1;2)(q22-23;p23)
ALK–TPM4TPM3–ALK
Low-grade fibromyxoid sarcoma
t(7;16)(q33;p11)t(11;16)(p11;p11)
FUS–BBF2H7CREB3L1–FUS
Myxoid round cell liposarcoma
t(12;16)(q13;p11)t(12;22)(q13;q12)
FUS–DDIT3EWSR1–DDIT3
Pericytoma with t(7;12)t(7;12)(p22;q13)
ACTB–GLI
Pigmented villonodular synovitis
t(1;2)(p13;q37) COL6A–CSF1
Soft tissue myoepithelioma
t(1;22)(q23;q12)t(19;22)(q13;q12)
EWSR1–PBX1EWSR1–ZNF444
Synovial sarcoma t(X;18)(p11;q11) SS18–SSX1SS18–SSX2SS18–SSX4
SUPPORTING DIAGNOSIS IN NON-CANONICAL CLINICAL PRESENTATION
The combination of morphological criteria and genetics validates the recognition of rare diseases even when arising at non-canonical anatomic locations.
This is particularly true for referral centers wherein challenging cases tend inevitably to concentrate.
Molecular genetics has greatly Contributed to the identification of
primary Ewing sarcoma of the skin, kidney, and dura mater, as well as of viscerally located synovial sarcomas.
DISTINGUISHING TRUE SARCOMAS FROM BENIGN MIMICS
Morphological appearance of mesenchymal lesions does not always reflect the clinical behavior.
The distinction of sarcomas from benign mimics most often relies on morphology.
In a minority of cases molecular genetics may also prove diagnostically helpful.
Fibroid and myxoid areas.Swirling whorled growth
pattern.Low to moderate cellularity.
Bland cells.Minimal nuclear pleomorphism.
LOW-GRADE FIBROMYXOID SARCOMA Deceptively bland-looking spindle cell
mesenchymal malignancY with an aggressive clinical behavior.
The differential diagnosis of LGFMS includes benign lesions such as perineurioma, neurofibroma, cellular myxoma, and nodular fasciitis, as well locally aggressive neoplasms such as desmoid fibromatosis.
MUC4 expression is regarded as key
diagnostic feature.Identification of
FUS rearrangement via FISH or
identification of FUS-CREB3L2
transcript via PCR is very useful.
B- Catenin
THE ROLE OF MOLECULAR GENETICS IN THE ASSESSMENTOF PROGNOSIS:
Several attempts have been made to determine the prognostic value of molecular genetic findings.
Focused on Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma.
No meaningful molecular prognostic stratification can be foreseen for now.
A notable exception is represented by a molecular signature named CINSARC, which allows better separation of grade 2 sarcomas. This attempt is based on the use of a complex technique (CGH-array) and requires availability of fresh material, which hampers a large scale clinical application of CINSARC.
GASTROINTESTINAL STROMAL TUMORS (GIST),
Type of mutations involving both the KIT and PDGFRA genes are associated with distinctive outcomes.
Deletions occurring at the exon 11 of the KIT gene are associated with more aggressive disease, whereas mutations of exon 18 of the PDGFRA gene generally identify a more indolent clinical course.
THE ROLE OF MOLECULAR GENETICS IN PREDICTING THERESPONSE TO TREATMENT
Distinct mutation types in GIST reflect different objective response rates (greater for KIT exon 11 mutation and much lower for so-called wild-type GIST).
presence of specific mutations in the exon 18 of the PDGFRA gene (D842V) predict primary resistance to tyrosine kinase inhibitors.
Molecular assessement in GIST assumes a central role in clinical decision making.
TRABECTEDIN INMYXOID LIPOSARCOMA The identification of the two
specific fusion products of CHOP/DDIT3 gene with FUS and more rarely with EWSR1 is extremely helpful in distinguishing challenging examples of myxoid liposarcoma from other myxoid sarcomas and therefore to apply the adequate therapeutic regimen.
Another examples is use of crizotinib in inflammatory myofibroblastic tumors wherein assessment of the ALK gene may represent an important diagnostic confirmatory finding as well as a key biomarker of prediction.
Molecular Genetics represents the most valuable tool to identify and validate new
therapeutic targets.
Good examples are represented by MDM2, amplified in dedifferentiated liposarcoma and potentially targetable by Nutlin-A3, the mTOR pathway in malignant PEComa and lymphangioleiomyomatosis, PDGFB in DFSP, and KDR in angiosarcoma.
PITFALLS IN CLINICAL APPLICATIONS OF MOLECULAR PATHOLOGY
Promiscuity
Molecular pathology/genetics does not represent an alternative but a complement to surgical diagnostic pathology.
Considering the degree of molecular promiscuity of EWSR1 gene aberrations, the results of FISH analysis need to be mandatorily evaluated in context with morphology as EWSR1 aberrations are described in a variety of unrelated entities.
GENETIC OVERLAP AMONG UNRELATED MALIGNANCY
ETV6–NTRK3/t(12;15) Infantile fibrosarcomaAcute myeloid leukemiaSecretory breast carcinoma
ALK gene fusions Inflammatorymyofibroblastic tumorAnaplastic large celllymphomaSubsets of lungadenocarcinoma
FUS–ERG/t(16;21) Ewing sarcomaAcute myeloid leukemia
ASPL–TFE3/t(X;17) Alveolar soft part sarcomaSubset of pediatric renal
EWS–ATF1/t(12;22) and EWS-CREB1/t(2;22)
Clear cell sarcomaAngiomatoid fibroushistiocytoma
THE DEFINITION OF DISEASE ENTITIES AND VALIDATION OCLASSIFICATION SCHEM
Genetics has certainly played a key role in allowing a better understanding of many lesions.
The unification of myxoid and round cell liposarcoma within a single tumor entity represents one of the best examples.
Genetics has greatly helped in recognizing the close relationships between several tumors such as giant cell fibroblastoma and DFSP, LGFMS and epithelioid sclerosing fibrosarcoma, and hemosiderotic fibrolipomatous tumor and myxoinflammatory fibroblastic sarcoma.
The definition of new entities has been strongly supported by genetics.
The identification of t(7;19)(q22;q13) translocation in pseudomyogenic (epithelioid sarcoma-like) hemangioendothelioma.
The future classifications will include more genetic observation.
Genetic aberrations will also contribute to the definition of the specific tumor entities.
CONCLUSION
The marriage between Pathology and Genetics not only proved to be
fruitful but also to be stable.