Previously Unreported Somatic Variants in Two Patients with Pleuropulmonary Blastoma with Metastatic Brain Recurrence

To the editor:

Pleuropulmonary blastoma (PPB) is the most common primary lung tumor of childhood with 96% of tumors diagnosed prior to seven years of age and are associated with pathogenic somatic or germline DICER1 variants in most cases [1,2]. First described as an entity in 1988, its initial feature is a multi-locular cyst whose septa are composed of primitive mesenchymal cells which either progress to a primitive multi-patterned sarcoma with overgrowth of these cysts into a high grade neoplasm with anaplasia or undergoes regression [3]. DICER1 germline pathogenic variants were discovered to harbor an increased risk for development of a variety of neoplasms, with PPB highlighted as the archetype [4–6]. Brain metastases, especially in recurrence, are difficult to treat and the survival rate in children remains low [7,8], emphasizing the need to understand additional genomic drivers to develop novel treatments. Here we present two cases that document additional distinct, uncharacterized molecular findings in recurrent type III PPBs with brain metastases.

The first patient presented at 2 years of age with a persistent cough. Subsequent computerized topography (CT) scan revealed a 10 × 9.8 × 8.1cm heterogeneous, hypodense mass occupying the left hemithorax. Biopsy revealed a high grade sarcoma with rhabdomyoblastic features and anaplasia characteristic of type III PPB (see Figure 1). There was no family history of cancer or thyroid problems. Staging brain CT and bone scan were negative for metastatic disease. The patient received 12 cycles of ifosfamide, vincristine, actinomycin-D and doxorubicin (IVADo), underwent a left lower lobe resection at week 12, and received intra-cavitary cisplatin for local control. At 36 months post-treatment a 3cm mass arising from the left ventricle was discovered by echocardiogram. The mass was resected and found to be recurrent PPB. He underwent re-induction chemotherapy with autologous stem cell rescue. Subsequently, he developed brain metastases two years later, prompting a Pediatric Precision Genomics consultation for tumor molecular analysis. He received irinotecan and pazopanib based on FGFR1 gene amplification and overexpression of TOPO1 protein. Additional genomic analysis was performed on another brain metastasis which demonstrated an ETV6-NTRK3 fusion. He participated in a larotrectinib trial for 4 cycles before progression of disease and subsequent death.

Figure 1.

Histologic images of initial diagnostic tumor samples. A. 20x view of the first patient tumor which shows scattered large bizarre-appearing anaplastic tumor cells are surrounded by smaller primitive malignant cells. This pattern is a common one in DICER1-associated neoplasms. B. 20x view of the second patient tumor nodule displaying neoplastic cartilage surrounded by a field of primitive tumor cells with a subpopulation of rhabdomyoblasts. This pattern characterizes both pulmonary and extrapulmonary DICER1 neoplasms.

The second patient presented at 3 years of age with a persistent cough and was found to have a solid heterogeneous mass in the left chest measuring 10 × 8.4 × 10.5cm on CT. Of note when reviewing family history, mother revealed that she had a thyroid goiter and an abdominal desmoid tumor that had been removed. Biopsy revealed a sarcoma with primitive multi-pattern of PPB type III (see Figure 1). Brain MRI and bone scan showed no metastatic disease, but an echocardiogram demonstrated a mass attached to the left atrium concerning for local extension. This patient subsequently underwent resection of both the atrial and intrathoracic masses, completed IVADo chemotherapy, and received intra-cavitary cisplatin for local control due to tumor spill. Two months later, multiple brain metastases were discovered and subsequently underwent gamma knife radiotherapy. Two cycles of ifosfamide, carboplatin, and etoposide followed and this patient then proceeded with metronomic chemotherapy consisting of thalidomide, celecoxib, fenofibrate, oral cyclophosphamide alternating with oral etoposide, and every two week bevacizumab, per Nakano et al, which she continues on without recurrence [7].

All cancer relevant genomic findings for both cases are summarized in Table 1 which include the previously unreported changes in PPB of an ETV6-NTRK3 fusion and a PIK3CA mutation. In the first patient, tumor whole genome sequencing (WGS) paired with tumor RNAseq and germline sequencing was performed for the first sample by NantHealth^™. At recurrence whole exome sequencing (WES) again paired with RNAseq and germline analysis was performed by NantHealth^™. For the second patient, germline analysis was performed shortly after diagnosis at Ambry Genetics Laboratory which identified a pathogenic DICER1 variant, p.R676*. The tumor sample from the second patient’s lung at initial diagnosis was sequenced using the FoundationOne^™ Heme panel.

Table 1.

Germline and Tumor Sequencing Results from Recurrent PPB Patients

Case No.	Tumor Sample	Sequencing Company	Pathogenic Somatic Mutations/Fusions	Pathogenic Germline Variants	Tumor Copy Number Variation	Tumor Mutation Burden	Tumor VUS of known oncogenic drivers
1#	Brain Recurrence	NantHealth™	TP53 p. R273H ETV6-NRTK3&	DICER1 WT	FGFR1 (7x)	2.7 mut/MB	KMT2C p.R2211T TCEB3 p.A22E
	2nd Brain Recurrence	NantHealth™	TP53 p. R273H ETV6-NRTK3	DICER1 WT	FGFR1 (7x)	2.7 mut/MB	KMT2C p.R2211T KCNT2 p.Y331CfsTer37
2	Primary	FoundationOne™ Heme Panel; Ambry Genetics Laboratory+	PIK3CA p.Q546P PPP2R1A p.R183W NOTCH2 Truncation Exon 24	DICER1 p.R676Ter	MDM2 amp (30x) C17orf39 (10x) FRS2 (93x)	4 mut/MB	ATM p.I1688V EPHB1 p.R84H HDAC1 p.M295T

^#No pathogenic DICER1 variants were found either tumor sample despite analysis of new tumor and germline samples 12 months after the initial sample sequencing.

^&The ETV6-NRTK3 gene fusion in the initial sequencing was later identified upon reanalysis of the sequencing data.

⁺Germline analysis was performed at Ambry Genetics Laboratory as FoundationOne^™ testing does not offer germline analysis

Definitions: VUS = Variant of Unknown Significance. A variant of unknown significance is an allele, or variant form of a gene, whose significance to the normal function of the encoded protein and any corresponding phenotype, is unknown. WT = Wild Type. Ter = termination codon.

For the first patient, the lack of either a DICER1 somatic or germline variant is unique despite pathologic findings characteristic of PPB though low-level mosaicism may provide an explanation for a DICER1-negative PPB [9]. Identification of the ETV6-NTRK3 fusion led us to question if this tumor may represent an infantile fibrosarcoma but brain metastases are an extremely unusual manifestation [10]. Additionally, the tumor continued to progress despite known high response rates of NTRK-fused infantile fibrosarcomas to larotrectinib [11,12]. A TP53 pathogenic variant, p.R273H, was identified in this case which have been observed in PPB and could have modulated response to larotrectinib [13]. Recent evidence from Gatalica et al. showed TP53 is the most commonly co-mutated gene in NTRK-fused neoplasms and another report showed an impressive response to larotrectinib in a refractory high-grade glioma despite tumoral TP53 loss [14,15]. Additionally, the AGO2 p.H443R variant could have promoted resistance given AGO2 is required for the efficient functioning of DICER1 though this particular mutational change in AGO2 has not been interrogated making its tumor impact unclear [16].

The sequencing results for the second patient are unique due to the identification of a PIK3CA mutation and MDM2 amplification in addition to a DICER1 germline variant identified around initial diagnosis. In the largest published study, exome sequencing was performed on 15 PPBs with no PIK3CA mutations or MDM2 amplification identified [13]. MDM2 is a negative regulator of TP53 [17,18] and its amplification promotes therapeutic resistance in a variety of cancers, including sarcomas leading to poor prognosis [19–22]. PIK3CA mutations have been reported as oncogenic drivers in embryonal rhabdomyosarcoma, which is associated with germline DICER1 variants. [23–26]. Similar to MDM2, PIK3CA mutations are reported to promote therapeutic resistance in breast cancer, germ cell tumors, and sarcoma [27–30]. Early phase trials are currently underway in recurrent pediatric cancers utilizing both MDM2 and PIK3CA inhibitors.

To date, comprehensive molecular analyses of PPBs are limited. Due to the histologic complexity and heterogeneity of these tumors, both cases highlight the importance of sequencing tumors to identify additional oncogenic drivers that promote early and novel therapeutic interventions for PPB recurrence.

Abbreviations:

PPB

Pleuropulmonary Blastoma

FGFR1

Fibroblast Growth Factor Receptor 1

CT

Computed Tomography

TOPO1

Topoisomerase I

ETV6

ETS Variant Transcription Factor 6

NTRK

Neurotrophic Tyrosine Receptor Kinase

MRI

Magnetic Resonance Imaging

AGO2

Argonaute RISC Catalytic Component 2

MDM2

Mouse double minute 2 homolog

PIK3CA

Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha

PPP2R1

Protein Phosphatase 2, scaffold subunit A, alpha

VUS

Variant of Unknown Significance

WES

Whole Exome Sequencing

WGS

Whole Genome Sequencing

WT

Wild Type