Sarcomas

Diagnosis

Histopathology and characteristics of NTRK+ tumours

NTRK-rearranged spindle cell tumours encompass a histologically diverse spectrum spanning low and high cellularity and represent an emerging group of molecularly defined rare sarcoma still under refinement [13].

A recent ad hoc analysis of patients from three larotrectinib clinical trials (NCT02122913, NCT02576431, NCT02637687) examined the concordance between independent histological diagnosis and local diagnostic assessment concluded that for patients with NTRK fusion-positive sarcomas, there was high concordance between the independent and local pathologist review for infantile fibrosarcoma and approximately 50% for other NTRK rearranged soft tissue sarcomas, although only H&E-digitized slides without any IHC staining were provided for independent assessment [16].

The presence of specific fusion partners may not necessary be a good surrogate for prognosis. TFG-NTRK3 sarcomas are amongst the rare NTRK3-fused sarcoma cases with a rather favourable histological and clinical picture, however cases with aggressive behaviour and poor clinical outcome have also been reported [17]. A study involving a series of 70 sarcoma cases screened for NTRK gene fusions using IHC, found seven NTRK fusion positive tumours including, two alveolar rhabdomyosarcomas, one epithelioid angiosarcoma, one malignant peripheral nerve sheath tumor (MPNST), one osteosarcoma and two spindle cell sarcomas not otherwise specified (NOS); all but one (a spindle cell sarcoma NOS) were reported to be high grade. Subsequent targeted RNA-NGS revealed that only two out of these tumours harboured an NTRK gene fusion (ETV6- NTRK3), both cases presenting with spindle cell morphology and nuclear IHC positivity.

In a series of 15 cases of NTRK-rearranged uterine sarcomas, the majority arose in the uterine cervix (n=14) and all but 2 were organ-confined at diagnosis. Tumors were composed of an infiltrative, fascicular proliferation of spindle cells and most showed mild-to-moderate cytologic atypia. All were pan-TRK positive by immunohistochemistry (13/13); S100 (11/13) and CD34 (6/10) positivity was also observed [18]

Current testing algorithms

Methods used for screening sarcomas for NTRK fusions include different molecular pathology techniques such as FISH, RT-PCR, and massive parallel sequencing (MPS), while TRK protein expression is assessed by IHC (see Module 2). Different testing algorithms, including those proposed by the ESMO and JSCO-ESMO-ASCO-JSMO-TOS [19, 20], Memorial Sloan Kettering Cancer Center [21], Penault-Llorca et al. [22], the Canadian Consensus [23], and the Singapore Task Force [24] have been proposed for screening for NTRK fusions in sarcomas including also paediatric sarcoma. The World Sarcoma Network has elaborated a set of practical guidance to aid the management of patients with sarcoma harbouring NTRK gene fusions [25], as shown in the figure below.

 The World Sarcoma Network recommended approach for testing patients with sarcoma for NTRK gene fusions (Adapted from [25]).

^a For patients at high risk of relapse, NTRK gene fusion testing might provide clinically actionable information for later in the disease course. ^b If histology is typical then confirmation by MPS is recommended. ^c Treatment may be considered concurrently with confirmatory MPS. ^d Consider parallel validation by MPS or RT-PCR to confirm that fusion is in-frame. ^e Avoid IHC screening in cases with myogenic and neural differentiation due to the high rate of false positivity.

GIST: gastrointestinal stromal tumor; IFS: infantile fibrosarcoma; IHC: immunohistochemistry; IMT: inflammatory myofibroblastic tumor; LPS: liposarcoma; MPS: massive parallel sequencing; NTRK: neurotrophic tyrosine receptor kinase; RT-PCR: reverse transcription polymerase chain reaction; TRK: tropomyosin receptor kinase.

Despite some differences among different algorithms, the principle remains the same. For tumours known to have a high prevalence of NTRK fusions (e.g., IFS), direct confirmation with molecular techniques is recommended, as it is also relevant to confirm diagnosis. Alternatively, IHC with a pan-TRK antibody can be performed as an enrichment strategy. For positive IHC results, a confirmation with molecular techniques is necessary. The preferred molecular technique in most cases is RNA-based NGS [22, 26]. If IHC results are negative, NGS confirmation would be still recommended for cases with histological features suspicious for NTRK gene fusions [24].

In the case of IFS, conventional cytogenetic/molecular assays (FISH or RT-PCR) have been considered the gold standard for confirming diagnosis [14]. One of the most commonly used commercial probes is an ETV6 break-apart probe that has shown efficacy in confirming ETV6-NTRK3 rearrangements. However, since the identification of the EML4–NTRK3 rearrangement in IFS, this testing strategy warrants revision [8]. NTRK1, NTRK2, and NTRK3 break-apart probes are also used to identify NTRK fusions. While a positive FISH result with a break-apart probe means that there is a structural variant involving the tested gene, it is not clear whether this results in a translated fusion nor which the fusion partner is [21]. Importantly, false negative results may occur more commonly than generally thought. IHC using a pan-TRK rabbit monoclonal antibody (EPR17341) has proven to be a sensitive marker, showing nuclear and/or cytoplasmic staining in cases with ETV6-NTRK3 fusions [14].

As for other tumour types, for sarcoma with a low prevalence of NTRK gene fusions, IHC screening is the recommended initial approach. However, positive IHC results in such histologies should be interpreted with caution in case of myogenic and neural differentiation because of the high rate of false positivity arising from the detection of wild-type TRK proteins [24]. If IHC results are negative, routine confirmatory NTRK testing is not recommended, though it can be considered on a case-by-case basis. The sensitivity of pan-TRK IHC has been reported to be relatively limited for NTRK3 fusions (55-79%) compared with that for NTRK1 (88–96%) [17, 24, 25].

Challenges

The rarity of sarcomas and rarity of NTRK gene fusions in certain sarcoma types present several challenges, including the cost of testing, limited resources, limited tumour tissue, and the complexities of integrating a new molecular test into the current diagnostic workup. Nevertheless, the overall benefit of molecular testing in the diagnosis and clinical management of patients with sarcoma has been demonstrated in multicentre studies (see Treatment section). Starting with a less expensive and quicker option such as IHC for initial screening has its own caveats including variable staining patterns and intensities depending on the sarcoma type. Although TRK antibody staining appears to have 100% specificity in certain carcinomas including colon, lung, pancreas and thyroid, the specificity in sarcomas is much lower, where also false‐positive staining is especially frequent in tumours with smooth muscle and neural differentiation [5, 24].

Given the robust efficacy and favourable safety profiles of TRK inhibitors demonstrated in patients with TRK fusion sarcomas, testing for NTRK gene fusions may be incorporated into the clinical management of these patients with and prioritization of specific stages and histologies or to aid differential diagnosis [27, 28].

References

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