PTEN Deficiency Is Fully Penetrant for Prostate Adenocarcinoma in C57BL/6 Mice via mTOR-Dependent Growth
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.080055
ISSN1525-2191
AutoresJorge Blando, Melisa J. Portis, Fernando Benavides, Angela Alexander, Gordon B. Mills, Bhuvanesh Dave, Claudio J. Conti, Jeri Kim, Cheryl L. Walker,
Tópico(s)Polyamine Metabolism and Applications
ResumoThe tumor suppressor phosphatase and tensin homolog (PTEN) is frequently involved in human prostate carcinoma. PTEN is therefore an attractive target for the development of preclinical animal models. Prostate intraepithelial neoplasia lesions develop in mice with Pten heterozygosity, but disease progression has been reported only in combination with either other tumor suppressor gene alterations or the conditional inactivation of both Pten alleles in prostate epithelial cells. We report that on a C57BL/6 background, in contrast to previous studies on mixed 129 genetic backgrounds, Pten locus heterozygosity is fully penetrant for the development of prostate adenocarcinoma. Grossly observable tumors were detected at 6 months of age, and, by 10 to 12 months, 100% of examined mice developed adenocarcinoma of the anterior prostate. Furthermore, double heterozygotes carrying both Pten and Tsc2-null alleles showed no increase relative to Pten+/− heterozygotes in either lesion development or progression. Lesions in both Pten+/−; Tsc2+/−, and Pten+/− mice exhibited loss of PTEN expression and activation of PI3K signaling. PI3K activation occurred early in prostate intraepithelial neoplasia lesion formation in these animals, consistent with loss of PTEN function, and contributed to the etiology of tumors that developed in Pten+/− mice. Furthermore, prostate lesion growth in Pten+/− mice was dependent on mTOR, as evidenced by a reduction in both phospho-S6 levels and proliferative index after rapamycin treatment. The tumor suppressor phosphatase and tensin homolog (PTEN) is frequently involved in human prostate carcinoma. PTEN is therefore an attractive target for the development of preclinical animal models. Prostate intraepithelial neoplasia lesions develop in mice with Pten heterozygosity, but disease progression has been reported only in combination with either other tumor suppressor gene alterations or the conditional inactivation of both Pten alleles in prostate epithelial cells. We report that on a C57BL/6 background, in contrast to previous studies on mixed 129 genetic backgrounds, Pten locus heterozygosity is fully penetrant for the development of prostate adenocarcinoma. Grossly observable tumors were detected at 6 months of age, and, by 10 to 12 months, 100% of examined mice developed adenocarcinoma of the anterior prostate. Furthermore, double heterozygotes carrying both Pten and Tsc2-null alleles showed no increase relative to Pten+/− heterozygotes in either lesion development or progression. Lesions in both Pten+/−; Tsc2+/−, and Pten+/− mice exhibited loss of PTEN expression and activation of PI3K signaling. PI3K activation occurred early in prostate intraepithelial neoplasia lesion formation in these animals, consistent with loss of PTEN function, and contributed to the etiology of tumors that developed in Pten+/− mice. Furthermore, prostate lesion growth in Pten+/− mice was dependent on mTOR, as evidenced by a reduction in both phospho-S6 levels and proliferative index after rapamycin treatment. The tumor suppressor gene phosphatase and tensin homolog (PTEN), also known as MMAC1 (for mutated in multiple advanced cancers),1Steck PA Pershouse MA Jasser SA Yung WK Lin H Ligon AH Langford LA Baumgard ML Hattier T Davis T Frye C Hu R Swedlund B Teng DH Tavtigian SV Identification of a candidate tumour suppressor gene. 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Importantly, progression to adenocarcinoma generally is not observed in Pten+/− mice, possibly due to age-dependent morbidity associated with the high incidence of thyroid lymphomas that occur in these animals. It has recently been suggested that genetic background and/or modifier genes may influence the development of lesions in Pten-haploinsufficient animals.23Freeman D Lesche R Kertesz N Wang S Li G Gao J Groszer M Martinez-Diaz H Rozengurt N Thomas G Liu X Wu H Genetic background controls tumor development in PTEN-deficient mice.Cancer Res. 2006; 66: 6492-6496Crossref PubMed Scopus (94) Google Scholar In that study, both onset and spectrum of lesions at several anatomical sites observed with a Pten null allele placed on a 129;BALB/c mixed background differed from that observed with Pten null alleles on either a 129;C57BL/6 or 129;CD-1 mixed backgrounds. In contrast to Pten heterozygotes, conditional Pten knockout mice with complete loss of Pten in the prostate develop invasive prostate carcinoma, with variable latency.24Backman SA Ghazarian D So K Sanchez O Wagner KU Hennighausen L Suzuki A Tsao MS Chapman WB Stambolic V Mak TW Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten.Proc Natl Acad Sci USA. 2004; 101: 1725-1730Crossref PubMed Scopus (138) Google Scholar, 25Trotman LC Niki M Dotan ZA Koutcher JA Di Cristofano A Xiao A Khoo AS Roy-Burman P Greenberg NM Van Dyke T Cordon-Cardo C Pandolfi PP Pten dose dictates cancer progression in the prostate.PLoS Biol. 2003; 1: E59Crossref PubMed Scopus (563) Google Scholar, 26Wang S Gao J Lei Q Rozengurt N Pritchard C Jiao J Thomas GV Li G Roy-Burman P Nelson PS Liu X Wu H Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.Cancer Cell. 2003; 4: 209-221Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 27Ma X Ziel-van der Made AC Autar B van der Korput HA Vermeij M van Duijn P Cleutjens KB de Krijger R Krimpenfort P Berns A van der Kwast TH Trapman J Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer accompanied by increased epithelial cell proliferation but not by reduced apoptosis.Cancer Res. 2005; 65: 5730-5739Crossref PubMed Scopus (160) Google Scholar, 28Ratnacaram CK Teletin M Jiang M Meng X Chambon P Metzger D Temporally controlled ablation of PTEN in adult mouse prostate epithelium generates a model of invasive prostatic adenocarcinoma.Proc Natl Acad Sci USA. 2008; 105: 2521-2526Crossref PubMed Scopus (76) Google Scholar Inactivation of Pten in combination with other mutations can also promote cancer progression. Double heterozygous mice that carry p27 (now Cdkn1b) and Pten defects (Pten+/−, p27+/−) develop invasive carcinoma.29Di Cristofano A De Acetis M Koff A Cordon-Cardo C Pandolfi PP Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse.Nat Genet. 2001; 27: 222-224Crossref PubMed Scopus (403) Google Scholar Similarly, when crossed with Nkx3–1 knockout mice (a homeobox gene expressed in prostate epithelium) Nkx3–1+/−, Pten+/− mice also develop metastatic prostate carcinoma.30Kim MJ Cardiff RD Desai N Banach-Petrosky WA Parsons R Shen MM Abate-Shen C Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis.Proc Natl Acad Sci USA. 2002; 99: 2884-2889Crossref PubMed Scopus (264) Google Scholar, 31Abate-Shen C Banach-Petrosky WA Sun X Economides KD Desai N Gregg JP Borowsky AD Cardiff RD Shen MM Nkx3.1;Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases.Cancer Res. 2003; 63: 3886-3890PubMed Google Scholar Heterozygosity at the Pten locus also promotes prostate cancer progression in the transgenic murine prostate cancer model (TRAMP)32Kwabi-Addo B Giri D Schmidt K Podsypanina K Parsons R Greenberg N Ittmann M Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression.Proc Natl Acad Sci USA. 2001; 98: 11563-11568Crossref PubMed Scopus (273) Google Scholar and conditional Pten knockouts crossed with p53 (Trp53) knockouts develop highly lethal invasive prostate carcinoma at a young age.33Chen Z Trotman LC Shaffer D Lin HK Dotan ZA Niki M Koutcher JA Scher HI Ludwig T Gerald W Cordon-Cardo C Pandolfi PP Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.Nature. 2005; 436: 725-730Crossref PubMed Scopus (1539) Google Scholar Recently, other alterations that regulate PTEN/PI3K signaling at the level of mTOR have also been shown to contribute to prostate carcinogenesis: Pten haploinsufficiency cooperates with Rheb overexpression to promote prostate tumorigenesis34Nardella C Chen Z Salmena L Carracedo A Alimonti A Egia A Carver B Gerald W Cordon-Cardo C Pandolfi PP Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events.Genes Dev. 2008; 22: 2172-2177Crossref PubMed Scopus (97) Google Scholar and Lkb1 deficiency, a tumor suppressor and upstream kinase for AMP-activated kinase signaling to TSC2, leads to the development of PIN lesions.35Pearson HB McCarthy A Collins CM Ashworth A Clarke AR Lkb1 deficiency causes prostate neoplasia in the mouse.Cancer Res. 2008; 68: 2223-2232Crossref PubMed Scopus (61) Google Scholar TSC2 has not been previously implicated in prostate tumorigenesis, but alterations in this tumor suppressor gene do predispose to genitourinary tumors, primarily renal cell carcinoma.36Gomez MR Sampson JR Whittemore VH Harris JC Tuberous Sclerosis Complex. Oxford University Press, Oxford1999: 3-46Google ScholarTsc2 knockout mice develop renal cell carcinoma and vascular lesions, primarily liver hemangiomas, but do not develop prostate lesions, even at older ages.37Kobayashi T Minowa O Kuno J Mitani H Hino O Noda T Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice.Cancer Res. 1999; 59: 1206-1211PubMed Google Scholar, 38Onda H Lueck A Marks PW Warren HB Kwiatkowski DJ Tsc2(+/−) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background.J Clin Invest. 1999; 104: 687-695Crossref PubMed Scopus (318) Google Scholar Interestingly, it was recently reported that Pten+/−;Tsc2+/− double heterozygous mice develop invasive prostate carcinoma with 100% penetrance.39Ma L Teruya-Feldstein J Behrendt N Chen Z Noda T Hino O Cordon-Cardo C Pandolfi PP Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression.Genes Dev. 2005; 19: 1779-1786Crossref PubMed Scopus (96) Google Scholar In this study, tumors were reported to arise as early as 5 months of age and were found in all lobes of the prostate (dorsolateral prostate, anterior prostate, and ventral prostate). Pten expression from the normal allele was reported to be retained in these tumors, suggesting that the Tsc2 defect was responsible for progression of Pten-dependent prostate cancer in these animals. However, a similar study using Pten+/−;Tsc2+/− mice reported no progression of PIN lesions in double heterozygotes.40Manning BD Logsdon MN Lipovsky AI Abbott D Kwiatkowski DJ Cantley LC Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2.Genes Dev. 2005; 19: 1773-1778Crossref PubMed Scopus (212) Google Scholar Thus the potential for Tsc2 defects to contribute to the development of Pten-dependent prostate carcinoma requires further study. We report here that in contrast to previous studies in which Pten null alleles were placed on mixed 129 genetic backgrounds, Pten haploinsufficiency is fully penetrant for development of prostate carcinoma on a C57BL/6 background. 100% of Pten+/− mice developed prostate adenocarcinoma by 10–12 months of age. Furthermore, double heterozygotes carrying both Pten and Tsc2 null alleles did not exhibit any acceleration in lesion development or progression. Activated mTOR signaling that could be reversed with rapamycin was observed in PIN lesions and adenocarcinomas that developed in Pten+/− animals, with adenocarcinomas from both Pten+/−;Tsc2+/− and Pten+/− mice exhibiting loss of PTEN expression. Mice were housed in suspended polycarbonate cages or individually ventilated cages (Lab Products, Maywood, NJ) on autoclaved hardwood bedding (PJ Murphy Forest Products Corp., Montville, NJ) in an AAALAC-accredited facility (M. D. Anderson Cancer Center, Science Park–Research Division). Room conditions included temperature (20–22°C), humidity (60–70%), and light (14/10 hours; light/dark). Commercial rodent pelleted food (Harlan Teklad, Madison, WI) and autoclaved water were available ad libitum. All procedures were in compliance with the Public Health Service Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The protocol involving use of these mice was approved by the M. D. Anderson Cancer Center Institutional Animal Care and Use Committee. Male mice were euthanized at various ages from 7 to 12 months by CO2 asphyxiation, and tissues were harvested and either snap-frozen in liquid N2 and stored at −80°C or fixed in 10% neutral buffered formalin and paraffin embedded. The intact male reproductive system was transversely sectioned and then paraffin-embedded for histopathological and immunohistological analysis. The Pten+/− mice were a kind gift from Dr. Ramone Parsons (Columbia University)20Podsypanina K Ellenson LH Nemes A Gu J Tamura M Yamada KM Cordon-Cardo C Catoretti G Fisher PE Parsons R Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems.Proc Natl Acad Sci USA. 1999; 96: 1563-1568Crossref PubMed Scopus (833) Google Scholar and the Tsc2+/− mice were obtained from Dr. David Kwiatkowski (Brigham and Women's Hospital).38Onda H Lueck A Marks PW Warren HB Kwiatkowski DJ Tsc2(+/−) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background.J Clin Invest. 1999; 104: 687-695Crossref PubMed Scopus (318) Google Scholar Pten+/− mice 12 to 14 months old were treated with rapamycin (LC Laboratories, Woburn, MA) (0.15 mg/kg) (n = 4) or vehicle (n = 3) for 14 days daily (i.p) and sacrificed at the end of the study. The vehicle was Tween 80, polyethylene glycol, and ethanol). Tissues were harvested and fixed as described above. We performed a genetic background characterization of our C57BL/6.129S1/v-Pten congenic strain containing a Pten targeted mutation (Ptentm1Rps).20Podsypanina K Ellenson LH Nemes A Gu J Tamura M Yamada KM Cordon-Cardo C Catoretti G Fisher PE Parsons R Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems.Proc Natl Acad Sci USA. 1999; 96: 1563-1568Crossref PubMed Scopus (833) Google Scholar For this, we selected 92 microsatellite markers (simple sequence length polymorphism) evenly distributed over all of the 19 autosomal chromosomes (genome scan) and polymorphic between C57BL/6J and 129S1/Sv inbred strains. The average marker spacing was 15 cM. D19Mit88 on chromosome 19 was the only marker showing 129S1/C57BL/6 heterozygosity for all Pten+/− mice. This is expected since the Pten gene is localized in the same region on chromosome 19 as D19Mit88, and the targeted allele is 129S1/Sv (W9.5 ES cells) in origin. In agreement with the number of backcross generations performed in our Pten null colony (>N6), the background strain characterization showed that 91 of 92 markers (98.9%) were homozygous C57BL/6. Tissues were stained with hematoxylin and eosin, and prostates were examined microscopically by two study pathologists (J.B. and C.J.C.) blinded as to age and genotype of study animals. Hematoxylin and eosin sections were evaluated for precursor lesions identified as hyperplastic, low-grade PIN, or high-grade PIN and adenocarcinoma whose severity was described by Shapell et al.41Shappell SB Thomas GV Roberts RL Herbert R Ittmann MM Rubin MA Humphrey PA Sundberg JP Rozengurt N Barrios R Ward JM Cardiff RD Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee.Cancer Res. 2004; 64: 2270-2305Crossref PubMed Scopus (478) Google Scholar To detect downstream targets of the PTEN signaling pathway, immunohistochemistry was performed on paraffin-embedded prostate tissue sections using primary antibodies against AKT (1:100; Santa Cruz no. sc-1619, Santa Cruz, CA), phospho-AKT (Ser 473) (1:50; Cell Signaling Technologies, Beverly, MA, no. 3787), S6 (1:50; Cell Signaling Technologies no. 2217), and phospho-S6 (S235/236) (1:50; Cell Signaling Technologies no. 2211). Ki-67 (1:50 Santa Cruz no.15402) was used to determine proliferative index in vehicle and rapamycin treated mice. Primary antibodies were detected with biotinylated secondary antibodies, including anti-goat IgG for AKT, and an Envision plus labeled polymer, anti-rabbit-horseradish peroxidase (Dako Laboratories, Carpinteria, CA), for phospho-AKT, S6 and phospho-S6. This was followed by peroxidase-conjugated avidin/biotin (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA) and DAB substrate (Dako Laboratories). Intensity of immunohistochemistry staining was graded on a scale as follows: “-” indicating no apparent staining, “+” indicating weak staining, “++” indicating moderate staining and “+++” indicating strong staining. The four point scale for grading immunohistochemistry was used as described previously.42Maxwell P McCluggage WG Audit and internal quality control in immunohistochemistry.J Clin Pathol. 2000; 53: 929-932Crossref PubMed Scopus (24) Google Scholar, 43Adams EJ Green JA Clark AH Youngson JH Comparison of different scoring systems for immunohistochemical staining.J Clin Pathol. 1999; 52: 75-77Crossref PubMed Scopus (110) Google Scholar For detection of PTEN, after deparaffinization, sections were pretreated with microwave irradiation in 0.01 mol/L citrate buffer (pH 6.0), followed by blocking 20 minutes with 10% normal donkey serum. Sections were incubated with anti-PTEN antibody (Neomarkers, 1:50 dilution, Freemont, CA) overnight at 4°C. After washing with phosphate-buffered saline, the sections were incubated with the secondary fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). The slides were examined using a Fluoroview laser confocal microscope (Olympus America, Melville, NY). Normal and tumorigenic anterior prostate tissue lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk and separately incubated with phospho-AKT (Thr 308) (1:1000, Cell Signaling Technologies), phospho-AKT (Ser 473) AKT (1:1000, Cell Signaling Technologies), (1:1000, Cell Signaling Technologies), phospho-S6 (1:2000, Cell Signaling Technologies), S6 (1:1000, Cell Signaling Technologies), phospho-TSC2 (Ser 939) (1:1000, Cell Signaling Technologies), and TSC2 (1:1000, Epitomics; Burlingame, CA) for 2 hours, followed by streptavidin horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for one hour at room temperature. Lumiglo (KPL) was used for detection on X-ray film (BioMax, Eastman Kodak, Rochester, NY). As a loading control, blots were stripped and reprobed with an antibody to γ-tubulin (1:5000; Sigma, St. Louis, MO). In-house colonies of Pten+/−20Podsypanina K Ellenson LH Nemes A Gu J Tamura M Yamada KM Cordon-Cardo C Catoretti G Fisher PE Parsons R Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems.Proc Natl Acad Sci USA. 1999; 96: 1563-1568Crossref PubMed Scopus (833) Google Scholar and Tsc2+/−38Onda H Lueck A Marks PW Warren HB Kwiatkowski DJ Tsc2(+/−) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background.J Clin Invest. 1999; 104: 687-695Crossref PubMed Scopus (318) Google Scholar mice maintained on a C57BL/6 genetic background and confirmed by microsatellite analysis to be homozygous C57BL/6 (see Materials and Methods) were used to generate double heterozygous mice (Pten+/−; Tsc2+/−) for analysis. Prostate lesions were evaluated in wild-type, Pten+/− single heterozygotes, Pten+/−; Tsc2+/− double heterozygotes and Tsc2+/− single heterozygotes with groups of male mice examined at 7–9 and 10–12 months of age. Whole prostates were removed from mice and processed using conventional histology techniques. At necropsy, grossly observable prostate lesions were often noted, in one case in a moribund animal at 6 months of age. As shown in Supplemental Figure 1 (found at ), enlargement and changes in the coloration and texture of the anterior prostrate were the most common observations in these animals. Large, solid masses were also observed, often adjacent to the bladder, which were later confirmed to be tumors arising from the ventral prostate. Although at a lower frequency, grossly observable lesions were also identified in the dorsolateral prostate and seminal vesicle. Histological analysis confirmed the neoplastic nature of grossly observable lesions and allowed the identification of a variety of microscopic lesions in all three lobes of the prostate. The adenocarcinomas observed in both the 7–9 and 10–12 month groups were moderately to well differentiated, forming in some cases large neoplastic masses with ill-defined glandular structures. Tumors with neuroendocrine differentiation or poorly differentiated histology were not observed. As shown in Table 1 and Figure 1, adenocarcinomas were observed in the anterior prostates of 100% of the Pten+/− male mice by 12 months of age, whereas no wild-type or Tsc2+/− mice developed these lesions. While complete penetrance for adenocarcinoma was observed in the anterior prostate of Pten+/− mice, adenocarcinomas also arose in the ventral and dorsolateral prostates of 33% and 67% of heterozygous mice, respectively, and in seminal vesicles of 14% of Pten+/− and 33% of Pten+/−;Tsc2+/− mice (Figure 1, A–F). Seminal vesicles with invasive adenocarcinoma and intraepithelial neoplasia exhibited similar features and cytological characteristics as lesions arising in other lobes of the prostate (Figure 1, G–J). Double heterozygous Pten+/−;Tsc2+/− animals failed to exhibit any increased incidence of adenocarcinoma relative to Pten+/− mice. Thus, in the anterior prostate the Pten null allele was fully penetrant for development of prostate adenocarcinoma, and the presence of a Tsc2 null allele did not accelerate lesion development throughout the prostate.Table 1Adenocarcinoma Incidence in the ProstatewtPten+/−Pten Tsc2Tsc2+/−nNo. of lesionsnNo. of lesionsnNo. of lesionsnNo. of lesion7–9 months AP400%11764%9333%1000.0% VP500%10220%9333%1000.0% DLP200%10330%9333%1000.0% SV500%1100%9001000.0%10–12 months AP500%88100%9889%1400.0% VP600%6233%9220%1400.0% DLP600%6467%9333%1400.0% SV600%7114%9333%1400.0%In two age groups, 7–9 months and 10–12 months, the number of lesions was counted in four regions of the male reproductive system. AP, anterior prostate; VP, ventral prostate; DLP, dorsolateral prostate; SV, seminal vesicle. n = number of prostates examined that contained the region of interest. Open table in a new tab In two age groups, 7–9 months and 10–12 months, the number of lesions was counted in four regions of the male reproductive system. AP, anterior prostate; VP, ventral prostate; DLP, dorsolateral prostate; SV, seminal vesicle. n = number of prostates examined that contained the region of interest. Precursor lesions for prostate adenocarcinoma were also evaluated microscopically. As shown in Table 2 the anterior prostates of wild-type, Pten+/− and Pten+/−;Tsc2+/−, and Tsc2+/− mice presented with hyperplasia, characterized by increased number of acini and increased epithelial tufting (Figure 1). In addition, a significant number of animals carrying the Pten-null allele showed atypical glandular structures with epithelial stratification and a cribriform pattern, essentially identical to the PIN lesions described previously in Pten+/− mice and other murine models of prostate cancer. These lesions were then further classified as low grade or high-grade PIN (Figure 1, D–F) according to the severity as described by Shapell et al (Bar Harbor Consensus Report).41Shappell SB Thomas GV Roberts RL Herbert R Ittmann MM Rubin MA Humphrey PA Sundberg JP Rozengurt N Barrios R Ward JM Cardiff
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