European Archives of Oto-Rhino-Laryngology (2022) 279:3679–3684 https://doi.org/10.1007/s00405-021-07223-4 HEAD AND NECK Relationship between the transcriptional expression of PIM1 and local control in patients with head and neck squamous cell carcinomas treated with radiotherapy Xavier León1,2,3 · Jacinto García4 · Albert Pujol1 · Julia de Juan1 · Rosselin Vásquez1 · Miquel Quer1,3 · Mercedes Camacho5 Received: 5 September 2021 / Accepted: 13 December 2021 / Published online: 6 January 2022 © Crown 2022 Abstract Purpose Proviral integration site for Moloney murine leukemia virus (PIMs) are proto-oncogenes encoding serine/threo- nine kinases that phosphorylate a variety of substrates involved in the regulation of cellular processes. Elevated expression of PIM-1 has been associated with poor prognosis in several types of cancer. There are no studies that have analyzed the response to radiotherapy in patients with head and neck squamous cell carcinoma (HNSCC) according to the expression of PIM-1. The aim of our study was to analyze the relationship between the transcriptional expression of PIM-1 and local response to radiotherapy in HNSCC patients. Methods We determined the transcriptional expression of PIM-1 in 135 HNSCC patients treated with radiotherapy, includ- ing patients treated with chemoradiotherapy (n = 65) and bioradiotherapy (n = 15). Results During the follow-up, 48 patients (35.6%) had a local recurrence of the tumor. Patients with local recurrence had a higher level of PIM-1 expression than those who achieved local control of the disease (P = 0.017). Five-year local recurrence- free survival for patients with a high expression of PIM-1 (n = 43) was 44.6% (95% CI 29.2–60.0%), and for patients with low expression (n = 92) it was 71.9% (95% CI 62.5–81.3%) (P = 0.007). According to the results of multivariate analysis, patients with a high PIM-1 expression had a 2.2-fold increased risk of local recurrence (95% CI 1.22–4.10, P = 0.009). Conclusion Patients with elevated transcriptional expression levels of PIM-1 had a significantly higher risk of local recur- rence after radiotherapy. Keyword PIM-1 · Head and neck carcinoma · Radiotherapy · Local control · Biomarker Introduction Radiotherapy is one of the therapeutic options for patients * Xavier León with head and neck squamous cell carcinoma (HNSCC). xleon@santpau.cat After treatment with radiotherapy, a variable percentage 1 of patients, depending on the location and extension of the Otorhinolaryngology Department, Hospital de La Santa tumor, have a local recurrence. The availability of biomark- Creu I Sant Pau, Universitat Autònoma de Barcelona, C/Mas Casanovas, 90, 08041 Barcelona, Spain ers with the capacity to discriminate tumor radiosensitivity 2 would make it possible to offer radiotherapy to those patients UVIC. Universitat Central de Catalunya, Barcelona, Vic, Spain with tumors with a greater probability of response, reducing 3 the percentage of local recurrence and the need for salvage Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales Y Nanomedicina (CIBER-BBN), Madrid, surgery. Spain Proviral integration site for Moloney murine leukemia 4 Otorhinolaryngology Department, Hospital del Mar, virus (PIMs) are proto-oncogenes encoding serine/threonine Universitat Autònoma de Barcelona, Barcelona, Spain kinases that phosphorylate a variety of substrates involved 5 Genomics of Complex Diseases, Research Institute Hospital in the regulation of cellular processes such as proliferation, Sant Pau, IIB Sant Pau, Barcelona, Spain survival, apoptosis, migration, and cell cycle regulation. The 13 Vol.:(0123456789) 3680 European Archives of Oto-Rhino-Laryngology (2022) 279:3679–3684 PIM family consists of three members, PIM-1, PIM-2, and Table 1  Characteristics of the patients included in the study PIM-3 [1]. N (%) Elevated expression of PIM-1 has been associated with poor prognosis in patients with leukemia [2] or lymphoma Mean age (standard deviation) years 63.6 (11.7) [3], as well as in patients with squamous cell carcinomas Gender located in the esophagus [4] or the lung [5], or adenocarci- Men 120 (88.9) nomas located in the breast [6], lung [5], pancreas [7], colon Women 15 (11.1) [8], stomach [9] or prostate [10]. Age Studies in experimental models of lung carcinoma [11] or <65 years 75 (55.6) pancreatic carcinoma [12] have shown that PIM-1 expres- ≥65 years 60 (44.4) sion is associated with a reduction in radiosensitivity. In a Toxic consumption study performed with HNSCC cell lines, Peltola et al. [13] None 14 (10.4) demonstrated that PIM-1 expression protects tumor cells Moderate 20 (14.8) from radiotherapy-induced damage. To our knowledge, there Severe 101 (74.8) are no studies that have analyzed the response to radiother- Location apy in patients with HNSCC according to the expression Oropharynx 63 (46.7) of PIM-1. Hypopharynx 16 (11.8) The aim of our study was to evaluate the relationship Larynx 56 (41.5) between PIM-1 transcriptional expression and sensitivity to Local extension radiotherapy in a cohort of patients with HNSCC. cT1–T2 74 (54.8) cT3–T4 61 (45.2) Regional extension cN0 81 (60.0) Materials and methods cN1 22 (16.3) cN2 30 (22.2) Patients cN3 2 (1.5) Histologic grade We carried out a retrospective study based on the analysis Well differentiated 13 (9.6) of biopsies obtained from the primary location of the tumor Moderately differentiated 109 (80.7) prior to any type of treatment in a cohort of 135 patients Poorly differentiated 13 (9.6) with a histologically confirmed squamous cell carcinoma Treatment located in the oropharynx, hypopharynx or larynx, and Radiotherapy 55 (40.7) treated with radiotherapy during the period 2008–2016. Chemoradiotherapy 65 (48.2) Patients who received concomitant treatment with chemo- Bioradiotherapy 15 (11.1) therapy (chemoradiotherapy) or cetuximab (bioradiotherapy) were also included in the study. Clinical data were obtained from a database that prospectively collects information on tumors was reclassified according to the 8th edition of the all patients with a malignant head and neck tumor treated at TNM. our center since 1985 [14]. Treatment with radiotherapy consisted of the adminis- All patients included in the study were evaluated by the tration of a dose of 70–72 Gy on the primary location of Oncologic Committee of our institution, which proposed the tumor, 50 Gy on the lymph node areas at risk in cN0 the treatment according to the center's clinical guidelines. patients, and 70–72 Gy on the affected lymph node areas Table 1 shows the characteristics of the patients included in cN + patients. Radiotherapy planning was done using 3D in the study. Given the interaction in tobacco and alcohol conformal fields until 2010, and IMRT (Instensity_Modu- consumption, a combined variable of toxics consumption lated Radiation Therapy) from 2011 onwards, with a lin- was created with three categories: no consumption; moder- ear accelerator as the radiation source. Most patients were ate consumption (< 20 cigarettes/day and/or < 80 g alco- treated with standard fractionation (2 Gy/fraction, 1 frac- hol/day); and severe consumption (≥ 20 cigarettes/day and/ tion/day, 5 days/week). Thirteen patients (9.6%) followed or ≥ 80 g alcohol/day). The HPV status of patients with oro- a hyperfractionation regimen (1.2 Gy/fraction, 2 fractions/ pharyngeal carcinoma was determined by viral DNA detec- day, 5 days/week). Sixty-five patients had a concomitant tion with SPF-10 RT-PCR, using the LiPA25_vl reverse treatment with chemoradiotherapy and 15 bioradiotherapy hybridization assay for genotyping. The loco-regional exten- with cetuximab. Patients treated with chemoradiotherapy sion category of patients with HPV-positive oropharyngeal received between two and three cycles of cisplatin at a dose 13 European Archives of Oto-Rhino-Laryngology (2022) 279:3679–3684 3681 of 100 mg/m2 every three weeks (n = 55), or carboplatin 1.5 the histological grade, as well as the expression category of AUC every week (n = 10). Those treated with bioradiother- PIM-1 as independent variables. apy received cetuximab 400 mg/m2 on day 1 of the week preceding radiotherapy, and a weekly dose of 250 mg/m 2 cetuximab during radiotherapy. Seventeen patients were Results treated with uni or bilateral neck dissections after complet- ing radiotherapy. There were no significant differences in the transcriptional All patients included in the study had a follow-up of expression of PIM-1 according to sex (P = 0.941), age more than 2 years. The mean follow-up period was 5.4 years (P = 0.060), toxics consumption (P = 0.301), location of (standard deviation 3.3 years). the primary tumor (P = 0.092), local (P = 0.326) or regional The study was approved by the Institutional Review Com- (P = 0.896) extension of the tumor, or histological grade mittee and was conducted following the principles estab- (P = 0.781). Information regarding HPV status was avail- lished in the Declaration of Helsinki. able for 47 of the patients with oropharyngeal carcinomas. The percentage of patients with HPV-positive oropharyngeal Transcriptional analysis carcinomas was 25.5%. No significant differences appeared in PIM-1 expression values as a function of HPV status The biopsy samples obtained from each patient were imme- (P = 0.137). Table 1 of the supplementary material shows diately included in RNA-later (Quiagen GmbH, Hilden, Ger- the median value of the transcriptional expression of PIM-1 many) to prevent RNA degradation and stored at −80 °C according to the variables analyzed. until processing. Total RNA was extracted using Trizol During the follow-up period, 48 patients (35.6%) had (Invitrogen, Carlsbad, USA) according to the manufacturer's a local recurrence of the tumor, 16 patients (11.9%) had instructions. The cDNA was obtained by reverse transcrip- a regional recurrence, and 15 patients (11.1%) had distant tion of 1 µg of RNA with the High-Capacity cDNA Archive metastases. Patients with a local tumor recurrence had sig- Kit (Applied Biosystems, Foster City, USA), and the tran- nificantly higher PIM-1 transcriptional expression values scriptional expression of PIM-1 and beta-actin as an endog- than patients in which treatment with radiotherapy achieved enous control was evaluated by RT-PCR in an ABI Prism local control of the disease (P = 0.017). Figure 1 in the sup- 7000 using validated pre-designed assays (TaqMan Gene plementary material shows the distribution of the PIM-1 Expression Assays; Applied Biosystems). transcriptional expression values according to the local con- trol of the tumor after treatment with radiotherapy. Statistical analysis The recursive partitioning analysis classified the patients into two groups according to the local control We compared the expression levels of PIM-1 according to of the tumor after treatment with radiotherapy. Patients gender, age, toxics consumption, location of the primary with a high PIM-1 expression value (n = 43, 31.9%) had tumor, local and regional extension of the tumor, histological a significantly higher rate of local tumor recurrence than grade, and the local control of the tumor after treatment with radiotherapy. The distribution of the PIM-1 expression val- ues did not meet the normality criteria, hence the non-par- ametric techniques of Mann–Whitney and Kruskal–Wallis were used in the comparison of the PIM-1 expression levels. For patients with oropharyngeal tumors, PIM-1 expression was analyzed in relation to the HPV status. The continuous value of PIM-1 expression was categorized according to the local disease control after treatment with radiotherapy with a recursive partitioning analysis, using the CRT (Classifica- tion and Regression-Tree) model. We calculated the local recurrence-free survival according to the categories defined by the recursive partitioning analysis with the Kaplan–Meier method, using the log-rank test in the comparison of the survival curves. A multivariate analysis was carried out with the Cox proportional hazards model, considering the local recurrence-free survival as the dependent variable, and the location of the primary tumor, the local (cT1-2 versus cT3- Fig. 1  Local recurrence-free survival according to the PIM-1 expres- 4) and regional (cN0 versus cN +) extension of the tumor, sion category 13 3682 European Archives of Oto-Rhino-Laryngology (2022) 279:3679–3684 patients with low expression (n = 92, 68.1%). Five-year Discussion local recurrence-free survival for patients with a high PIM-1 expression was 44.6% (95% CI 29.2–60.0%), and According to our results, the transcriptional expression of for patients with low expression it was 71.9% (95% CI PIM-1 was significantly related to the local control of the 62.5–81.3%). Figure 1 shows local recurrence-free sur- tumor in patients with HNSCC treated with radiotherapy, vival according to the PIM-1 expression categories. There chemoradiotherapy, or bioradiotherapy. Those patients were significant differences in the local recurrence-free with elevated expression levels of PIM-1 had a 2.24-fold survival after treatment with radiotherapy as a function of increased risk of local recurrence. the PIM-1 expression category (P = 0.007). Immunohistochemical studies carried out in patients There were no significant differences in specific-dis- with HNSCC have found a significant increase in the ease survival according to the PIM-1 expression cate- expression of PIM-1 in tumor tissue, opposite to healthy gory. Five-year specific-disease survival for patients with mucosa [13, 15, 16]. In agreement with the results a high PIM-1 expression category was 61.1% (95% CI obtained by other authors that have analyzed the immu- 44.3–77.9%), and for patients with a low expression it was nohistochemical expression of PIM-1 in HNSCC tumors 71.5% (95 CI 62.1–80.9%) (P = 0.810). There were also [13, 15, 16], we did not find a relationship between the no significant differences in regional (P = 0.50) or distant transcriptional expression of PIM-1 and clinicopathologi- disease (P = 0.275) recurrence-free survival as a function cal variables such as tumor extension or histological grade. of the PIM-1 expression. Regarding the prognostic capacity of PIM-1 in patients Table 2 shows the results of multivariate analysis in with HNSCC, the studies performed so far have mostly which local recurrence-free survival after radiotherapy included patients treated surgically. In a study of 36 was considered as the dependent variable. The variables patients with oral cavity carcinomas treated with surgery, that were associated with an increased risk of local recur- Chiang et al. [15] found that patients with elevated PIM-1 rence were the location of the tumor in the hypopharynx, expression at transcriptional or immunohistochemical an advanced local extension (cT3–T4), and an elevated level had worse survival, but the differences did not reach PMI-1 transcriptional expression category. Considering statistical significance. Similarly, in a study by Peltola those patients with a low expression of PIM-1 as the ref- et al. [13] involving 71 patients with HNSCC treated with erence category, patients with high expression had a 2.2- surgery and/or radiotherapy, those patients with elevated fold increased risk of local recurrence (95% CI 1.22–4.10, immunohistochemical expression of PIM-1 tended to have P = 0.009). poorer survival (P = 0.09). Finally, in 39 patients with car- cinoma of the tongue treated with surgery, Tanaka et al. [17] found that immunopositivity to PIM-1 correlated with regional and distant recurrences along with a decrease in Table 2  Results of a multivariate analysis considering the local con- survival. trol after treatment with radiotherapy as the dependent variable In vitro studies performed with HNSCC cell lines have HR CI 95% HR P associated PIM-1 with increased tumor aggressiveness. PIM-1 expression has been associated with increased cell Location proliferation [18, 19], increased migration and invasion Oropharynx 1 capacity [17, 20], and increased cell viability and resist- Hypopharynx 2.98 1.14–7.80 0.026 ance to apoptosis [18, 19]. In an in vivo experimental Larynx 1.58 0.64–3.91 0.315 study with a murine model, it was shown that inhibition of Local extension PIM-1 led to a significant reduction in tumor growth [18]. cT1-2 1 In nasopharyngeal carcinoma cell lines, PIM-1 inhibition cT3-4 2.92 1.39–6.10 0.004 decreased cell proliferation and migration capacity [21]. Regional extension In tumor models of lung carcinoma [11] or pancre- cN0 1 atic carcinoma [12], PIM-1 expression has been associ- cN + 1.98 0.85–4.61 0.110 ated with a decrease in radiosensitivity. Peltola et al. [13] Treatment observed that in patients with HNSCC treated with pre- Radiotherapy 1 operative radiotherapy, a high expression of PIM-1 corre- Chemoradiotherapy 0.95 0.33–2.74 0.930 lated with a reduced response to radiotherapy. In HNSCC Bioradiotherapy 1.44 0.52–4.00 0.479 cell lines, these authors demonstrated that inhibition of PIM-1 PIM-1 expression with siRNA induced radiosensitization Low 1 of tumor cells, therefore supporting the concept that PIM-1 High 2.24 1.22–4.10 0.009 13 European Archives of Oto-Rhino-Laryngology (2022) 279:3679–3684 3683 protects cells from radiotherapy-induced damage. Simi- Supplementary Information The online version contains supplemen- larly, in prostate carcinoma cell lines it has been shown tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00405-0​ 21-0​ 7223-4. that the increased sensitivity to radiotherapy mediated by Author contributions All authors contributed substantially to the paper miR-124 and miR-144 was a consequence of PIM-1 down- in conception, design, and writing. The publication has been approved regulation [22]. by all (co-)authors. In addition, an association has been described between PMI-1 expression and resistance to chemotherapy in pros- Funding Open Access Funding provided by Universitat Autonoma tate carcinoma models [23]. There is also evidence that de Barcelona. This study was supported by a grant from Instituto de Salud Carlos III (FIS PI19/01661 to XL). Fondo Europeo de Desarrollo downregulation of PIM-1 activity increased sensitivity to Regional (FEDER), A Way to Build Europe. chemotherapy in the colon [24] or bladder carcinoma cell lines [25]. Declarations In our study, we found a significant relationship between PIM-1 transcriptional expression and the risk of local Conflict of interest The authors declare that they do not have any con- recurrence in patients treated with radiotherapy, includ- flict of interest. ing patients treated with chemoradiotherapy and bioradio- Ethical approval The study has been performed in accordance with the therapy. Figure 2 of the supplementary material shows the ethical standards as laid down in the 1964 Declaration of Helsinki and local recurrence-free survival as a function of the PIM-1 its later amendments. The study was approved by the Hospital de Sant expression category depending on the type of treatment Pau Ethics Committee for Scientific Research (IIBSP-CCC-14–93). used. According to the results of multivariate analysis, the Informed consent Informed consent was obtained from all individual variables that were significantly associated with an increased participants included in the study. risk of local recurrence after radiotherapy were hypopharyn- geal tumor location, advanced local extension (cT3-4), and Open Access This article is licensed under a Creative Commons Attri- high PIM-1 expression. bution 4.0 International License, which permits use, sharing, adapta- These differences in local disease control were not ulti- tion, distribution and reproduction in any medium or format, as long mately reflected in specific survival, basically due to the as you give appropriate credit to the original author(s) and the source, possibility of carrying out salvage surgeries after a local provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are recurrence of the tumor. Salvage surgery entails a higher included in the article's Creative Commons licence, unless indicated frequency of postoperative complications and morbidity otherwise in a credit line to the material. If material is not included in versus similar surgery performed as an initial treatment. the article's Creative Commons licence and your intended use is not The availability of a biomarker with the capacity to assess permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a the sensitivity to radiotherapy would make it possible to copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. personalize the type of treatment according to the tumor's sensitivity profile. 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