DICER 1 syndrome and pleuropulmonary blastoma : a report from the International Pleuropulmonary Blastoma Registry

K.A.P. Schultz , G.M. Williams , D.Yu. Kachanov, S.R. Varfolomeeva, A.D. Hill, L.P. Dehner, Y.H. Messinger 2 International Pleuropulmonary Blastoma Registry, Children’s Minnesota, Minneapolis, MN; Department of Cancer and Blood Disorders, Children’s Minnesota, Minneapolis, MN; Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia, Moscow, Russia; Division of Pathology Children’s National Medical Center, Washington, DC; Lauren V. Ackerman Laboratory of Surgical Pathology, Barnes-Jewish and St. Louis Children’s Hospitals, Washington University Medical Center, St. Louis, MO


Introduction
Pleuropulmonary blastoma (PPB, OMIM #601200) 1 is the most common primary lung tumor of infancy and early childhood 2 and heralds a unique spectrum of familial disease. 3,4The International PPB Registry has enrolled individuals with PPB and related conditions from all over the world including Russia.Early reports linked PPB to additional tumor types. 4Linkage analysis of affected families by the PPB Registry first localized the associated gene to chromosome 14 and sequencing confirmed DICER1 gene mutations underlie familial PPB and related conditions. 5DICER1 DICER 1 (OMIM #606241) 1 is a highly conserved gene encoding Dicer, an endonuclease which cleaves precursor microRNAs into mature miRNAs.DICER1 seems to function as a two hit tumor suppressor in PPB and most other DICER1 related conditions.In most patients, a germline loss-of-function mutation is seen.Most tumors in individuals with germline DICER1 mutations also display a second, tumor specific "hotspot" mutation in one of 5 codons in the RNase IIIb domain.This results in altered Dicer function and improperly cleaving 5p miRNAs leaving a hairpin structure that is then targeted for destruction.An abnormal 5p to 3p miRNA ratio in the cell leads to altered expression of mRNAs and increased risk of tumor formation. 6n analysis of loss-of-function germline DICER1 variation in the Cancer Genome Atlas component of the Exome Aggregation Consortium showed a prevalence of presumed pathogenic DICER1 variants of 1:10,600.
DICER1 testing should also be considered for individuals with multinodular goiter or differentiated thyroid carcinoma in two or more relatives or in an index patient with a family history consistent with DICER1associated conditions.
Two or more of the following conditions in an individual's personal or family history may also prompt consideration of testing: lung cysts, renal cysts, multinodular goiter or differentiated thyroid carcinoma, Wilms tumor, embryonal rhabdomyosarcoma, poorly differentiated neuroendocrine tumors or undifferentiated sarcomas.Macrocephaly may also be seen though its utility as an indication for testing is unknown.It should be noted that many of the aforementioned conditions may also be seen in alternate cancer predisposition syndromes including Cowden syndrome, Peutz Jegher syndrome and others, thus pre and post testing genetic counseling and consideration of other genetic predisposition syndromes is strongly recommended.

Pleuropulmonary Blastoma (PPB)
PPB is pathognomonic for DICER1 gene mutations and also one of the most aggressive and potentially fatal manifestations of this syndrome.PPB is believed to arise from primitive pleuropulmonary mesenchyme.It is the pulmonary analog of more familiar dysembryonic childhood tumors such as Wilms tumor (nephroblastoma), embryonal rhabdomyosarcoma, retinoblastoma and others. 2,7The biology of PPB is unique among these embryonal tumors in that clinically and pathologically PPB follows an agerelated progression over the first 7.5 years of life. 7,8The relatively innocuous-appearing early cystic manifestation of PPB (Type I) in very young children may progress to cystic/solid Type II PPB and then solid Type III PPB, both of which are overt aggressive mixed-pattern sarcomas (Fig. 1). 7,8In rare circumstances individuals older than 7 have been diagnosed with PPB; one even at 36 years of age. 9ung cysts later pathologically diagnosed as PPB have been observed by prenatal ultrasound as early as 31 weeks gestation.8 Cystic PPB can be unifocal, unilateral, and limited to the affected lobe, but in many cases it is multifocal and/or bilateral (Fig. 2). 8Type I PPB is often indistinguishable on imaging from congenital cystic adenomatoid airway malformation (also called congenital pulmonary airway malformation).[12]    Type II PPB is a mixed cystic and solid lesion.The residual cystic portions of Type II PPB feature the delicate cystic structures of type I PPB with its population of primitive small cells with or without rhabdomyoblastic differentiation and nodules of fetal cartilage (Fig. 3) whereas the solid foci of type II PPB have histological features that overlap with the exclusively solid type III PPB.Type III PPB is a complex sarcoma with embryonal 4 2017 ТОМ 4 О р и г и н а л ь н ы е и с с л е д о в а н и я rhabdomyosarcoma, high-grade spindle cell features, scattered large, bizarre-appearing anaplastic cells, and nodules of sarcomatous cartilage.The various patterns are not necessarily represented in any single Type III PPB.The median age of presentation (Type I, 8 months; Type II, 36 months; Type III, 41 months), the histologic features that overlap type I with type II and type II with type III and individual patients who demonstrate progression suggest that these 3 types represent a continuum (Fig. 5).Not all type I PPB will progress, some may actually regress or involute to type Ir PPB as noted by Hill et al. 13 Type Ir are cysts with the architecture of the type I PPB that lack the subepithelial primitive small cell population and have a hyalinized interstitium.Type Ir PPB has been observed at all ages.Unresected presumed type Ir PPBs in adult relatives of PPB patients have minimal potential to progress to a type II or III lesions. 8An asymptomatic lung cyst in a PPB family member after the age of 7 years (age at which >95% type II and III lesions are diagnosed 8 ) is likely a type Ir lesion (Fig. 6).The prognosis of PPB is correlated with the pathological type as a reflection of the increasingly aggressive nature of this neoplasm from a purely cystic to a solid neoplasm.Based on the PPB Registry retrospective series of 350 cases with nonstandardized treatment from many institutions, the long-term overall survival for type Ir is 100%, for type I PPB is 94%, for type II is 71%, and for type III is 53% (Fig. 7). 8The survival of Type I/Ir is significantly better than type II which is better than type III Management of Type I PPB If a type I PPB or type Ir lesion in a young child (<7 years of age) is suspected, complete cyst resection is recommended by performing cystectomy or lobectomy.The remaining lung and other unaffected lobes readily expand even when completely atelectatic before resection.Postoperative CT scan is recommended to look for residual cysts.
The major risk after type I PPB is its progression to the more advanced type II and III.A recent analysis by the PPB Registry of 115 type I and 40 type Ir PPBs has shown disease progression in 19/155 (12%) -all by 53 months of age.At last follow-up 95% of the 115 in this group with type I PPB are alive, and all deaths were preceded by progression to type II or III. 14Whether chemotherapy will prevent such progression is the most important unsolved question regarding the management of type I PPBs.Earlier retrospective study of type I PPB suggested that chemotherapy may improve outcome. 15In contrast a larger study by the PPB Registry of 89 type I and 26 type Ir patients did not show statistical advantage to chemotherapy. 8The PPB Registry currently suggests adjuvant chemotherapy for type I PPB with vincristine, actinomycin-D, and cyclophosphamide (VAC).The European EXPeRT group suggests vincristine and actinomycin-D. 16

Management of Type II and Type III PPB
Combined multimodal therapy including surgery and aggressive chemotherapy is necessary in cases of type II and III PPB. 8,16,17omplete surgical resection is recommended either primarily or following neoadjuvant chemotherapy for suspected type II and III PPB.Because these neoplasms are often large, a preoperative biopsy may be the initial step.Local control with re-resection should be attempted after no more than three rounds of chemotherapy because tumors have been demonstrated to recrudesce thereafter.Chemotherapy alone cannot be expected to produce complete disease eradication.At surgery, the type II or III tumor may appear encapsulated (Fig. 4b) or may be necrotic and friable with a tendency to rupture, requiring piecemeal removal.Intrathoracic spillage has been associated with poorer event-free survival for type II PPB, but not type III. 8 .Intrathoracic spread may involve direct invasion of pleural surfaces, and the diaphragmatic muscle, whereas, ribs and intercostal muscles are only rarely invaded.Occasionally, an extrapleural pneumonectomy maybe required to remove extensive tumor that involves the lung and both pleural surfaces.
8][19] The regimen of IVADo: ifosfamide, vincristine, actinomycin-D, and doxorubicin has been recommended by the PPB Registry since 2009. 20IVADo has been given in a semiprospective manner to 54 patients.An ad-hoc analysis shows relative advantage of this regimen compared with historical controls (Fig. 8).

Figure 8. Analysis in 2015 by the IPPBR of event-free-survival (EFS) and overall survival (OS) analysis of 54 cases of IVDo treated Type II&III compared to historical controls. A. The whole group. B. Only patients treated after 2000. In both cases survival benefit is noted
In contrast to type I PPB, type II and III PPB may present with metastatic disease.Brain metastasis is the most common site. 8Central nervous system (CNS) metastasis has been observed within 6 weeks of normal surveillance brain imaging, which indicates the potentially rapid growth of brain metastases.Therapy must be prompt and may include gross total resection followed by radiation therapy.Stereotactic radiosurgery followed by focal conformal radiotherapy would be a minimum for therapy although some children have had additional chemotherapy, including high-dose chemotherapy with stem cell rescue. 21Other metastatic sites include intrathoracic metastasis along pleural surfaces, bone and rarely liver metastasis.
Whether upfront radiation therapy in type II and III PPB is necessary is not clear.Radiation is commonly used for residual disease following surgery and in the context of intensive chemotherapy.It is clearly indicated for brain metastasis.Dosage appropriate for high-grade sarcoma (44 Gy or above) may be required. 18lapsed PPB In a recent PPB Registry analysis of individuals with recurrent PPB, the majority of relapses (59%) were brain metastasis; some had local chest relapse as well. 22n most of these cases metastasis to the brain occurred within 36 months of detection of the primary tumor and was independent of disease status in the chest. 8,23Spinal metastases may also occur.Therapy for relapsed type II and III PPBs is not standardized and the outcome is poor, with only 26% long-term survival (Fig. 9). 22Some have survived after relapse with multimodal therapy with surgery, radiotherapy.Some survivors have also undergone high-dose chemotherapy with autologous stem cell rescue.

Conclusions
The International PPB Registry and the International Ovarian and Testicular Stromal Tumor Registry represent a multi-institutional consortium of clinicians and research scientists based at Children's Minnesota, devoted to understanding the pathogenesis of PPB, Sertoli-Leydig cell tumor, and other DICER1-associated conditions and to improving their diagnosis and treatment.Founded in 1988 and 2011 respectively, the two linked registries have enrolled over 500 patients with PPB and more than 150 patients with ovarian stromal tumors and additional patients with other DICER1related conditions.The registries serve as coordinating centers for procurement and shared access to biological samples and clinical information.More information is available at PPBinfo@childrensmn.org or www.PPBregistry.organd OTST@childrensmn.org and OTSTregistry.org.
Through collaborative studies using these clinical and biological repositories, much progress has already been achieved.The clinical arms of the consortium have defined the spectrum of childhood cancers associated with germline DICER1 mutations and conducted the first clinical trials to refine PPB treatment protocols.
These discoveries are transforming the diagnosis and clinical management of children with PPB and other DICER1-associated solid tumors.We have shown that appropriately targeted genetic and clinical screening can lead to early detection and may increase likelihood of cure for children with early PPB.24

Figure 1 .
Figure 1.Age at presentation histograms: type I and type Ir PPB (left); type II and III PPB (right) .8

Figure 2 .
Figure 2. Chest film and CT images of type I PPB demonstrating typical presentation with pneumothorax and showing the multilocular cyst

Figure 3 .
Figure 3. Type II PPB.(a) Axial CT image of solid tumor encroaching upon residual air-filled cystic spaces.Thirty months earlier, a large right lower lobe cyst was apparent on an abdominal flat plate radiograph obtained for abdominal pain (not shown).This cyst was not appreciated.(b) Sarcomatous overgrowth of cyst septa (different patient from (a))

Figure 4 .
Figure 4. Type III PPB.(a) Axial chest CT image of large right hemithoracic mass.(b) Cut section of encapsulated solid PPB resected prior to chemotherapy.This child developed bifrontal cerebral metastases 12 months following chest PPB diagnosis.Chest disease did not recur.The child is alive 10 years from chest diagnosis

Figure 5 .
Figure 5. Classical progression of PPB type I to PPB type II and III

Brother 1 .Figure 6 .
Figure 6.Type Ir PPB are lung cysts which radiographically resemble PPB with delicate septa without any remnants of the primitive cells in the cyst walls.An older PPB family member with cysts most likely has Type Ir PPB.However even very young children can have Type Ir PPB

Figure 7 .
Figure 7. Outcome of PPB subtypes based on data from 350 PPB cases.8The survival of Type I/Ir is significantly better than type II which is better than type III

4 ОРис. 3 .Fig. 3 .Рис. 4 .Fig. 4 .
Fig. 3. Type II PPB: a -axial CT image of solid tumor encroaching upon residual air-filled cystic spaces.Thirty months earlier, a large right lower lobe cyst was apparent on an abdominal flat plate radiograph obtained for abdominal pain (not shown).This cyst was not appreciated; -sarcomatous overgrowth of cyst septa (different patient from (a))

4 ОFig. 7 .
Fig. 7. Outcome of PPB subtypes based on data from 350 PPB cases[8].The survival of Type I/Ir is significantly better than type II which is better than type III

4 ОFig. 8 . 9 .Fig. 9 .
Fig. 8. Analysis in 2015 by the IPPBR of event-free-survival (EFS) and overall survival (OS) analysis of 54 cases of IVDo treated Type II&III compared to historical controls.A. The whole group.B. Only patients treated after 2000.In both cases survival benefit is noted

DICER1 syndrome and pleuropulmonary blastoma: a report from the International Pleuropulmonary Blastoma Registry K.A.P. Schultz 1, 2 , G.M. Williams 1, 2 , D.Yu. Kachanov 3 , S.R. Varfolomeeva 3 , A.D. Hill 4 , L.P. Dehner 5 , Y.H. Messinger 1, 2 1
4nternational Pleuropulmonary Blastoma Registry, Children's Minnesota, Minneapolis, MN; 2 Department of Cancer and Blood Disorders, Children's Minnesota, Minneapolis, MN; 3 Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia, Moscow, Russia;4Division of Pathology Children's National Medical Center, Washington, DC; PPB) is the most common primary lung tumor of infants and young children and is known to be associated with pathogenic variants in the DICER1 gene.In addition to PPB, DICER1-associated conditions include a variety of other benign and malignant tumors including cystic nephroma and Wilms tumor, ovarian Sertoli-Leydig cell tumor and gynandroblastoma, multinodular goiter and thyroid carcinoma and certain childhood brain tumors among others.Early identification of individuals most at risk for DICER1-associated conditions may allow family education, targeted surveillance and identification of DICER1-related tumors and conditions in their earliest and most curable form.Given the rarity of these conditions, international collaboration is underway and encouraged.