The evolving role of precision medicine in the management of advanced sarcomas – A mini review

Conventional cytogenetic analysis

Conventional karyotyping is now only of historical importance in sarcoma management. Its disadvantages include a long turnaround time (TAT), the requirement of a cell culture medium for processing, low resolution, limited utility, and its inability to pick up cryptic mutations. These drawbacks have led to this tool being obsolete.

Fluorescent in situ hybridization (FISH)

This technique uses labeled complementary DNA, RNA, or modified nucleic acid strand probes to localize a gene of interest in a tumor sample. Different probes are used for FISH testing in STS, break-apart and dual-fusion probes are used for sarcomas with translocation. Locus specific probes are used to identify insertions and deletions. Some of the commonly used break apart probes clinically include

1. SS18 break apart probe in SS

2. MDM2 probe in well-differentiated liposarcoma/atypical lipomatous tumors (ALT/WDS), dedifferentiated liposarcoma, parosteal sarcoma, and malignant peripheral nerve sheath tumor (MPNST)

3. FUS/DDIT3 break apart probes in myxoid/round cell liposarcoma (MLPS)

4. ALK in IMFT and PDGB in DFSP, respectively.

Indication of dual fusion probes includes detection of EWSR1 and WT1 in ES, desmoplastic small round cell tumor (DSCRT)

FISH excels over karyotyping in that there is no culture required and the testing can be done on formalin-fixed tissue as well. It is also faster and can be done on minute tissue samples. Both FISH and polymerase chain reaction (PCR) have a capacity for high resolution (150–200 kb), with an ability to pick up even cryptic translocations. The advantages over PCR are in assessment of tissue with a mixed cell population, wherein only a specific subset of tumor cells might harbor the target gene translocation. Furthermore, in a given translocation with a common break point, but involving multiple fusion partners (such as in ES), FISH can diagnose all known rearrangements whereas PCR would require multiple primers to cover all possible fusion partners. The disadvantages of FISH are its low specificity and its ability to detect only known genetic abnormalities.

PCR

This technique uses specific primers to amplify known DNA or RNA sequences. Added to its greater sensitivity and high resolution, it also has a conveniently short TAT. Its disadvantages include its greater expense, lack of wide availability, and an inability to diagnose cryptic mutations. With both FISH and PCR, care should be taken while interpreting borderline values, and in such unequivocal cases, the reports from one modality should be confirmed with the other.

Massive parallel sequencing (MPS)

MPS uses a comprehensive whole exome/genome/ transcriptome approach to diagnose mutations at the nucleotide level. For instance, they can identify novel mutations such as BCOR–CCNB3 in undifferentiated small-cell sarcoma,^[8] CNVs, and point mutations. Sequencing techniques include the traditional Sanger method, which takes 1–2 weeks for results, and next-generation sequencing (NGS), which is a more rapid, high throughput technique, but requires trained staff for the operation and interpretation of data. Sequencing is more useful than PCR or FISH for the diagnosis of unknown mutations. While both fresh and fixed tissues can be used, adequate genetic material (DNA or RNA) is needed for proper interpretation. NGS sequencing includes the following approaches.^[9]

Whole genome sequencing (WGS)

Most comprehensive sequencing technique, but is clinically not practical as it is cumbersome to perform and analyze the large amount of data created, not cost effective, the functional significance of most of the variants detected by WGS is not known; hence, it is still considered as experimental.

Targeted sequencing (whole exome sequencing [WES] or cancer gene panels)

Commercially available gene panels sequence for a limited number of genes 100–300 and hence limit the general cost of sequencing, but the disadvantage being it might miss on the novel gene mutations in most of the cases. Exome accounts for about 2% of the entire genome. Therefore, WES has the obvious advantage of screening the entire exome and being easier and faster to perform, interpret and being more cost effective with screening of clinically relevant mutations. Some examples of application of WES include NAB–STAT6 in solitary fibrous tumor (SFT).^[10] It can also detect point mutations and insertions and deletions (indels) such as KIT/ PDGFRA and SDHA/B in gastrointestinal stromal tumors (GIST), CTNNB1 in desmoid tumors, IDH1 or IDH2 in enchondroma/chondrosarcoma, and NF1 in MPNST. Because most of the large gene deletions and structural translocations occur at various sites of introns, WEScan miss them for instance in BRCA1 and BRCA 2 can be missed out on WES.

RNA sequencing (transcriptome sequencing)

The attractive part of transcriptome sequencing in STS is in its applicability to detect structural rearrangements, large genomic deletions which are missed out by WES. They, hence, also detect novel fusion genes. Some of the clinical break through achieved by RNA sequencing includes, WHAE-NUTM2A/B in high-grade endometrial stromal sarcoma (ESS), WWTR1-CAMTA1 in epithelioid hemangioendothelioma, and BCOR-CCNB3 in undifferentiated round cell sarcoma. However, RNA sequencing is still not used wide spread as it is more technically challenging to be performed as they require high quality RNA.

Small round cell tumors

Small blue round cell tumors (SBRCT) may be mesenchymal, epithelial or lymphoreticular in origin. They are often difficult to differentiate by light microscopy or IHC alone. Sarcomas presenting with small round cell phenotype include ES, RMS, poorly differentiated SS, DSCRT, and MLPS. As an illustrative example, poorly differentiated SS is often confused with extra skeletal ES. Although they can share a few clinical and histological similarities, they can be differentiated molecularly based on the presence or absence of SS18–SSX fusions or SS18– rearrangement which is specific for SS.^[12] Similarly, there is a subset of SBRCT which behaves clinically similar to ES (Ewing like) but, unlike ES, have distinct rearrangements between EWSR1 and non-ETS partner genes such as PATZ1, POU5F1, and SMARCA5. Some of these tumors behave aggressively.^[13] A special mention of two subtype of undifferentiated SBRCT is necessary, the first of them being CIC-DUX4 gene fusion associated sarcomas, it is the most common mutation found in EWSR1-negative SBRCTs. It, usually, occurs in the young adults between third to fourth decades of life and commonly affects the soft tissue rather than bone. It is associated with a more aggressive course and poor outcomes.^[14] BCOR-CCNB3 fusions associated round cell sarcoma are another subset of sarcomas, usually seen in adolescent and young males, commonly affecting the long bones and has relatively better prognosis.^[8]

Spindle cell tumors

Sarcomas presenting with spindle cell morphology include, fibrosarcoma, leiomyosarcoma, MPNST, and monophasic SS. Infantile fibrosarcoma is often confused morphologically with other spindle cell tumors, such as the adult-form of fibrosarcoma, infantile fibromatosis, and infantile myofibromatosis. Molecular testing for the ETV6– Neurotrophic tyrosine receptor kinase (NTRK3) fusion gene, specific for infantile fibrosarcoma,^[15] and enables us to confirm the diagnosis in perplexing cases. Molecular testing for NTRK in such tumors has therapeutic implications. On the same note, monophasic SS presenting in the pleural cavity can be differentiated molecularly from SFT, sarcomatoid malignant mesothelioma, and MPNST based on the presence of SS18-SSX gene fusion.

1. Differentiating between a lipoma and an ALT/ WDS is important as both of them have contrasting management principles. Dedifferentiated liposarcoma with high grades of dedifferentiation often cannot be differentiated from other high grade pleomorphic sarcoma or poorly differentiated sarcoma by histology alone, but the molecular demonstration of MDM2 (+CDK4) amplification in ALT/WDS or dedifferentiated LPS enables us to clinch the diagnosis^[16]

2. Clear cell sarcoma (CCS), often confused with melanoma, can be recognized by testing for t(12;22) (q13;q12) translocation and its associated EWSR1–ATF1 fusion gene, which are associated with CCS.^[17]

Table 1 summarizes the important molecular changes with appropriate tests for diagnosis of commonly encountered sarcomas.^{[41,43,44,52,53]}

GISTs

GIST is a prime example, where targeted therapy revolutionized management. Prior 1998 there was no effective therapy for advanced GIST. Discovery of KIT and PFGFRA changed the paradigm of treatment in GIST. Over 80% of GISTs have mutations in the KIT and PDGFRA genes, making them responsive to Imatinib, with a response rate of 69% and a PFS of up to 26 months.^[46] The particulars of these mutations, however, give us even more insight into their sensitivity to Imatinib. Patients with mutations in exons 4 or 12 of PDGFRA respond to imatinib, whereas exon 18 D842v mutations are imatinib-refractory.^[24] On progression to imatinib, several targeted options are available, including sunitinib, regorafenib, avapritinib, and ripretinib.^[47-50] Wild-type GISTs, which lack cKIT and PDGFRA mutations, are mostly SDH-deficient and exhibit O6-methylguanineDNA methyltransferase promoter methylation, but trials of alkylating agents in this subtype have failed to show any benefit.^[25] In wild-type GISTs that are SDH-competent, vandetanib (an inhibitor of VEGFR2, RET, and EGFT) has been tried in V600E-mutated cases with good results.^[26]

PEComas

PEComas are a group of related tumors that include angiomyolipoma of the kidney, clear cell sugar tumor of the lung, and lymphangioleiomyomatosis. These tumors are characterized by the presence of TSC1 and TSC2 mutations affecting the mammalian target of rapamycin (mTOR) pathway. mTOR inhibitors, such as sirolimus and temsirolimus, have been used in PEComas with good clinical responses in case reports.^[27]

DFSP

DFSP is a locally aggressive tumor most commonly seen on the trunk followed by proximal extremities. Fibrosarcomatous areas occasionally occur in DFSP, which are associated with increased chance of metastasis and P53 mutations.^[28] It is characterized by a t(17;22) translocation involving COL1A1 and PDGFB genes. The consequent hyperactivation of PDGFRB rendering them sensitive to targeted therapy with the tyrosine kinase inhibitors (TKI) imatinib.^[29]

Alveolar soft part sarcoma

It is a rare, slow-growing tumor with propensity to metastasize to the brain and lungs.^[45] It is chemoresistant, but responds to TKIs such as sunitinib and pazopanib. As MET is also overexpressed in these tumors, MET inhibitors, such as tivatinib, have also been found to be useful in their management.^[30,51]

Epithelioid sarcoma

ES is a rare aggressive malignancy, with dismal outcomes in advanced stage. About 90% of these tumors are characterized by INI/SMARCB1 deficiency which leads to elevated levels of enhancer of Zeste homolog 2 (EZH2) and finally promotes oncogenesis. Tazemetostat is an EZH2 inhibitor with Phase II basket trials demonstrating good activity of the drug.^[31]

IMFT

It is a tumor with intermediate malignant potential.^[42] It can occur anywhere but is most commonly seen in lungs, GIT, and retroperitoneum. The hallmark of this tumor is the coexistence of myofibroblastic tissue with immune-cell infiltrates. ALK fusion rearrangements, identified by FISH, are seen in about half of such tumors, more commonly in children. This is clinically targeted by the ALK inhibitor crizotinib.^[32] ALK fusion negative patients are to be tested for ROS 1 and NTRK as they are targetable mutations.

NF1-related plexiform neurofibroma

Plexiform neurofibromas are seen in 20–50% patients with NF1. They can lead to disfigurement, pain and high risk of transformation to MPNST. These tumors are dependent on MEK1 and 2 signaling for survival. Selumetinib, a MAP kinase inhibitor has shown clinical benefit in children treated with this drug.^[33]

TGCT

It is an aggressive tumor of the synovial tissue, usually affecting wrists. These tumors are characterized by pathognomonic gene fusions involving colony stimulating factor-1 (CSF-1). This has been clinically targeted by imatinib and nilotinib with modest responses.^[34] Pexidartinib is potent CSF-1 R inhibitor. Phase III trial (ENLIVEN) showed good results for the drug leading to its FDA approval and hence supporting the role of targeted therapies in these rare tumors.^[35]

CCS

CCS is a rare chemo sensitive and radiosensitive tumor. Its survival is dependent on MET signaling, tivatinib has been explored in the same setting and has failed to achieve to show clinical response.^[30] Crizotinib another MET inhibitor has also been tried and failed.^[36]

Angiosarcoma

This is a rare aggressive malignancy which is chemosensitive but outcomes are poor because it is locally aggressive and has metastatic potential. As the name indicates, they are characterized by increased expression of vascular endothelial growth factor (VEGF), which is explored clinically using anti VEGF agents such as pazopanib, sorafenib, and bevacizumab.^[37] Retrospective studies have also shown efficacy of propranolol, a non-selective beta blocker in treatment of angiosarcoma.^[38] Combination of propranolol, methotrexate, and vinblastine as metronomic therapy has shown 100% efficacy in one trial.^[39]

Role of TRK in advanced STS

NTRK gene fusions as tissue-agnostic oncogenic drivers are seen in subtypes of STS. This has been explored for targeted therapies in form of TRK inhibitors. Tumors with high NTRK gene fusion among STS include infantile fibrosarcoma and ALK, ROS 1 negative IMFT. Larotrectinib is the first in class TRK inhibitor, ORR was 87% in the STS subgroup with 71 patients bearing NTRK mutations. NTRK testing with therapy has shown great leaps in management of advanced STSs like infantile Fibrosarcoma and GIST.^[40]