Sequencing PEComas: Viewing Unicorns through the molecular looking glass

“Well, now that we have seen each other,” said the unicorn, “if you’ll believe in me, I’ll believe in you.”

— Lewis Carroll, Through the Looking-Glass and What Alice Found There

Three years ago, in a review on the role of next-generation sequencing (NGS) in sarcomas, we wrote “precision oncology” has been so far limited to few case reports in sarcomas”[1]. In just a short time we have witnessed an explosion of use cases justifying NGS in sarcomas. With the tissue agnostic approval of NTRK inhibitors (Larotrectinib, Entrectinib) in NTRK positive solid tumors (many of them sarcomas), pembrolizumab in microsatellite instability high/deficient mismatch repair (MSI-H/dMMR) cancers and metastatic tumor mutational burden-high (TMB-H≥10 mutations/megabase) cancers, NGS is becoming standard of care in sarcomas. In addition NGS has refined discussion on adjuvant therapy in gastrointestinal stromal tumors (GIST)[2], identified predictive biomarkers to immunotherapy in angiosarcomas[3], and become ever more relevant with emergence of targeted therapies in INI-1 gene loss/SMARCB1 deficient epithelioid sarcomas[4]. From research to practice, NGS is becoming our most powerful weapon in the war to divide and conquer sarcomas [5, 6].

“PEComa”s are a rare subtype of soft-tissue sarcoma arising from the perivascular epithelioid cell, hence PEComa. Malignant PEComas (mPEComas) are a rare subtype of the disease, highlighted by aggressive local and distant recurrences. A recent breakthrough in the treatment of this malignant variant came from the AMPECT trial, which evaluated nab-sirolimus (nano-particle albumin-bound sirolimus, an mTOR inhibitor) and showed a response rate of 39% (95% CI: 22%–58%). The median progression-free survival was 8.9 months (95% CI : 5.5 – not reached) and the 1 year overall survival rate was 89% [7]. In an exploratory analysis of the subset of patients with TSC2 mutations, the independently reviewed response rate was 89% (95% CI: 57%–99%).

In this issue Akumalla et al [8] perform an in-depth analysis of the genomic landscape of malignant PEComas using NGS. The analysis sheds further light not only on the pathogenesis of mPEComas but helps explain response to mTOR inhibitors as well. For over a decade we have known that inactivation of tuberous scerlosis 1 and 2 genes (TSC1/2) is common in PEComas, leading to activation of the mTOR pathway that can be successfully targeted by mTOR inhibitors[9]. What we did not appreciate until the current study is that the genomic landscape of mPEComas is diverse, yet still functions through the mTOR pathway.

The authors should be commended for collecting “only” 31 mPEComa patients, a number that seems tiny in the era of large-scale genomic projects. However, for an ultra-rare disease that even busy oncologists may go entire careers without ever seeing, this is a remarkable feat. Additionally, the small sample size did not impede the authors from creating a comprehensive genomic landscape. This last point reinforces what the rare tumor community has known for a long time: Rare tumors are rare because they are driven by a single genomic event and disproportionately benefit from targeted therapies[10].

Across the study, 100 genomic alterations were identified in 31 patients with an average of 3.2 genomic alterations per sample. Not surprisingly, TSC1/2 were commonly mutated as were genes commonly mutated in sarcomas as a disease group: TP53, CDKN2A, RB1, ATRX[11]. A simple, but useful analysis would have been to correlate a patient’s age with the number of genomic alterations. It is likely that older patients will have had more mutations, including some related to clonal hematopoiesis of indeterminate potential (CHIP), while younger patients may harbor fewer alterations. This may help explain co-occurring aberrations such as FLT4 mutation in patient 20.

This study prudently looked at advanced mechanisms of gene inactivation, such as loss of heterozygosity (LOH). While we have known that TSC1/2 is frequently aberrant in PEComas, this is the first study to show that the tumor has bi-allelic knockdown of these genes, predominately by LOH of the intact allele. Patients who are “wild-type” for TSC1/2 still have inactivation of mTOR pathway. This study discovers one such mechanism: FLCN mutation, the gene encoding folliculin, a positive regulator of the mTOR pathway. The other major group of mPEComas are mediated by TFE3 fusions and all cases in this study had a different fusion partner gene. Even this characteristic fusion is a mediator of the mTOR pathway, and likely is a driver for mPEComa in the same fashion as TSC1/2 mutations. Future work could combine what has been learned from this study with what was observed in the AMPECT trial, hopefully increasing our understanding of response to nab-sirolimus and most importantly resistance to mTOR inhibitors[12]

Unfortunately, this paper is only able to “explain” 20 out of the 31 cases they analyzed using CGP. The rest remain “wild-type”. Based on what is seen here, it is reasonable to hypothesize that the remaining unexplained mPEComas also have activation of mTOR pathway at their core. This may not have been observed because of limits in tissue quantity or quality that resulted in incomplete sequencing. The tumor may also be using alternative methods of gene silencing such as RNAi or epigenetic regulation. In addition to more comprehensive in-depth sequencing like whole exome or whole genome sequencing, future efforts may also require alternative methods by techniques such as single cell RNA sequencing.

This study highlights the growing body of evidence demonstrating utility of next-generation sequencing in sarcomas. Indeed, as a field we have come a long way from our early efforts at comprehensive genomic profiling of sarcomas [11]. However, future landscape and sequencing studies need to move away from “lumping” all sarcomas together. There are clear genomic differences that make sarcoma subtypes unique and by “splitting” up, we can arrive at a common pathway that accurately explains the pathogenesis of a particular subtype[6]. It is no accident that sarcoma landscape studies such as our own early work yielded little actionable knowledge and served only to generate initial hypothesis.

This brings us to important lesson number two from this work: large numbers of cases are not needed when dealing with a rare disease governed by a single pathway. Investigators should take whatever sample sets they may have from a rare sarcoma subtype and analyze the genomics. An added advantage is the ability to include clinical data into small sampler sets.

Lesson three from this study is that ultra-rare tumors such as mPEComas have a specific clear common pathway aberration. All efforts in designing novel therapeutic interventions need to focus on this pathway and optimizing the drugs we have to target it, including novel combinations of drugs. Targeting of the oncogene KIT in gastrointestinal stromal tumors is a prime example of how successful such a strategy can be.

Lesson four centers around the analysis of comprehensive genomic profiling. Too often, such genomic landscape papers simply report on the most commonly mutated genes in a disease. This may have yielded significant dividends in our early search for oncogenes (EGFR, KIT, RET, BRAF), but going forward these will be increasingly difficult to find. Investigators will have to dive deeper, looking at LOH and other methods of gene inactivation to explain oncogenic pathway activation.

The final lesson is philosophical. Since the first complete human genome was sequenced in 2003, we have captured the public imagination about unlocking cancer’s secrets using this DNA technology. The commercial availability of NGS circa-2005 and the resultant exponential decline in sequencing costs fueled this zeal further. Yet, another decade passed, and we had little to show for the clinical utility of this now seasoned technology. The technology may have existed, but we had to learn how to use it effectively. This “experience” is now showing what can be done with NGS. The same need be kept in mind for upcoming nascent technologies such as single cell RNA sequencing. Such efforts will pave the way in unravelling the molecular underpinnings of these ultra-rare unicorns of cancer.