Mild hyperthermia upregulates PD-L1 in the tumor microenvironment and enhances antitumor efficacy of PD-L1 blockade in murine squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) has a low five-year survival rate because of its high rate of recurrence and metastasis. After surgical resection or radiation, the main treatments for HNSCC, patients sometimes experience functional or aesthetic disorders. Therefore, there is a great demand for the development of non-surgical treatment strategies to improve clinical outcomes and patients' quality of life. One such non-surgical treatment is mild hyperthermia (mHT). Many studies have investigated combination treatments with mHT and immune checkpoint inhibitors in preclinical settings. However, there have been no detailed reports on the effects of mHT on immune checkpoint molecules. Here, we investigated the effects of mHT on the tumor microenvironment (TME), particularly on programmed cell death receptor-1 (PD-1)/programmed cell death ligand-1 (PD-L1), in SCCVII cells and a squamous cell carcinoma mouse model. First, we found that PD-L1 mRNA levels and surface PD-L1 expression significantly increased after mHT. Second, a single tumor model was used to determine the effect of HT on the TME. mHT enhanced the accumulation of CD4+ and CD8+ T cells, elevated PD-L1 expression in the TME, and decreased the PD-1 positive rate of CD4+ T cells. Finally, using a bilateral tumor model, we found that anti-PD-L1 monotherapy and combination therapy resulted in longer survival than the isotype control or mHT monotherapy. Moreover, the combination therapy resulted in a significantly higher survival rate than anti-PD-L1 monotherapy. In conclusion, our findings elucidate changes in PD-L1 expression in the TME and strengthen the rationale for mHT and PD-L1 blockade combination therapy.

Systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus).

Systemic amyloid light-chain (AL) amyloidosis is an infrequent disease in which amyloid fibrils derived from the immunoglobulin light chain are deposited in systemic organs, resulting in functional impairment. This disease has been notably uncommon in animals, and nonhuman primates have not been reported to develop it. In this study, we identified the systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus) and analyzed its pathogenesis. Amyloid deposits were found severely in the submucosa of the large intestine, lung, mandibular lymph nodes, and mediastinal lymph nodes, with milder lesions in the liver and kidney. Mass spectrometry-based proteomic analysis revealed an abundant constant domain of the immunoglobulin kappa chain in the amyloid deposits. Immunohistochemistry further confirmed that the amyloid deposits were positive for immunoglobulin kappa chains. In this animal, AL amyloidosis resulted in severe involvement of the gastrointestinal submucosa and lymph nodes, which is consistent with the characteristics of AL amyloidosis in humans, suggesting that AL amyloid may have a similar deposition mechanism across species. This report enhances the pathological understanding of systemic AL amyloidosis in animals by providing a detailed characterization of this disease based on proteomic analysis.

Identification of apolipoprotein E-derived amyloid within cholesterol granulomas of leopard geckos (Eublepharis macularius).

Apolipoprotein E (ApoE) is involved in cholesterol transport among cells and also plays an important role in amyloid formation, co-depositing with amyloid fibrils in various types of amyloidosis. Although the in vivo amyloidogenicity of ApoE has not been previously demonstrated, this study provides evidence of ApoE amyloidogenicity in leopard geckos (Eublepharis macularius), belonging to the class Reptilia. Histologically, amyloid deposits were localized within cholesterol granulomas and exhibited positive Congo red staining, with yellow to green birefringence under polarized light. On mass spectrometry-based proteomic analysis, ApoE was detected as a dominant component of amyloid; of the full length of the 274 amino acid residues, peptides derived from Leu185-Arg230 were frequently detected with non-tryptic truncations. Immunohistochemistry with anti-leopard gecko ApoE antibody showed positive reactions of amyloid deposits. These results show that ApoE is an amyloid precursor protein within the cholesterol granulomas of leopard geckos. Although further investigations are needed, the C-terminal region of ApoE involved in amyloid formation is a lipid-binding region, and there should be a relationship between amyloidogenesis and the development of cholesterol granulomas in leopard geckos. This study provides novel insights into the pathogenesis of ApoE-related diseases.

A Japanese multi-institutional phase II study of moderate hypofractionated intensity-modulated radiotherapy with image-guided technique for prostate cancer.

BACKGROUND: The aim of this multi-institutional phase II study was to confirm the safety and the potential efficacy of moderately hypofractionated intensity-modulated radiotherapy (IMRT) with prostate-based image-guidance for Japanese patients. METHODS: Patients with low- or intermediate-risk localized prostate cancer were eligible. Patients with a part of high risk (having only one of the following factors, cT3a, 20 < PSA ≤ 30, or GS = 8 or 9) were also included. Hypofractionated IMRT using daily image-guided technique with prostate matching was performed with a total dose of 70 Gy in 28 fractions. Neoadjuvant hormonal therapy for 4-8 months was mandatory for patients with intermediate or high-risk prostate cancer. RESULTS: From 20 institutions, 134 patients enrolled. The median follow-up was 5.16 years (range, 1.43-6.47 years). The number of patients with low, intermediate, and high-risk prostate cancer was 20, 80, and 34, respectively. The 5-year overall, biochemical failure-free, and clinical failure-free survival was 94.5%, 96.0%, and 99.2%, respectively. The 5-year biochemical failure-free survival for patients with low-, intermediate-, and high-risk disease was 94.1%, 97.4%, and 93.9%, respectively. The incidences of grade 2 gastrointestinal (GI) and genitourinary (GU) late toxicities at 5 years were 5.3% and 5.3%, respectively. There are no acute or late toxicities ≥ grade 3. Of 124 patients who were followed for up to 5 years, the grade 2 late GU or GI toxicities were 10.5% (90% confidence intervals, 6.3-16.2%, p = 0.0958). CONCLUSION: The safety and efficacy of moderately hypofractionated IMRT with prostate-based image-guidance was confirmed among Japanese patients with prostate cancer.

Apolipoprotein C-III amyloidosis in white lions (Panthera leo).

Apolipoprotein C-III (ApoC-III) amyloidosis in humans is a hereditary amyloidosis caused by a D25V mutation in the APOC3 gene. This condition has only been reported in a French family and not in animals. We analyzed a 19-year-old white lion (Panthera leo) that died in a Japanese safari park and found renal amyloidosis characterized by severe deposition confined to the renal corticomedullary border zone. Mass spectrometry-based proteomic analysis identified ApoC-III as a major component of renal amyloid deposits. Amyloid deposits were also positive for ApoC-III by immunohistochemistry. Based on these results, this case was diagnosed as ApoC-III amyloidosis for the first time in nonhuman animals. Five additional white lions were also tested for amyloid deposition retrospectively. ApoC-III amyloid deposition was detected in 3 white lions aged 19 to 21 years but not in 2 cases aged 0.5 and 10 years. Genetic analysis of white and regular-colored lions revealed that the APOC3 sequences of the lions were identical, regardless of amyloid deposition. These results suggest that ApoC-III amyloidosis in lions, unlike in humans, may not be a hereditary condition but an age-related condition. Interestingly, lion ApoC-III has a Val30 substitution compared with other species of Panthera that have Met30. Structural predictions suggest that the conformation of ApoC-III with Met30 and ApoC-III with Val30 are almost identical, but this substitution may alter the ability to bind to lipids. As with the D25V mutation in human ApoC-III, the Val30 substitution in lions may increase the proportion of free ApoC-III, leading to amyloid formation.

Local hyperthermia with built-in endoscopy for radioresistant cervical cancer: a case series.

We evaluated the efficacy and safety of an endoscope-embedded transvaginal laser hyperthermia system for superficial cervical cancer that remained in the cervix after radiotherapy. We developed an innovative endoscope-embedded hyperthermia system consisting of a diode laser device, a temperature control unit, an endoscope control unit, and a transvaginal probe. Superficial lesions of recurrent or residual cervical cancer on the uterine cervix or vaginal wall after radiotherapy were eligible for this study. A total of four cases of three patients were eligible for this treatment. Case 1: The post-chemoradiotherapy residual tumor of a patient with stage IIB squamous cell carcinoma of the cervix was treated with the device. Two months after the laser hyperthermia treatment, the tumor's disappearance was confirmed. Case 2: A post-hysterectomy persistent tumor on the vaginal stump of a patient with stage IIB adenocarcinoma of the cervix was subjected to the laser hyperthermia treatment. Two months after the treatment, the stump's cytology was false positive. Case 3: As in case 2, this patient's recurrence in the anterior vaginal wall was subjected to laser hyperthermia treatment, but the tumor's growth was not controlled. Case 4: A tumor at the vaginal margin was identified during a salvage hysterectomy in a patient with stage IIB squamous cell carcinoma of the cervix who underwent chemoradiotherapy. After laser hyperthermia treatment, the tumor's disappearance was confirmed. Our new endoscope-embedded laser hyperthermia system can be a candidate for treating residual superficial cervical cancer after radiotherapy by accurately capturing superficial lesions.

Fibrinogen Aα-chain amyloidosis outbreaks in Japanese squirrels (Sciurus lis): a potential disease model.

Fibrinogen Aα-chain amyloidosis is a hereditary systemic amyloidosis characterized by glomerular amyloid depositions, which are derived from the fibrinogen Aα-chain variant in humans. Despite its unique pathology, the pathogenic mechanisms of this disease are only partially understood. This is in part because comparative pathological studies on fibrinogen Aα-chain amyloidosis are currently unavailable as there is a lack of reported cases in animals other than humans. In this study, mass spectrometry-based proteomic analyses of Japanese squirrels (Sciurus lis) that died in five Japanese zoos showed that they developed glomerular-associated fibrinogen Aα-chain amyloidosis with an extremely high incidence rate (29/38 cases, 76.3%). The condition was found to be age-dependent in the Japanese squirrels, with 89% of individuals over 4 years of age affected. Mass spectrometry revealed that the C-terminal region of the fibrinogen Aα-chain was involved in amyloidogenesis in Japanese squirrels as well as humans. No gene variations were identified between amyloid-positive and amyloid-negative squirrels, which contrasted with the available data for humans. The results indicate that fibrinogen Aα-chain amyloidosis is a senile amyloidosis in Japanese squirrels. The results have also provided comparative pathological support that the amyloidogenic C-terminal region of the fibrinogen Aα-chain is involved in the characteristic glomerular pathology, regardless of the animal species. This study elucidates the potential causes of death in Japanese squirrels and will contribute to future comparative pathological studies of fibrinogen Aα-chain amyloidosis. © 2023 The Pathological Society of Great Britain and Ireland.

Optimized radiotherapy treatment strategy for early glottic carcinoma.

The local control rates of T1 bulky and T2 glottic carcinoma treated via radiation therapy alone are unsatisfactory; thus, we aimed to evaluate the efficacy and safety of our treatment protocol for early glottic carcinoma. Patients with early glottic squamous cell carcinoma treated via radiation therapy from January 2007 to November 2019 were reviewed. Patients were treated with: 63-67.5 Gy/28-30 fractions of radiation therapy alone for T1 non-bulky; concurrent chemoradiotherapy with S-1 and 60 Gy/30 fractions for T1 bulky and T2 favorable; and concurrent chemoradiotherapy with high-dose cisplatin and 66-70 Gy/33-35 fractions for T2 unfavorable glottic carcinoma. Local failure rates were estimated using the cumulative incidence function, overall and disease specific survival rates were estimated using Kaplan-Meier analysis, and adverse events were evaluated. Eighty patients were analyzed; the median age was 69.5 (range, 26-90) years, the median follow-up time for survivors was 40.1 (range, 1.9-128.4) months, and the 3-year local failure, disease specific survival, and overall survival rates were 5.8%, 98.3%, and 94.4%, respectively. In T1 bulky and T2 cases, the local failure rate was significantly lower in the concurrent chemoradiotherapy than in the radiation therapy alone group. Grade 3 acute dermatitis and mucositis were noted in nine and four patients, respectively. There were no acute adverse events of Grade 4 or higher, or late adverse events of Grade 2 or higher. The treatment protocol was effective and well-tolerated; thus, the efficacy of concurrent chemoradiotherapy was suggested in T1 bulky and T2 cases.

Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication.

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

Identification of Ameloblastin as an Amyloid Precursor Protein of Amyloid-Producing Ameloblastoma in Dogs and Cats.

Amyloid-producing ameloblastoma (APAB) is characterized by abundant amyloid deposits in ameloblastoma, but the amyloid precursor protein is unknown. To explore this, we conducted histopathologic and proteomic analyses on formalin-fixed and paraffin-embedded samples from five cases of APAB (three dogs and two cats). Histologically, the samples exhibited a proliferation of the odontogenic epithelium, with moderate to severe interstitial amyloid deposits. By using Congo red and polarized light, the amyloid deposits were found to show characteristic birefringence. Amyloid deposits were dissected from tissue sections and analyzed by LC/MS/MS, and high levels of ameloblastin were detected in all tissues. Mass spectrometry also revealed that the N-terminal region of ameloblastin is predominantly present in amyloid deposits. Immunohistochemistry was performed using two anti-ameloblastin (N terminal, middle region) antibodies and showed that amyloid deposits were positive for ameloblastin N terminal but negative for ameloblastin middle region. These results suggest that ameloblastin is the amyloid precursor protein of APABs in dogs and cats, and the N-terminal region may be involved in the amyloidogenesis of ameloblastin.

Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation.

Mammary tumor-associated amyloidosis (MTAA) in dogs is characterized by amyloid deposition in the stroma of mammary adenoma or carcinoma; however, the amyloid precursor protein remains unknown. We attempted to identify an amyloid precursor protein and elucidated its etiology by characterizing 5 cases of canine MTAA. Proteomic analyses of amyloid extracts from formalin-fixed paraffin-embedded specimens revealed α-S1-casein (CASA1) as a prime candidate and showed the N-terminal truncation of canine CASA1. Both immunohistochemistry and immunoelectron microscopy showed that amyloid deposits or fibrils in MTAA cases were positive for CASA1. Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction revealed the complete mRNA sequence encoding CASA1, whose expression was significantly higher in the amyloid-positive group. The recombinant protein of the N-terminal-truncated canine CASA1 and the synthetic peptides derived from canine and human CASA1 formed amyloid-like fibrils in vitro. Structural prediction suggested that the N-terminal region of CASA1 was disordered. Previously, full-length CASA1 was reported to inhibit the amyloidogenesis of other proteins; however, we demonstrated that CASA1 acquires amyloidogenicity via excessive synthesis followed by truncation of its disordered N-terminal region. By identifying a novel in vivo amyloidogenic protein in animals and revealing key mechanistic details of its associated pathology, this study provides valuable insights into the integrated understanding of related proteopathies.

＜Editors' Choice＞ Evaluation of system-related magnetic resonance imaging geometric distortion in radiation therapy treatment planning: two approaches and effectiveness of three-dimensional distortion correction.

We propose two methods to evaluate system-related distortion in magnetic resonance imaging (MRI) in radiation therapy treatment planning (RTP) and demonstrate the importance of three-dimensional (3D) distortion correction (DC) by quantitatively measuring the distortion magnitude. First, a small pin phantom was scanned at multiple positions using an external laser guide for accurate phantom placement and combined into one image encompassing a large area. Direct plane images were used for evaluating in-plane distortion and multiplanar reconstruction images for through-plane distortion with no DC, two-dimensional (2D) DC, and 3D DC. Second, a large grid sheet was scanned as the direct plane of the phantom placement. The distortion magnitude was determined by measuring the displacement between the MRI and reference coordinates. The measured distortions were compared between in- and through-plane when applying DC and between the two methods. The small pin phantom method can be used to evaluate a wide range of distortions, whereas data from the entire plane can be obtained with a single scan using the grid sheet without a laser guide. The mean distortion magnitudes differed between the methods. Furthermore, the 3D DC reduced in- and through-plane distortions. In conclusion, the small pin phantom method can be used to evaluate a wide range of distortions by creating a combined image, whereas the grid sheet method is simpler, accurate, repeatable, and does not require a special-order phantom or laser guide. As 3D DC reduces both in- and through-plane distortions, it can be used to improve RTP quality.

Proposal for personalized treatment of early glottic cancer with radiation therapy.

The preservation of both the organ and its function is important for the treatment of early stage glottic cancer, and radiation therapy is an important and useful option. However, treatment with radiation therapy alone is insufficient. Therefore, to improve the local control rate even for early stage glottic cancer, attempts have been made to individualize treatment with radiation therapy (±chemotherapy) based on T stage and morphological characteristics. This individualized treatment greatly improved the local control rate for early glottic cancer. In the future, more suitable individualization can be achieved by investigating the radiosensitivity of biomarkers using biopsy materials before radiation therapy, in addition to T stage and morphological characteristics. Currently, many biomarkers are being investigated; however, appropriate biomarkers for predicting local control remain unknown.

Poor local control of ulcerative T1 glottic cancer treated with 2.25-Gy per fraction radiotherapy.

The Tokai Study Group for Therapeutic Radiology and Oncology (TOSTRO) started managing T1 glottic cancer using 2.25 Gy/fraction radiotherapy in 2011. The aim was to evaluate the local control (LC) rate and toxicity with 2.25-Gy radiotherapy in clinical practice and identify prognostic factors.The eligibility criteria were T1 glottic squamous cell carcinoma patients with age ≥20 years, treated with 2.25 Gy/fraction without chemotherapy between 2011 and 2017. LC rates were evaluated based on age, performance status, sex, T-category, tumor type (ulcerative or non-ulcerative), presence of anterior commissure invasion, tumor size, X-ray beam energy, and overall treatment time. Acute and late adverse events were evaluated using CTCAE version 4.0. A total of 202 patients were enrolled. The median follow-up period was 34.2 months. The 2- and 4-year LC rates were 93.8% and 93.1%, respectively. There was a significant difference in the LC rate between non-ulcerative type and ulcerative type (95.2% vs. 74.1% at 2 years, 94.4% vs. 74.1% at 4 years; p = 0.01). On univariate analysis, only tumor type was significantly correlated with a poor LC rate (hazard ratio 4.3; 95% confidence interval 1.2-15.4; p = 0.03). Acute grade 3 adverse events occurred in 17 patients. However, no late adverse events of grade 3 or higher have occurred to date. T1 glottic cancer treatment outcomes using hypofractionated radiotherapy with 2.25 Gy/fraction in clinical practice were comparable to previously reported results. However, ulcerative type tumor was associated with a poor LC rate.

Development of an x-ray-opaque-marker system for quantitative phantom positioning in patient-specific quality assurance.

PURPOSE: We developed an x-ray-opaque-marker (XOM) system with inserted fiducial markers for patient-specific quality assurance (QA) in CyberKnife (Accuray) and a general-purpose linear accelerator (linac). The XOM system can be easily inserted or removed from the existing patient-specific QA phantom. Our study aimed to assess the utility of the XOM system by evaluating the recognition accuracy of the phantom position error and estimating the dose perturbation around a marker. METHODS: The recognition accuracy of the phantom position error was evaluated by comparing the known error values of the phantom position with the values measured by matching the images with target locating system (TLS; Accuray) and on-board imager (OBI; Varian). The dose perturbation was evaluated for 6 and 10 MV single-photon beams through experimental measurements and Monte Carlo simulations. RESULTS: The root mean squares (RMSs) of the residual position errors for the recognition accuracy evaluation in translations were 0.07 mm with TLS and 0.30 mm with OBI, and those in rotations were 0.13° with TLS and 0.15° with OBI. The dose perturbation was observed within 1.5 mm for 6 MV and 2.0 mm for 10 MV from the marker. CONCLUSIONS: Sufficient recognition accuracy of the phantom position error was achieved using our system. It is unnecessary to consider the dose perturbation in actual patient-specific QA. We concluded that the XOM system can be utilized to ensure quantitative and accurate phantom positioning in patient-specific QA with CyberKnife and a general-purpose linac.

The importance of choosing the right strategy to treat small cell carcinoma of the cervix: a comparative analysis of treatments.

BACKGROUND: Standard treatments for small cell carcinoma of the cervix (SCCC) have not been established. In this study, we aimed to estimate the optimal treatment strategy for SCCC. METHODS: This was a multicenter retrospective study. Medical records of patients with pathologically proven SCCC treated between 2003 and 2016 were retrospectively analyzed. Overall survival (OS) was plotted using the Kaplan-Meier method. Log-rank tests and Cox regression analysis were used to assess the differences in survival according to stage, treatment strategy, and chemotherapy regimen. RESULTS: Data of 78 patients were collected, and after excluding patients without immunohistopathological staining, 65 patients were evaluated. The median age of the included patients was 47 (range: 24-83) years. The numbers of patients with International Federation of Gynecology and Obstetrics (FIGO) 2018 stages I-IIA, IIB-IVA, IVB were 23 (35%), 34 (52%), and 8 (12%), respectively. Of 53 patients who had undergone chemotherapy, 35 and 18 received SCCC and non-SCCC regimens as their first-line chemotherapy regimen, respectively. The 5-year OS for all patients was 49%, while for patients with FIGO stages I-IIA, IIB-IVA, IVB, it was 60, 50, and 0%, respectively. The 5-year OS rates for patients who underwent treatment with SCCC versus non-SCCC regimens were 59 and 13% (p < 0.01), respectively. This trend was pronounced in locally advanced stages. Multivariate analysis showed that FIGO IVB at initial diagnosis was a significant prognostic factor in all patients. Among the 53 patients who received chemotherapy, the SCCC regimen was associated with significantly better 5-year OS in both the uni- and multivariate analyses. CONCLUSION: Our results suggest that the application of an SCCC regimen such as EP or IP as first-line chemotherapy for patients with locally advanced SCCC may play a key role in OS. These findings need to be validated in future nationwide, prospective clinical studies.

Early glottic cancer treatment with concurrent chemoradiotherapy with once-daily orally administered S-1.

Glottic carcinoma is the most common laryngeal cancer. The outcomes for T1 bulky Glottic carcinoma and T2N0 Glottic carcinoma after radiation therapy alone are unsatisfactory. This study was conducted to evaluate the efficacy and safety of unique concurrent chemoradiotherapy regimen using S-1 for early glottic cancer. Concurrent chemoradiotherapy consisted of 60 Gy in 30 fractions with once-daily, orally administered S-1 exclusively within three to six hours prior to each irradiation. Twenty-one consecutive patients treated with this regimen were retrospectively reviewed. Initial complete remission was achieved in all patients without any subsequent local and/or regional recurrences to the last follow-up. The 4-year local control, overall survival, and disease-free survival rates were all 100%. No significant toxicities were observed, except for three cases with Grade 3 acute dermatitis.This regimen is highly effective and well-tolerated, and these results encourage further research to long-term efficacy and functional preservation.

Dosimetric impacts of beam-hardening filter removal for the CyberKnife system.

PURPOSE: Equipment refurbishment was performed to remove the beam-hardening filter (BHF) from the CyberKnife system (CK). This study aimed to confirm the change in the beam characteristics between the conventional CK (present-BHF CK) and CK after the BHF was removed (absent-BHF CK) and evaluate the impact of BHF removal on the beam quality correction factors kQ. METHODS: The experimental measurements of the beam characteristics of the present- and absent-BHF CKs were compared. The CKs were modeled using Monte Carlo simulations (MCs). The energy fluence spectra were calculated using MCs. Finally, kQ were estimated by combining the MC results and analytic calculations based on the TRS-398 and TRS-483 approaches. RESULTS: All gamma values for percent depth doses and beam profiles between each CK were less than 0.5 following the 3%/1 mm criteria. The percentage differences for tissue-phantom ratios at depths of 20 and 10 cm and percentage depth doses at 10 cm between each CK were -1.20% and -0.97%, respectively. The MC results demonstrated that the photon energy fluence spectrum of the absent-BHF CK was softer than that of the present-BHF CK. The kQ values for the absent-BHF CK were in agreement within 0.02% with those for the present-BHF CK. CONCLUSIONS: The photon energy fluence spectrum was softened by the removal of BHF. However, no remarkable impact was observed for the measured beam characteristics and kQ. Therefore, the previous findings of the kQ values for the present-BHF CK can be directly used for the absent-BHF CK.