Gene Mutation and Possible Malignancy-Induction Mechanism in PPB

In mid-2009, the International PPB Registry and collaborators from Children’s National Medical Center, Washington, D.C., the Siteman Cancer Center of Washington University St. Louis, Cincinnati Children’s Hospital and Children’s Hospital of Wisconsin-Milwaukee reported that loss-of-function germline mutations in the DICER1 gene were found in 11 of 11 families studied (AACR abstract LB-91, below). The PPB Family Tumor and Dysplasia Syndrome is the first human disease complex to be associated with a DICER1 mutation. Because the syndrome appears to be inherited as an autosomal dominant phenomenon (with reduced penetrance and marked pleiotropy), all somatic cells have one normal and one mutant DICER1. (see Registry Publications Hill Science 2009)

Also included was the important observation that, by immunohistochemical staining, lung epithelial cells, present in Types I and II PPB and histologically benign, show focal or segmental loss of DICER1 protein, whereas overtly sarcomatous cells retained staining for DICER1. It is postulated that dysregulated autocrine signals from the epithelial cells may induce in mesenchymal cells cyst formation and malignant transformation. It is known that, in embryonic mouse lung, epithelium and mesenchyme interact in a complex way (epithelial/mesenchymal 'cross-talk') for proper branching lung morphogenesis (Chuang; Trends in Cell Biology 2003; 13:86-91) and that normal dicer1 (mouse dicer gene) is required in mouse lung epithelium for normal branching lung morphogenesis (Harris; PNAS 2006; 103:2208-13). In mice with dicer1-mutant lung epithelium, branching morphogenesis is impaired and cystic dilatations result (Harris). A related phenomenon may be represented by the observation that epithelial dicer1 is required in epithelium of mouse mammary tissue for normal mammary development: during puberty: mice with dicer1-mutant epithelium in mammary glands exhibited inhibition of ductal elongation and branching (Xinjuang; AACR 2009 meeting abstract #1937).

AACR Abstract Denver, April 20,2009 #LB-91:

Inherited DICER1 mutations in familial pleuropulmonary blastoma

Dana Ashley Hill, Jennifer Ivanovich, John R. Priest, Christina A. Gurnett, Louis P. Dehner, David Desruissea, Jason A. Jarzembowski, Kathryn A. Wikenheiser, Brian K. Suarez, Alison J. Whelan, Gretchen Williams, Dawn Bracamontes, Yoav Messinger, Paul J.Goodfellow. Children's National Medical Center, Washington, DC, Washington University Medical Center, St. Louis, MO, International PPB Registry, Minneapolis, MN, Children's Hospital of Wisconsin, Milwaukee, WI, Children's Hospitals and Clinics of Minnesota, Minneapolis-St. Paul, MN, Cincinnati Children’s Hospital, Cincinnati, OH

Introduction: Pleuropulmonary blastoma (PPB) is a rare lung sarcoma that arises during fetal development. The majority of patients with PPB are diagnosed before 6 years of age. The early stage of PPB is characterized by cysts lined by benign epithelial cells and small numbers of subepithelial, uncommitted mesenchymal cells. In later stages the mesenchymal cells transform and become solid high grade, multipatterned sarcomas. Sarcomatous overgrowth by the mesenchymal component dictates clinical outcome. Approximately 25% of children with PPB have a personal or family history suggestive of underlying inherited cancer susceptibility. Some children with PPB have multifocal lung tumors and/or synchronous or metachronous embryonal cancers. Family members are at increased risk for developing PPB, Wilms tumor-related neoplasms, rhabdomyosarcoma and a number of other conditions.

Methods: Review of family histories for sixty-five PPB probands ascertained through the International PPB Registry (www.ppbregistry.org) revealed 11 families with apparent inherited predisposition to PPB as evidenced by two or more relatives with PPB, lung cysts, cystic nephroma and/or embryonal rhabdomyosarcoma. Blood and/or saliva specimens were collected as a source of genomic DNA. All research subjects provided written consent for molecular and family history studies as approved by the Human Research Protection Office at Washington University in St. Louis.  We used Affymetrix 6.0 human SNP arrays to genotype 49 individuals from four families (14 affecteds). 4117 SNPs were selected for the analysis. Genome wide parametric linkage analysis was performed using the Genehunter v2.1r5 algorithm. Subsequent sequencing, PCR and immunohistochemistry were performed using standard methods. DICER1 immunohistochemistry was performed with a rabbit polyclonal anti-DICER1 antibody (HPA000694, rabbit anti-human, Sigma-Aldrich, St. Louis, MO). 

Results: Linkage analysis suggested a PPB locus on a 7 Mb interval on chromosome 14q31-32 containing 72 genes. Sequencing identified germline DICER1 mutations in the four families included in the linkage analysis and seven additional PPB families. 10 mutations resulted in premature stop codons proximal to the functional RNase III domains. The 11th mutation resulted in a leucine to arginine change in a conserved amino acid between the two functional RNase domains. All 11 sequence defects appear to be loss-of-function mutations. RT-PCR and direct sequencing demonstrated marked reductions in levels of mutant DICER1 transcripts compared with wild-type suggestive of nonsense-mediated decay. Immunohistochemical analysis of DICER1 in PPB tumors from mutation carriers showed retention of staining in the mesenchymal/sarcomatous components of the tumors. In contrast, focal or segmental loss of DICER1 staining was seen specifically in the benign epithelial component of the tumors.

Conclusions: The discovery of germline DICER1 mutations in affected members of PPB kindreds, coupled with absence of DICER1 protein in tumors, suggests DICER1 loss is responsible for familial PPB. DICER1 is responsible for generating active miRNAs (and siRNAs). Our immunohistochemical studies, in which we show loss of DICER1 in the epithelial component of tumors but retention of DICER1 expression in the transformed mesenchymal cells, provide evidence DICER1 loss promotes malignant transformation through a non-cell autonomous mechanism. In the mouse, loss of Dicer1 in the epithelium of the developing lung alters epithelial-mesenchymal signaling, resulting in a lung phenotype that mimics early PPB (PNAS 103, 2208 (2006)). Our study of hereditary PPB suggests the primary defect does not occur in the mesenchymal cell as was long suspected, but rather in the epithelial cell and extends the findings in mice to human tumorigenesis. We hypothesize that loss of DICER1 in lung epithelium promotes both cyst formation and tumor initiation through dysregulation of developmental genes that are normally regulated by miRNAs. Further study of PPB families and the tumors that arise in DICER1 mutation carriers represent a unique opportunity to learn about the cellular processes in the borderland between development and neoplasia and to study how tissue-specific loss of DICER1 (and the miRNAs it regulates) manifests in human disease.

Much remains to be learned about the implications of DICER1 mutations for PPB and the associated conditions in the PPB Family Tumor Susceptibility Syndrome. PPB families have many phenotypically normal obligate carriers. The inheritance pattern suggests an autosomal dominant susceptibility with reduced penetrance. DICER1 haploinsufficiency does not appear to be the sole cause for disease in these families, but may be “the first hit”, with another event required for disease expression. Observations reported in the study suggest that the second hit is in the benign-appearing epithelium. The extent to which DICER1 mutations are found in familial PPB and in apparently sporadic PPB must be defined. Additional PPB patients and families are under study by the Registry and our collaborators to explore further the implications of DICER1 mutation. Referrals of PPB families are appreciated.

The PPB Registry intends to establish a CLIA-approved laboratory to offer clinical tests for use by physicians and genetic counselors when PPB patients and their families seek such testing.

Biology Summary:  Dicer, Drosha, and Silencing RNA

DICER1 codes for DICER1 protein  - a key enzyme in the production of micro-RNA (miRNA) and short interfering RNA (siRNA), known collectively as small silencing RNAs.  DICER1 is a cytoplasmic endoribonuclease III enzyme discovered in 2001(Bernstein; Nature 2001; 409:363); DICER1 cleaves ~70 nucleotide stem-loop precursor molecules yielding miRNA and siRNA (typically ~22 nucleotides). Small silencing RNAs are non-coding cytoplasmic RNAs and are critical regulators of messenger RNA (mRNA) by interfering with (silencing, quenching, suppressing) mRNA. More than 95% of cytoplasmic RNA does not code for protein synthesis (Nelson; Brain Pathol 2008; 18:130). The importance of non-coding RNAs in controlling mRNA expression by suppression has been elaborated since ~1992 (Bernstein; RNA 2001; 7:1509). The many unique small silencing RNAs target specific mRNAs and guide them to an effector complex known as the “RNA-induced silencing complex” (RISC). In addition to DICER1’s role cleaving precursors into small RNAs, DICER participates with RISC in suppressing mRNA expression by either degrading it or preventing its transcription.

“Drosha” enzyme is another critical endoribonuclease III required for production of small non-coding RNAs. Drosha was discovered in 2003 (Lee; Nature 2003; 425: 415). Located in the nucleus, it processes early small RNA precursors. Drosha products are then actively transported to the cytoplasm where DICER1 protein acts.

DICER1 and drosha and the small RNA mRNA-silencing activities are highly conserved across plant and animal species. This gene-regulating schema is now considered essential for normal development and function of cells and is being implicated in disease (Alvarez-Garcia; Development 2005; 132: 4653). The PPB Family Tumor Susceptibility Syndrome is the first human disease in which DICER1 malfunction is implicated.

The PPB Family Tumor and Dysplasia Syndrome

Pleuropulmonary blastoma is a genetic and familial disease in some cases. The International PPB Registry currently estimates that approximately 40% of cases have a genetic basis, based on the presence of multiple associated conditions. This is the PPB Family Tumor and Dysplasia Syndrome; it may be manifested in the patient or family or both (see Registry Publications: Priest 1996; Boman 2006; Priest 2009, Hill Science 2009 and Literature Sources, focus words: “familial/constitutional” and “dicer mutation”). In general this syndrome involves neoplastic and dysplastic disease appearing in the first 5-6 years of life, but some conditions appear up to the late teens or early 20’s. Although the full phenotypic expression of this syndrome is not yet known, there do not appear to be neoplasias in later adult years associated with PPB. This syndrome does not appear to overlap with other known family tumor or cancer syndromes.

The conditions most prominent in this syndrome are as follows. The most frequently observed conditions are in bold, but a genetic basis for associating most of these conditions to PPB remains to be investigated.

PPB -     may be bilateral or multifocal, may be more than one PPB in a family

Lung cysts –  (when other family members’ cysts have been available for pathologic study, they are Type I or Type Ir PPB)

Cystic nephroma – observed in 9-10% of PPB patients or kindreds

Dysplasias – such as nasal chondromesenchymal hamartoma; also, small bowel (and rarely large bowel) hamartomatous polyps, cystic hepatic hamartoma

Any childhood cancer – including rhabdomyosarcoma, other sarcomas, leukemias, medulloblastoma and other CNS tumors

Gonadal tumors – especially Sertoli-Leydig cell tumors, ovarian dysgerminoma, testicular seminoma and perhaps germ cell tumors

Thyroid nodular hyperplasia or cancer – especially in teen years (because thyroid disease is common in the general population, it may be coincidental that nodular thyroid hyperplasias are frequent in PPB families)

A few children with PPB have had what appear to be treatment-related second malignant neoplasms, but despite their apparent propensity to neoplasia, the frequency of treatment-related neoplasms in PPB survivors does not appear to be different from other childhood cancer survivors.