<div>Abstract</div><div>Pleuropulmonary blastoma (PPB) is the most common primary lung tumor of
childhood and is associated with somatic or germline DICER1 variants.
Recurrent PPB, especially with brain metastases, are difficult to treat
and survival is poor. Comprehensive genomic analyses of PPB have been
limited in number and depth. The cases presented here identified
additional oncogenic drivers from tumor sequencing that could be
modulating tumor progression and response to therapy outside of known
DICER1 mutations highlighting the need for upfront genomic analysis on
all patients with PPB.</div><div>Introduction</div><div>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 <i>DICER1</i>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 the cysts into a high grade
neoplasm with anaplasia and p53 mutations or undergoes regression
[3]. From this observation, familial predisposition, <i>DICER1</i>germline mutation and the recognition of a family of<i>DICER1</i> -associated neoplasms emerged [3].</div><div>The current study documents other molecular aspects of PPB which are the
emergence of distinct genomic findings in recurrent type III PPBs in two
children.</div><div>Case Descriptions</div><div>Case 1 – A 2-year-old male initially presented with a persistent cough
with subsequent computerized topography (CT) scan revealing a 10 x 9.8 x
8.1cm heterogeneous, hypodense mass occupying the left hemithorax. A
biopsy revealed a high grade sarcoma with the primitive multi-pattern of
PPB type III. Brain CT and bone scan were negative for metastatic
disease at that time. The patient received 12 cycles of ifosfamide,
vincristine, actinomycin-D and doxorubicin (IVADo) and underwent a left
lower lobe resection at week 12. He subsequently received intra-cavitary
cisplatin for local control. At 36 months post-treatment a 3cm mass
arising from the left ventricle was discovered by routine
echocardiogram. The mass was resected and found to be recurrent
(metastatic) PPB. He underwent re-induction chemotherapy with autologous
stem cell rescue and was without recurrence for two years. Subsequently,
he developed brain metastases, prompting a Precision Genomics
consultation for tumor molecular analysis of these brain metastases. He
then received irinotecan and pazopanib due to FGFR1 gene amplification
and overexpression of TOPO1 protein. Genomic analysis was performed on a
new brain metastasis and demonstrated an ETV6-NTRK fusion. He
participated in a larotrectinib trial for 4 cycles before progression of
disease and subsequent death nearly 7 years after initial diagnosis.</div><div>Case 2 – A 3-year-old female was admitted with a history of persistent
cough and found to have a solid heterogeneous mass in the left chest
measuring 10 x 8.4 x 10.5cm on CT. A biopsy revealed a primitive sarcoma
with rhabdomyoblastic features and anaplasia whose features were those
of type III PPB. Brain MRI and bone scan failed to demonstrate
metastatic disease, but an echocardiogram showed a multi-lobulated mass
attached to the left atrium. She subsequently underwent resection of the
cardiac mass. She then completed IVADo chemotherapy and received
intra-cavitary cisplatin. Two months post-completion of intra-cavitary
cisplatin, she was found to have multiple brain metastases for which she
received gamma knife radiotherapy. She then received 2 cycles of
ifosfamide, carboplatin, and etoposide post radiation. Utilizing
metronomic chemotherapy, consisting of fenofibrate, thalidomide,
alternating oral cyclophosphamide and etoposide, and every 2 week
bevacizumab, she is continuing on therapy without recurrence [4].</div><div>Genetic Analysis and Results</div><div>Case 1. DNAseq, RNAseq and limited proteome analysis was performed on a
recurrent brain tumor sample and whole-exome DNAseq and RNAseq was
repeated from a second tumor sample from a subsequent recurrence
(NantHealth™) with matching germline sequencing. The first tumor sample
showed amplification of FGFR1 (7x), TP53 p.R273H and high expression of
TOP1 protein. From the sequencing of the second specimen, the presence
of an additional ETV6-NRTK3 gene fusion was identified. Reanalysis of
the DNA and RNA data from the first tumor sample revealed that the
ETV6-NRTK3 fusion was present, but was not initially identified by
NantHealth™. A somatic AGO2 variant, AGO2 p.H443R, classified as unknown
significance was identified in both tumor samples. DICER1 mutation was
not found in the somatic and germline specimens.</div><div>Case 2. The tumor sample from the initial diagnosis was sequenced using
the FoundationOne™ Heme panel. A number of known oncogenic alterations
were identified in the sample: MDM2 amplification (30x), PIK3CA,
p.Q546P, and PPP2R1A p.R183W. Germline analysis was performed at Ambry
Genetics Laboratory and identified a pathogenic DICER1 variant, p.R676*.</div><div>Additional cancer relevant genomic findings are summarized in Table 1.</div><div>Discussion</div><div><i>DICER1</i> germline pathogenic variants were discovered to harbor an
increased risk for development of a variety of neoplasms, with PPB
highlighted as the archetype [5-7]. Brain metastases, especially in
recurrence, are difficult to treat and the survival rate in children
remains low [4,8], emphasizing the need to understand additional
genomic drivers to develop novel treatments.</div><div>In the first case, the lack of either a <i>DICER1</i> somatic or
germline variant is unique though the pathologic findings were
characteristic of PPBs and mosaicism may provide an explanation for
these <i>DICER1</i> -negative PPBs [9]. Identification of the
ETV6-NTRK3 fusion argues this tumor could be molecularly related to an
infantile fibrosarcoma but given the brain metastasis, would be an
unusual manifestation since the brain is the most common metastatic site
for PPB [10]. The tumor continued to progress until his untimely
death despite the high response rates of NTRK-fused infantile
fibrosarcoma to larotrectinib observed in multiple clinical trials
[11,12]. TP53 and NRAS pathogenic variants have also been reported
to occur frequently in PPB [13] and a TP53 pathogenic variant,
p.R273H, was identified in this case. One could hypothesize the TP53
variant modulated response to larotrectinib. 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. However, this particular mutational change in AGO2 has not
been interrogated at the cellular level making its tumoral impact
unclear [16]. In this case, earlier identification of the NTRK
fusion prior to multiple recurrences and large tumor burden may have
improved the chance for a response.</div><div>The second case is unique due to the identification of a PIK3CA mutation
and MDM2 amplification in addition to a DICER1 germline variant. In the
largest published study, exome sequencing was performed on 15 PPBs and
none had a PIK3CA mutation or MDM2 amplification [13]. MDM2 is a
negative regulator of TP53 [17,18] and its amplification promotes
therapeutic resistance leading to poor prognosis in a variety of
cancers, including sarcomas, similar to the one in our patient
[19-22]. PIK3CA mutations have been reported as potential oncogenic
drivers in pediatric rhabdomyosarcoma, a tumor type seen in individuals
with pathogenic germline DICER1 variations. [23-26]. Like MDM2,
PIK3CA mutations have been shown to promote therapeutic resistance
particularly in breast cancer, but also in germ cell tumors and sarcoma
[27-30]. Early phase trials are currently underway in recurrent
pediatric cancers utilizing MDM2 and PIK3CA inhibitors, which stress the
need for early identification of these variants to define the utility of
these drugs in recurrent PPB.</div><div>To date, comprehensive molecular analyses of PPBs are limited. Due to
the histologic complexity and heterogeneity of these tumors, these two
cases highlight the importance of sequencing all tumors to identify
additional oncogenic drivers that promote discovery of early therapeutic
interventions for PPB recurrence.</div><div>Conflicts of Interest</div><div>The authors of this manuscript have no relevant conflicts of interest to
disclose.</div><div>Acknowledgements</div><div>The Precision Genomics Team at Indiana University School of Medicine is
supported by U54HD16014 (Renbarger) - Indiana University Center for
Pediatric Pharmacology and Precision Medicine (ICPPPM).</div><div>The authors would like to acknowledge D. Ashley Hill, MD of Children’s
National Hospital and the International PPB Registry for reviewing the
accuracy of these cases and manuscript.</div><div>References</div><div>1. Manivel J. C. et al. Pleuropulmonary Blastoma. The so-called
pulmonary blastoma of childhood. Cancer. 1988. 62:1516-1526.</div><div>2. Messinger Y et al. Pleuropulmonary blastoma: a report on 350 central
pathology-confirmed pleuropulmonary blastoma cases by the
International Pleuropulmonary Blastoma Registry. 2015.
Cancer. 2015.
121(2):276-85.</div><div>3. Dehner LP et al. Pleuropulmonary blastoma: evolution of an entity as
an entry into a familial tumor predisposition syndrome. Pediatr Dev
Pathol. 2015. 18(6):504-511.</div><div>4. Nakano Y, et al. Successful treatment of metastatic cerebral
recurrence of pleuropulmonary blastoma. Pediatr Blood Cancer. 2019
May;66(5):e27628</div><div>5. Hill D.A. et al. DICER1 mutations in familial pleuropulmonary
blastoma. Science. 2009. 325:965.</div><div>6. Schultz KA et al. DICER1 and associated conditions: Identification of
at-risk individuals and recommended surveillance strategies. Clinical
Cancer Research 2018. 1-11.</div><div>7. Stewart D.R. et al. Neoplasm risk among individuals with a pathogenic
germline variant in DICER1. Journal of Clinical Oncology.
10;37(8)668-676.</div><div>8. Priest JR, et al. Cerebral metastasis and other central nervous
system complications of pleuropulmonary blastoma. Pediatr Blood Cancer.
2007 Sep;49(3):266-73.</div><div>9. Brenneman M, et al. Temporal order of RNase IIIb and loss-of-function
mutations during development determines phenotypes in pleuropulmonary
blastoma/DICER1 syndrome: a unique variant of the two-hit tumor
suppression model. F1000Res. 2015. Jul 10:4:214</div><div>10. Parida L, et al. Clinical management of infantile fibrosarcoma: a
retrospective single-institution review. Pediatr Surg Int. 2013
Jul;29(7):703-8.</div><div>11. Drilon A, et al. Efficacy of Larotrectinib in TRK Fusion-Positive
Cancers in Adults and Children. N Engl J Med. 2018 Feb
22;378(8):731-739.</div><div>12. Laetsch TW, et al. Larotrectinib for paediatric solid tumours
harbouring NTRK gene fusions: phase 1 results from a multicentre,
open-label, phase 1/2 study. Lancet Oncol. 2018 May;19(5):705-714.</div><div>13. Pugh TJ et al. Exome sequencing of pleuropulmonary blastoma reveals
frequent biallelic loss of TP53 and two hits in DICER1 resulting in
retention of 5p-derived miRNA hairpin loop sequences. Oncogene. 2014.
33:5295-5302.</div><div>14. Gatalica Z, et al. Molecular characterization of cancers with NTRK
gene fusions. Mod Pathol. 2019 Jan;32(1):147-153.</div><div>15. Ziegler DS, et al. Brief Report: Potent clinical and radiological
response to larotrectinib in TRK fusion-driven high-grade glioma. Br J
Cancer. 2018 Sep 11; 119(6): 693–696.</div><div>16. Koscianska E, et al. The role of Dicer protein partners in the
processing of microRNA precursors. PLoS One. 2011;6(12):e28548</div><div>17. Momand J, et al. The mdm-2 oncogene product forms a complex with the
p53 protein and inhibits p53-mediated transactivation. Cell. 1992 Jun
26;69(7):1237-45.</div><div>18. Hou H, et al. The role of MDM2 amplification and overexpression in
therapeutic resistance of malignant tumors. Cancer Cell Int. 2019 Aug
22;19:216.</div><div>19. Kato S, et al. Analysis of MDM2 Amplification: Next-Generation
Sequencing of Patients With Diverse Malignancies. JCO Precis Oncol.
2018;2018.</div><div>20. Dembla V, et al. Prevalence of MDM2 amplification and coalterations
in 523 advanced cancer patients in the MD Anderson phase 1 clinic.
Oncotarget. 2018 Sep 4;9(69):33232-33243.</div><div>21. Sawada R, et al. MDM2 copy number increase: a poor prognostic,
molecular event in esophageal squamous cell carcinoma. Hum Pathol. 2019
Jul;89:1-9.</div><div>22. Bill KLJ, et al. Degree of MDM2 Amplification Affects Clinical
Outcomes in Dedifferentiated Liposarcoma. Oncologist. 2019
Jul;24(7):989-996.</div><div>23. Shukla N, et al. Oncogene mutation profiling of pediatric solid
tumors reveals significant subsets of embryonal rhabdomyosarcoma and
neuroblastoma with mutated genes in growth signaling pathways. Clin
Cancer Res. 2012 Feb 1;18(3):748-57.</div><div>24. Kikuchi K, et al. MDM2 Amplification and PI3KCA Mutation in a Case
of Sclerosing Rhabdomyosarcoma. Sarcoma. 2013;2013:520858.</div><div>25. Agaram NP, et al. MYOD1-mutant spindle cell and sclerosing
rhabdomyosarcoma: an aggressive subtype irrespective of age. A
reappraisal for molecular classification and risk stratification. Mod
Pathol. 2019 Jan;32(1):27-36.</div><div>26. Doros L, et al. DICER1 mutations in embryonal rhabdomyosarcomas from
children with and without familial PPB-tumor predisposition syndrome.
Pediatr Blood Cancer. 2012 Sep;59(3):558-60.</div><div>27. Lobil S, et al. PIK3CA mutations are associated with reduced
pathological complete response rates in primary HER2-positive breast
cancer: pooled analysis of 967 patients from five prospective trials
investigating lapatinib and trastuzumab. Ann Oncol. 2016
Aug;27(8):1519-25.</div><div>28. Rimawi MF, et al. Low PTEN levels and PIK3CA mutations predict
resistance to neoadjuvant lapatinib and trastuzumab without chemotherapy
in patients with HER2 over-expressing breast cancer. Breast Cancer Res
Treat. 2018 Feb;167(3):731-740.</div><div>29. Feldman DR, et al. Presence of somatic mutations within PIK3CA, AKT,
RAS, and FGFR3 but not BRAF in cisplatin-resistant germ cell tumors.
Clin Cancer Res. 2014 Jul 15;20(14):3712-20.</div><div>30. Jiang Y, et al. Novel secondary somatic mutations in Ewing’s sarcoma
and desmoplastic small round cell tumors. PLoS One. 2014 Aug
13;9(8):e93676.</div><div>Table 1 Legend</div><div>*No pathogenic DICER1 variants were found either tumor sample despite
reanalysis.</div><div>**The ETV6-NRTK3 gene fusion in the initial sequencing was later
identified upon reanalysis of the sequencing data.</div><div>***Germline analysis was performed at Ambry Genetics Laboratory as
FoundationOne™ testing does not offer germline analysis</div><div>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</div>