Upregulation of pakt(ser473) expression in progression of HPV-positive oropharyngeal squamous cell carcinoma

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1 Received: 4 December 2016 Revised: 6 June 2017 Accepted: 6 July 2017 DOI: /hed ORIGINAL ARTICLE Upregulation of pakt(ser473) expression in progression of HPV-positive oropharyngeal squamous cell carcinoma Dominik Horn, MD, DMD 1 Christian Freudlsperger, MD, DMD 1 Dana Holzinger, PhD 2 Kevin Kunzmann, MSc 5 Peter Plinkert, MD 3 Gerhard Dyckhoff, MD 3 J urgen Hoffmann, MD, DMD 1 Kolja Freier, MD, DMD 1 Jochen Hess, PhD 3,4 1 Department of Oral and Cranio- Maxillofacial Surgery, Heidelberg University Hospital, Heidelberg, Germany 2 Molecular Diagnostics of Oncogenic Infections, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Department of Otorhinolaryngology, Head and Neck Surgery, Heidelberg University Hospital, Heidelberg, Germany 4 Research Group Molecular Mechanisms of Head and Neck Tumors, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Institute for Medical Biometry and Informatics, Heidelberg University Hospital, Heidelberg, Germany Correspondence Dominik Horn, Department of Oral and Cranio-Maxillofacial Surgery, Heidelberg University Hospital, Im Neuenheimer Feld 400, Heidelberg, Germany. Dominik.horn@med.uni-heidelberg. de Abstract Background: PIK3CA alterations have been shown to be a frequent event in oropharyngeal squamous cell cancer (SCC), especially in human papillomavirus (HPV)- related tumors. Methods: Tissue microarrays (TMAs) were used to evaluate pakt(ser473)/ (Thr308), total protein kinase B (AKT)(pan) and phosphatase and tensin homolog (PTEN) expression in primary tumors and corresponding nodal disease in oropharyngeal SCC. The HPV status was determined in regard of HPV16 DNA and RNA. Survival analysis was performed by using Kaplan-Meier curves, log-rank testing, and multivariate Cox regression analysis. Results: HPV16 is a prognostic predictive marker for advanced oropharyngeal SCC. pakt(ser473) and PTEN are highly expressed in HPV-related oropharyngeal SCCs in contrast to pakt(thr308). The pakt(ser473) expression increased from primary tumors to progressive nodal disease (21.1%; P <.011). Conclusion: Activation of phosphoinositide 3-kinase (PI3K)/pAKT(Ser473) frequently occurs in advanced HPV-positive oropharyngeal SCC and elevated pakt (Ser473) levels represent a feature during progression of oropharyngeal SCC, indicating a critical role of the mammalian target of rapamycin (mtor) complex. Further studies are required to evaluate specific drugs targeting PI3K/AKT/mTOR in consideration of PIK3CA alterations. KEYWORDS human papillomavirus, lymph node metastasis, oropharyngeal squamous cell carcinoma, protein kinase B (AKT), tissue microarray 1 INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer by incidence. It affects around patients worldwide. 1,2 Prognosis of advanced oropharyngeal squamous cell carcinoma (SCC) remains to be unfavorable with a 5-year survival <50%. 3 Locoregional recurrence in up to 60% of patients and development of resistance against established treatment modalities are a major challenge. 4 In general, oropharyngeal SCC is characterized by clinical, etiological, and molecular heterogeneity. Tobacco smoking and regular alcohol consumption are considered to be major risk factors for cancer development, particularly if Head & Neck. 2017;39: wileyonlinelibrary.com/journal/hed VC 2017 Wiley Periodicals, Inc. 2397

2 2398 HORN ET AL. both habits are combined. 5 7 Despite a growing number of prevention programs to reduce tobacco abuse, the number of patients with oropharyngeal SCC has increased in the past decades This subset is clinically characterized by younger age and less smoking habits in contrast with other patients with HNSCCs. Viral infection, in terms of human papillomavirus (HPV), was introduced as a further risk factor for oropharyngeal SCC explaining this trend in cancer etiology. 11 Since then, HPV infection has become a main risk factor for oropharyngeal SCCs and defines a new clinical and molecular subgroup. 12 One known mechanism of HPVrelated carcinogenesis is the inactivation of the p53 and Rb as tumor suppressors due to upregulation of viral oncoproteins E6 and E7. 13,14 Recent genomic studies confirmed the different mutational landscape of HNSCC. 15,16 The Cancer Genome Atlas recently presented the first results of a multiplatform analysis including 279 tumors. Within this analysis, HPV-positive oropharyngeal SCC frequently showed loss of tumor necrosis factor receptor-associated factor 3, amplification of E2F1, and activating mutations of PIK3CA. 17 The PIK3CA alterations were detected in 56% of HPV-positive and in 34% of HPV-negative tumors. The PIK3CA encodes the p110a catalytic subunit of phosphoinositide 3-kinase (PI3K) with corresponding downstream signaling of protein kinase B (AKT) and nuclear factor-kappa B (Supporting Information Figure S1). As a negative regulator of PI3K, phosphatase and tensin homolog (PTEN) shows genetic alterations in 12% of HPV-negative but only 6% in HPVpositive HNSCC. The purpose of this study was to analyze the protein expression patterns of the PI3K signaling in a large oropharyngeal SCC cohort, depending on the HPV16 status and the correlation with clinical features. Furthermore, we investigated changes in the expression of PI3Kdependent proteins during progression of oropharyngeal SCC based on paired histopathological samples of matched primary tumors and lymph node metastases. 2 MATERIALS AND METHODS 2.1 Patients The retrospective study cohort comprised patients with primary oropharyngeal SCC who were diagnosed and treated between 1990 and Tissue was harvested at the Department for Head and Neck Surgery of the University of Heidelberg during diagnostic or therapeutic procedures. Informed consent was given and the study permission was granted by the Ethics Committee of the Medical Faculty of the University of Heidelberg (Ethic votes: 176/2002 and 206/2005). Clinical and therapeutic follow-up was assessed retrospectively, and final analysis was based on patients with advanced oropharyngeal SCC (clinical stage III and IV; n 5 116) and available corresponding lymph node metastases (n 5 22). 2.2 HPV genotyping and HPV16 RNA analysis The molecular HPV status of the tumors was made available due to the assessed data from Holzinger et al. 22 Genomic DNA was isolated from fresh frozen tissue samples using Qiagen s QIAamp DNA Mini Kit (Venlo, The Netherlands), and RNA was isolated by using Qiagen s RNeasy Mini Kit (Venlo, The Netherlands), in accord with the manufacturer s instructions. To determine HPV DNA status, BSGP51/61- PCR/Multiplex HPV genotyping was used, including the amplification of 54 mucosal HPV types and the b-globin gene as a control for the quality and quantity of genomic DNA To determine HPV RNA status, HPV16 E6*I mrna transcripts were determined by a nucleic acid sequence-based amplification assay, as described recently. 22,26 A tumor was defined HPV-related in the presence of HPV16 DNA and HPV16 E6*I mrna. HPV DNA-positive but HPV RNA-negative tumors and HPV DNA-negative tumors were defined as non-hpv-related. 2.3 Tissue microarray Tissue microarrays (TMAs) were prepared as described previously. 27 Hematoxylin-eosin stained sections, which were representative for the corresponding paraffin-embedded tissue block, were analyzed by an experienced pathologist. Tumor areas on the slides were marked and tissue cores of the tumor areas were punched from the paraffin blocks using the tissue chip microarrayer (Beecher Instruments, Sun Prairie, WI). Tissue punches measuring 0.6 mm in diameter were implanted into a recipient block. After completing the paraffin-embedded TMA blocks, 2-3-lm thick sections were cut and applied on slides (Histo Bond, Marienfeld, Germany). 2.4 Immunohistochemistry The TMAs were stained with anti-pakt(ser473; Cell Signaling, Cambridge, UK), anti-pakt(thr308) (Cell Signaling), anti-akt(pan) (Cell Signaling), and anti-pten (Abcam, Cambridge, UK) monoclonal antibodies. Incubation was performed according to the manufacturer s instruction. Diaminobenzidine substrate incubation visualized the localization and intensity of antibodies. The TSA System (Perkin Elmer, Waltham, MA) was used for signal amplification for pakt(ser473), pakt(thr308), and AKT(pan), as described before. 28

3 HORN ET AL TABLE 1 cohort Descriptive analysis of clinical features of the patient Clinical feature No. of patients (%) Sex Female 29 (25) Male 87 (75) Tobacco use Nonsmoker 15 (18.9) Smoker/ex-smoker 101 (87.1) Alcohol use No/occasional 26 (22.5) Yes/regular 90 (77.5) Tumor size (T) T1 7 (6) T2 38 (32.8) T3 31 (26.7) T4 40 (34.5) Lymph node (N) 0 15 (12.9) 1 23 (19.8) 2 71 (61.2) 3 7 (6) Stage III 27 (23.3) IV 89 (76.7) Localization Tonsils 65 (56) Tongue base 24 (20.7) Soft palate 16 (13.8) Extending several anatomic regions 11 (9.5) HPV16 DNA - /RNA - 52 (46.4) DNA 1 /RNA - 32 (28.6) DNA 1 /RNA 1 28 (25) Missing 4 Abbreviation: HPV, human papillomavirus. Staining intensity and number of stained cells of healthy mucosal specimens were regarded as baseline expression. Values for intensity and percentage total of stained tumor cells were assessed by 3 independent observers and subsequently grouped into an ordinal scale (1 5 no staining; 2 5 low; 3 5 medium; and 4 5 high intensity; 1 5 no stained cells, 0%; >2 5 <33%; 33% >3 5 <66%; and 4 5 >66% stained cells). Median scores between all observers scores were calculated. Values for staining intensity and number of stained cells were multiplied. Expression scale for each protein ranged from 1 to 16 and they were grouped into high and low staining patterns. 2.5 Statistics SPSS 21 (SPSS for Windows, SPSS, Chicago, IL) was used for statistical analysis. Clinical features were analyzed descriptively to outline the cohort. Correlation analysis was performed using cross tables and chi-square test. Overall and progression-free survival data were plotted by Kaplan-Meier survival curves. Differences between groups were assumed using log-rank testing. Multivariate Cox proportional hazard models were applied and adjusted to the molecular HPV status to validate survival data by including relevant clinical covariates. The difference between HPV-related and non- HPV-related tumors regarding the increase of protein expression from primaries to lymph node metastases was analyzed by using independent t tests. Means of HPV-related (n 5 8) und non-hpv-related (n 5 14) primaries with available lymph node metastasis were compared. 3 RESULTS 3.1 Patient cohort is valid in consideration of clinical and molecular features We analyzed our patient cohort descriptively considering clinical features and expression levels of key nodes within the PI3K/AKT/PTEN signaling cascade. We identified 116 patients diagnosed with advanced oropharyngeal SCC. The majority of the patients were men (75%), alcohol consumers (77.5%), and smokers (87.1%; Table 1). The anatomic distribution varied from the tonsils (56%), base of tongue (20.7%), and soft palate (13.8%), to tumors extending over several anatomic regions (9.5%). Molecular analysis showed HPV16-related tumors in 25%, whereas non-hpv-related was assessed in 75%. HPV-related tumors occurred exclusively in the tonsils (56%) or base of tongue (20.7%), whereas non-hpv-related tumors derived from all oropharyngeal subsites (Table 1). The descriptive analysis of protein expression is summarized in Supporting Information Table S High pakt(ser473), high phosphatase and tensin homolog expressions, absence of tobacco abuse, and reduced tumor size correlate with HPV16 status A further purpose of this study was to analyze expression patterns of key regulators of the PI3K/AKT/PTEN signaling cascade in the context of the HPV16 status. We performed an exploratory correlation analysis that showed a significant dependence between HPV-related tumors and high PTEN expression (P <.01) as well as high pakt (Ser473) expression (P <.02; Table 2). In contrast, pakt (Thr308) and AKT(pan) did not show a correlation with

4 2400 HORN ET AL. TABLE 2 Correlation of clinical and molecular variables with HPV16 status Characteristics Non-HPV16-related (%) HPV16-related (%) P value Sex Female 17 (15.2) 11 (9.8) Male 67 (59.8) 17 (15.2).08 Tobacco use Nonsmoker 2 (1.8) 11 (9.8) Smoker/ex-smoker 82 (73.2) 17 (15.2).01 a Alcohol use No/occasional 20 (17.8) 4 (3.6) Yes/regular 63 (56.2) 24 (21.4).47 T classification 1/2 26 (23.2) 17 (15.2) 3/4 58 (51.8) 11 (9.8).01 a N classification Negative 13 (11.6) 2 (1.8) Positive 71 (63.4) 26 (23.2).26 Localization Tonsils 41 (36.6) 22 (19.6) Tongue base 16 (14.3) 6 (5.4) Soft palate 16 (14.3) 0 Extending several anatomic regions 11 (9.8) 0.02 a pakt(ser473) Low 23 (25.3) 1 (1.1) High 48 (52.7) 19 (17.9).02 a pakt(thr308) Low 33 (36.3) 14 (15.4) High 36 (39.6) 8 (8.7).23 AKT(pan) Low 33 (37.5) 7 (8.0) High 36 (40.9) 12 (13.6).45 PTEN Low 50 (51.5) 8 (8.3) High 23 (23.7) 16 (16.5).01 a Abbreviations: AKT, protein kinase B; HPV, human papillomavirus; PTEN, phosphatase and tensin homolog. a Indicates significant results of independent variables. the HPV status (P <.23 and P <.45). In order to validate the exploratory correlation analysis, we included relevant clinical parameters. We found significant positive correlation to the HPV16 status and known predisposed anatomic sites (tonsil and base of tongue; P <.02) in contrast to all other sites. In addition, HPV16-related oropharyngeal SCCs were significantly correlated with absence of tobacco abuse (P <.01) and reduced tumor size (P <.04) as compared to the non-hpv-related counterparts. There was no correlation between HPV status and sex (P <.08), presence of lymph node metastasis (P <.26), or alcohol consumption (P <.47). 3.3 pakt(ser473) and molecular HPV16 status are independent prognostic markers for survival in advanced oropharyngeal squamous cell carcinomas We performed survival analysis for HPV16 status and key regulators of the PI3K/AKT/PTEN pathway by using

5 HORN ET AL TABLE 3 Univariate and multivariate analysis of 10-year survival and progression-free survival in consideration of relevant clinical variables and protein expression 10-year survival Progression-free survival Univariate Multivariate Univariate Multivariate Variables HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value HPV16 (DNA 1 /RNA 1 ) 0.32 ( ).01 a 0.38 ( ) ( ).01 a 0.3 ( ).02 a pakt (Ser473) 1.8 ( ) ( ) ( ) ( ).02 a PTEN 0.56 ( ) ( ) ( ).01 a 0.5 ( ).06 Tumor size (T) 2.32 ( ).01 a 2.61 ( ).04 a 1.84 ( ).03 a 1.11 ( ).79 Lymph node (N) 1.11 ( ) ( ).04 a 1.04 ( ) ( ).23 Smoking 0.44 ( ) ( ) ( ) ( ).62 pakt(thr308) 0.75 ( ) ( ).93 AKT(pan) 0.65 ( ) ( ).56 Sex 0.77 ( ) ( ).63 Grading 0.63 ( ) ( ).63 Abbreviations: AKT, protein kinase B; CI, confidence interval; HPV, human papillomavirus; HR, hazard ratio; PTEN, phosphatase and tensin homolog. a Indicates significant results of independent variables. Kaplan-Meier curves and log-rank testing. Patients diagnosed with HPV-related tumors showed a better 10-year survival and progression-free survival compared with patients with non-hpv-related (OS 5 P <.005; PFS 5 P <.001; Supporting Information Figure S2). High PTEN expression was associated with significant improved progression-free survival (P <.004) and a clear tendency for an improved 10-year survival (P <.056; Supporting Information Figure S3). In contrast, high pakt(ser473) and low AKT(pan) revealed only a trend toward worse 10-year survival and progression-free survival (P <.1 and P <.14; Supporting Information Figure S3). Stratification of patients according to pakt (Thr308) expression patterns showed no significance (P <.34 and P <.93). Univariate survival analysis of HPV status and pakt (Ser473), pakt(thr308), AKT(pan), and PTEN expression levels were performed considering relevant clinical covariates. For 10-year survival, we found a significant survival benefit in terms of HPV-related tumors (P <.01) and smaller tumor size (T1/2; P <.01; Table 3). In addition, patients with HPV-related (P <.01), high PTEN (P <.01) or smaller tumor size (P <.03) showed a significantly better progression-free survival (Table 3). Next, we performed multivariate analysis, including expression patterns for pakt(ser473) and PTEN, and relevant clinical parameters (tumor size, lymph node metastasis, and smoking). For 10-year survival, we found the clinical T and N classifications to be significant prognostic parameters (both P <.04), whereas HPV status showed a clear trend (P 5.05) for a better survival. A high pakt(ser473) staining patterns revealed a trend (P <.11) for worse 10-year survival (Table 3). In terms of multivariate disease progression, HPV-related tumors showed significantly better progression-free survival (P <.01), whereas a high pakt(ser473) staining patterns was significantly associated with progression (P <.02). No statistically significant correlation was observed between the PTEN staining pattern and progression-free survival (P <.66). 3.4 Elevated pakt(ser473) expression in lymph node metastasis of HPV-related oropharyngeal squamous cell carcinoma In order to investigate the potential impact of pakt(473) and PTEN on tumor progression, we analyzed protein levels in specimens of primary tumors and their corresponding lymph node metastasis and grouped the samples based on the HPV16 status of the primary tumor. We found a significant increase in pakt(ser473) expression in lymph node metastasis as compared with matched primary tumors for the HPVrelated subgroup (121.1%; P <.011), whereas all other analyzed proteins did not show any significant difference (P >.09; Figure 1, Table 4). In contrast, no significant difference was observed in the subgroup of non-hpv-related tumors. 4 DISCUSSION Recent data from extensive genomic investigations of HNSCC provided new insights on mutations in the molecular

6 2402 HORN ET AL. FIGURE 1 Expression pattern of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) (upper row) in contrary to HPV-negative (lower row) oropharyngeal SCC regarding to pakt(ser473) during progression. The left images (A and C) are showing primary tumors. The right images (B and D) are showing progressive disease, whereas HPV-positive oropharyngeal SCCs show increased pakt(ser473) expression [Color figure can be viewed at wileyonlinelibrary.com] landscape of HPV-positive and HPV-negative cancers. 17,29 Among others, alterations of PIK3CA proved to be one of the most frequent events, especially in HPV-related HNSCC (22%-56%). 17,29,30 Activating mutations of PIK3CA can promote a malignant phenotype by affecting central hubs in cell cycle progression, metabolic transactions, and gene regulatory TABLE 4 T test to evaluate differences of the protein levels during progression depending on HPV16-related and non-hpv16-related samples Protein HPV16-related Mean expression Mean difference 95% CI P value Non-HPV16-related Mean expression Mean difference 95% CI P value pakt(ser473) 7.71 (p) (m) 3.38 (21.1%) a 9.58 (p) 9.18 (m) -0.4 (-2.5%) to PTEN 4.5 (p) 5.86 (m) 2.05 (12.8%) to (p) 9.54 (m) 2 (12.5%) to pakt(thr308) 7.71 (p) 8.17 (m) 0.14 (0.9%) to (p) 6.67 (m) -1.5 (-9.4%) to AKT(pan) 8.14 (p) 9.29 (m) 1.38 (8.6%) -1.7 to (p) 9.73 (m) 0.06 (0.06%) to Abbreviations: AKT, protein kinase B; CI, confidence interval; HPV, human papillomavirus; m, metastasis; p, primary; PTEN, phosphatase and tensin homolog. a Indicates significant results.

7 HORN ET AL networks. 17,31,32 PIK3CA encodes the p110a catalytic subunit of the PI3K and triggers activation of several downstream targets, including AKT and nuclear factor-kappa B Using PTEN as a negative regulator of PI3K shows alterations in 12% of HPV-negative but only in 6% of HPVpositive HNSCC. 17 The HPV-positive HNSCC that evokes mainly in anatomic localizations, such as the tonsils and base of tongue, is currently accepted as its own cancer entity with regard to etiology, molecular and cellular traits, and prognosis. 11,33 36 The scientific focus shifted to HPV-positive oropharyngeal SCC due to its increasing incidence over the past decades as well as the burden for patients and healthcare systems worldwide. 37 Oropharyngeal SCC can be treated curative at early stages, whereas diagnosis at later stages or recurrence remains the major challenge, in particular when it comes to the molecular modifications in cancer cell subclones and changes in the tumor microenvironment. Therefore, we focused on a patient cohort with advanced oropharyngeal SCC (stages III and IV) to investigate expression patterns of relevant proteins of the PI3K/AKT/PTEN pathway and their changes during progression. Our study cohort complied with published data of other cohorts and showed similar clinical features considering HPV status, history of smoking, tumor size, and nodal disease. Survival analysis showed a higher 10-year survival and progression-free survival for patients with HPV-positive cancer. In accordance with the current data situation, we found HPV status 38 as well as high expression levels of pakt (Ser473) 39,40 to be independent prognostic factors for survival in a multivariate setting; in contrary to pakt(thr308). Considering the frequency of PIK3CA mutations in HPV-related cancers, 17 we identified a positive correlation between the presence of an HPV-related tumor and high pakt(ser473) levels but not for high pakt(thr308) levels. These data indicate an indirect activation of AKT at Ser473 via the mammalian target of rapamycin (mtor) complex, which is supported by Molinolo et al 41 providing similar results in TMA expression analysis for HPV-positive cancer. However, they did not investigate pakt(thr308) staining patterns in their study. The frequent PIK3CA alterations in HPV-positive HNSCC but low activation of pakt(thr308) that we and others found 42 is unexpected as activating mutations would imply phosphorylation at Thr308. The high PTEN expression in our cohort might be one explanation for this observation and might indicate a negative feedback loop. The PI3K activation via PTEN loss is a widely accepted mechanism in several malignancies. Nevertheless, in HPV-positive HNSCC, PTEN loss is rare. 17 Further preclinical studies applying appropriate invivoandexvivomodelsarerequiredtounraveltheimpact of PIK3CA alterations in HPV-positive HNSCC. Another indication for the importance of the mtor signaling in HPV-positive oropharyngeal SCC was the detection of elevated pakt(ser473) levels in lymph node metastasis as compared with matched primary tumors. To the best of our knowledge, this is the first study showing an increase in pakt(ser473) levels during progression to a nodal disease in advanced HPV-positive oropharyngeal SCC. As a clinical consequence, mtor inhibitors might be more suitable in HPVpositive advanced oropharyngeal SCC in contrast to PI3K/ AKT inhibitors. It is hoped that ongoing clinical trials will provide further information (NCT ; NCT ). Nevertheless, long-term side effects of current therapies are increasingly included in oncologic discussions. 43 In regard to HPV-related oropharyngeal SCC, patients are younger and have a higher lifetime risk for radiation and medicationassociated complications. Among others, we could show that targeting the PI3K and mtor axis can lead to enhanced radiosensitivity. 39,44 46 Therefore, a potential future application of mtor or combined AKT/mTOR inhibitors could reduce the radiation dose in HPV-related oropharyngeal SCC and prevent secondary therapy-related complications in the long term. Furthermore, specific targeting of AKT/mTOR signaling could be useful as adjuvant treatment modality after surgical resection of positive lymph nodes with a high pakt(ser473) staining pattern in order to reduce the risk for locoregional recurrence. According to a recent clinical trial, individualized treatment strategies in dependency to the mutational landscape are required due to the inhomogeneity of HNSCC. 47 In this regard, further ex vivo and more sophisticated cell culture models derived from HPV-related cancers are urgently needed to individualize treatment strategies that depend on the mutational landscape and adjust radiation, chemotherapy, and antibody doses in this patient group. 5 CONCLUSION Increasing incidence of HPV-positive oropharyngeal SCC during the last centuries, particularly in younger patients, is a major challenge for healthcare systems in the Western world. Recent studies showed HPV-related HNSCC to be a distinct clinical and molecular entity. Elevated levels of pakt (Ser473) and PTEN is a frequent event in advanced HPVpositive oropharyngeal SCC in contrast to pakt(thr308). A further increase of pakt(ser473) in lymph node metastasis indicates a critical role of the mtor complex in this cancer entity. Further preclinical studies applying ex vivo cultures and in vivo models for HPV-related tumors in consideration of PIK3CA alterations are required to individualize therapy regimen. ORCID Dominik Horn MD, DMD

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