Euploid Tumors Research Articles

Striking heterogeneity of somatic L1 retrotransposition in single normal and cancerous gastrointestinal cells

Somatic LINE-1 (L1) retrotransposition has been detected in early embryos, adult brains, and the gastrointestinal (GI) tract, and many cancers, including epithelial GI tumors. We previously found numerous somatic L1 insertions in paired normal and GI cancerous tissues. Here, using a modified method of single-cell analysis for somatic L1 insertions, we studied adenocarcinomas of colon, pancreas, and stomach, and found a variable number of somatic L1 insertions in tumors of the same type from patient to patient. We detected no somatic L1 insertions in single cells of 5 of 10 tumors studied. In three tumors, aneuploid cells were detected by FACS. In one pancreatic tumor, there were many more L1 insertions in aneuploid than in euploid tumor cells. In one gastric cancer, both aneuploid and euploid cells contained large numbers of likely clonal insertions. However, in a second gastric cancer with aneuploid cells, no somatic L1 insertions were found. We suggest that when the cellular environment is favorable to retrotransposition, aneuploidy predisposes tumor cells to L1 insertions, and retrotransposition may occur at the transition from euploidy to aneuploidy. Seventeen percent of insertions were also present in normal cells, similar to findings in genomic DNA from normal tissues of GI tumor patients. We provide evidence that: 1) The number of L1 insertions in tumors of the same type is highly variable, 2) most somatic L1 insertions in GI cancer tissues are absent from normal tissues, and 3) under certain conditions, somatic L1 retrotransposition exhibits a propensity for occurring in aneuploid cells.

Acquisition of chromosome instability is a mechanism to evade oncogene addiction

Chromosome instability (CIN) has been associated with therapeutic resistance in many cancers. However, whether tumours become genomically unstable as an evolutionary mechanism to overcome the bottleneck exerted by therapy is not clear. Using a CIN model of Kras‐driven breast cancer, we demonstrate that aneuploid tumours acquire genetic modifications that facilitate the development of resistance to targeted therapy faster than euploid tumours. We further show that the few initially chromosomally stable cancers that manage to persist during treatment do so concomitantly with the acquisition of CIN. Whole‐genome sequencing analysis revealed that the most predominant genetic alteration in resistant tumours, originated from either euploid or aneuploid primary tumours, was an amplification on chromosome 6 containing the cMet oncogene. We further show that these tumours are dependent on cMet since its pharmacological inhibition leads to reduced growth and increased cell death. Our results highlight that irrespective of the initial CIN levels, cancer genomes are dynamic and the acquisition of a certain level of CIN, either induced or spontaneous, is a mechanism to circumvent oncogene addiction.

KRAS and aneusomy of chromosomes 4, 10 and 12 in colorectal carcinomas

AimsKRAS mutation status has predictive significance in EGFR-antibody treatment of colorectal adenocarcinoma. The aim of the study was the evaluation of KRAS mutation status in correlation to KRAS copy numbers and ploidy status. MethodsColorectal adenocarcinomas (n=52) were assembled into a TMA and analyzed by FISH. Probes for centromeres 4 and 10 were applied as surrogate markers for the ploidy status. In addition, a dual color FISH probe set for the centromere of chromosome 12 and the KRAS gene was applied to the TMA to analyze numerical alterations and KRAS gene copy numbers. Further we analyzed DNA sequence profiles of KRAS codons 12 and 13 to assess the allele status of the mutation within the tumor samples. ResultsKRAS mutation was confirmed in 24 cases, while 28 cases showed a wild-type KRAS status. The majority of cases showed diploid FISH signals for chromosomes 4 and 10. Near triploid FISH signals were observed in only 2 cases, 12 cases were hypodiploid, and 8 cases were hyperdiploid. In 6 cases, trisomy 12 could be ascertained. In total, aneuploidy could be detected in 28 cases, including cases with trisomy 12 and hyposomy 10. Tumors with aneuploid chromosomal content had a worse prognosis compared to euploid tumors, however, without reaching statistical significance (p=0.231). Hypodiploid carcinomas carried the worst prognosis. Specifically, monosomy 10 was significantly associated with reduced survival (p=0.039). Increased FISH signals of KRAS did not correlate significantly with relapse (p=0.916). ConclusionsFISH analysis can be used as a surrogate marker for the ploidy status. Loss of chromosome 10 may serve as a potential adverse prognostic marker being indicative for tumor progression.

Large‐scale genomic instability in colon adenocarcinomas and correlation with patient outcome

The purpose of this study was to evaluate the association between DNA content in colon adenocarcinomas using high-resolution image cytometry and patient outcome. Tumours from 219 patients operated for colon adenocarcinoma were analysed using high-resolution image cytometry. Proteins involved in cell cycle propulsion (cyclins A, D1, D3 and E) and cell proliferation (c-Myc and non-membranous beta-catenin) have previously been reported in the same cohort and were included in this study. The results were related to disease-free survival and to cancer-specific death. Patients with aneuploid tumours showed shorter relapse-free survival than patients with euploid tumours (univariate log-rank test, p = 0.004 and multivariate Cox regression model p = 0.009, HR 0.51, 95% CI 0.31-0.84). Also the risk of death from cancer was greater in patients with aneuploid tumours (log-rank test, p = 0.006 multivariate Cox regression model p = 0.014, HR 0.47, 95% CI 0.26-0.86). When analysing patients with Dukes stages A and B, nuclear expression of beta-catenin was highly significantly associated with both shorter relapse-free survival (p < 0.005, HR 5.0, 95% CI 1.6-15.5) and cancer-specific death (p = 0.036, HR 6.9, 95% CI 1.1-42.1). DNA content in colon adenocarcinomas measured by image cytometry is an independent predictor of prognosis in our patients operated for colon adenocarcinoma.

DNA content heterogeneity in neuroblastoma analyzed by means of image cytometry and its potential significance

The aim of this study was to determine the frequency and significance of the tumor DNA content heterogeneity in 33 previously untreated human neuroblastomas. We used image cytometry to selectively analyze neuroblasts by excluding karyorrhectic or stromal cells from cytometric measurements. DNA content heterogeneity with more than one clonal subpopulation on DNA histogram was found in 8 of 33 cases. Of these 8 cases, 4 showed MYCN amplification. Double labeling fluorescent in situ hybridization with probes for the centromeric region of chromosome 2 and MYCN gene was used to confirm the DNA content heterogeneity. DNA content heterogeneity was associated with poorer prognosis in this study (P<0.05). There was a significant correlation between euploidy (di- and tetraploidy) and worse prognosis, but only when heterogeneous neuroblastomas with euploid cell population were assigned to euploid tumors (P=0.006). Our results may explain the conflicting data in the literature regarding ploidy and suggest that DNA content heterogeneity and the presence of a euploid population may predict worse prognosis in neuroblastoma patients.

Increasing Sensitivity of Morphological Diagnosis in Hepatocellular Carcinoma (HCC) by Combination of Cytological and Fine-Needle Histological Examination after Ultrasound Guided Fine Needle Biopsy

Increasing sensitivity of morphological diagnosis by combination of cytological and fine-needle histological examination after ultrasound guided fine needle biopsy in hepatocellular carcinoma (HCC). We investigated the factors which may affect the sensitivity of morphological diagnostics (cytology and fine-needle histology) in HCC after ultrasound guided biopsy. In 62 patients, an ultrasound guided cytological (n = 62) and fine-needle histological (n = 45) biopsy was performed. In addition, the smears were investigated by cytophotometry (n = 62). Considering each method separately, the sensitivity of both cytological and histological diagnostics was about 80%. The sensitivity could be raised to 89% by combining the two methods. In seven patients, the HCC could not be primarily confirmed morphologically. In nine patients, the result of histological and cytological investigation was not congruent. With regard to the size of the biopsied lesion, tumor stage, cytophotometric finding and survival these 16 patients (group A) were compared with the 46 patients in whom the HCC could be primarily confirmed morphologically (group B). The statistical analysis did reveal a significantly lower percentage of tumors with diploid or tetraploid (euploid) cells in group B. Euploid tumors were significantly more highly differentiated. In patients with euploid tumors, the sensitivity of the cytological investigation was 67% and respectively 75% of the histological investigation. The sensitivity could be raised to 82% in this population by combining the two investigations. On the other hand in aneuploid tumors, the sensitivity could only be increased from 93% and 88% respectively to 97% by combining cytology and fine-needle histology. Our investigations show that the sensitivity of morphological diagnostics can be raised especially in the highly differentiated HCC with a physiological DNA content by combination of cytological and fine-needle histological investigations.

TP53 and p21WAF1/CIP1 Behave Differently in Euploid versus Aneuploid Bladder Tumours Treated with Radiotherapy

The aim of this study was to examine any relation between DNA ploidy and previously detected TP53 (p53) or p21WAF1/CIP1 expression in 94 patients with muscle-invasive transitional cell carcinoma of the urinary bladder and to associate these factors with survival. DNA ploidy was determined by image cytometry. In a subgroup of patients, the mutational status of the TP53 gene was assessed by temporal temperature gradient electrophoresis (TTGE) or perpendicular denaturant gradient gel electrophoresis (DGGE) and subsequent sequencing. Significantly more aneuploid than euploid tumours showed TP53 accumulation (p = 0.003). Patients with aneuploid tumours lived longer than patients with euploid tumours (p = 0.003). In the euploid, but not in the aneuploid group, TP53 and p21WAF1/CIP1 were associated with cancer-specific survival (p = 0.002 and 0.02, respectively). Patients with > 50% TP53 expression had the longest survival time. Mutation analyses showed acceptable concordance with TP53 expression. We conclude that DNA aneuploidy may confer increased radiosensitivity in bladder cancer patients and that TP53 accumulation may confer increased radiosensitivity, but its effect is detectable only in euploid tumours.

Relationship between flow cytometric DNA ploidy and nuclear grade with endocrine dysfunction in adrenal cortical adenomas

Objectives. To determine the relationship among the DNA ploidy, histopathologic features, and clinical syndrome in adrenal cortical adenomas, because the cells often show variability in nuclear size and configuration. Methods. Our study included 44 adenomas associated with primary aldosteronism and 23 adenomas associated with Cushing syndrome. Normal adrenal glands from patients with renal carcinoma served as the controls. Paraffin-embedded tissues were examined for DNA content by flow cytometry. The mean percentage of G 2/M (4C%) of the control samples was 3.8%. Tetraploid was represented by a histogram with both a 4C peak greater than 9% (mean + 2.4 SD of control samples) and a small 8C peak. Results. Flow cytometric analysis revealed diploidy in 30, tetraploidy in 27, and aneuploidy in 8 of the 67 adenomas; 2 adenomas could not be classified. All 17 normal adrenal glands showed diploidy. A significant relationship was noted between DNA ploidy and the clinical syndrome (ie, a larger proportion of adenomas with primary aldosteronism had a tetraploid DNA histogram compared with adenomas with Cushing syndrome, P <0.0001). Adenomas with primary aldosteronism had a significantly higher nuclear grade (III or IV) than did tumors with Cushing syndrome ( P = 0.033). A significant relationship was also observed between DNA ploidy and nuclear grade in 57 euploid tumors, with tetraploid tumors often showing the highest nuclear grade ( P = 0.037). Conclusions. Our results have demonstrated that adrenal cortical adenomas associated with primary aldosteronism often reveal severe nuclear pleomorphism, indicating that nuclear pleomorphism might be due to a tetraploid stemline.

DNA content and p53 protein expression in ductal breast cancer.

The DNA content of 85 ductal breast cancers of different histological grades was evaluated using static cytometry and correlated with immunocytochemical expression of p53 protein in tumour cells in cytological material. A statistically significant difference was observed between p53 protein expression and grade of malignancy (P < 0.001). The percentage of euploid tumours significantly decreased from grade I through grade II to grade III tumours (P < 0.001). Clonal DNA heterogeneity was observed in 26.6% of cases analysed and was correlated with p53 protein expression (P < 0.001). These changes probably reflect genomic alterations which may affect potential malignancy of breast cancer.

Flow cytometric evaluation of transferrin receptor in transitional cell carcinoma.

We evaluated flow cytometric (FCM) analysis of transferrin receptor (TFR) expression as a marker for the malignant potential in transitional cell carcinoma (TCC). TCCs from 55 patients were analyzed by FCM using an anti-TFR monoclonal antibody (CD71) and a TCC-specific monoclonal antibody (EH14), which recognizes most TCC cells irrespective of the grade. The cells were divided into subpopulations according to DNA ploidy determined simultaneously. TFR expression correlated well with the grade and the stage of the tumors. TFR expression of the aneuploid tumors was significantly higher than that of the euploid tumors in all subpopulations. EH14 expression did not correlate with the grade or the stage of the tumors. EH14 expression of the aneuploid tumors was significantly higher than that of the euploid tumors in the whole cell population but not in the subpopulations. In moderately differentiated tumors or in T1 tumors, TFR expression was higher in multiple or recurrent tumors than in simple tumors. The cell size or shape were not the primary reasons for the enhanced expression of TFR in the high-grade or the high-stage tumors; instead, overproduction of TFR may take place in these tumors. Clinically, many of the TCC tumors are grouped into G2 or T1 tumors, some of which will be invasive cancers. Quantitative analysis of TFR expression using FCM may be useful to predict the prognosis of these tumors.

DNA ploidy and pS2 protein expression in breast cancer.

The DNA content of ductal breast carcinomas of varying histological grade was measured using static image cytometry and correlated with pS2 expression in the tumour cells. Our study was performed on imprint of surgical biopsies of 60 women with ductal breast cancer. A statistically significant difference was observed between pS2+ expression and grade of malignancy (P < 0.001). The percentage of euploid tumours significantly decreased from grade I to grade II to grade III (P = 0.01). The percentage of aneuploid tumours increased from pS2+ to pS2- breast tumours (P < 0.001). These findings may be indicative of pS2 and DNA ploidy alterations and tumour aggressiveness.

Predictive value of the flow cytometric PCNA assay (proliferating cell nuclear antigen) in head and neck tumors after accelerated-hyperfractionated radiochemotherapy

Proliferation of tumor cells during radiotherapy may limit tumor control, especially in rapidly proliferating tumors such as head and neck carcinomas. We present a flow cytometric method for detection of PCNA in solid head and neck tumors and how these data correlate with outcome. Pretherapeutic biopsies of 20 inoperable patients with Stage IV squamous cell carcinoma were examined. Biparametric flow cytometry was done after anti-PCNA (PC10) and propidium iodine staining were performed. PCNA index (percentage PCNA positive cells), DNA index, and S phase fraction (SPF, euploid tumors only) were determined. The therapy consisted of an accelerated-hyperfractionated radiochemotherapy (66 Gy/5 weeks, concomitant boost of 1.6 Gy/day in weeks 4+5, Carboplatin 5 x 70 mg/m2 in weeks 1+5). The median follow-up time was 30 months. Fourteen patients suffered from disease progession and 12 died. Median actuarial, cause-specific survival, and disease-free survival (DFS) times were 17 and 9 months, respectively. PCNA indices ranged from 4 to 70% (median 9%); there were 7 aneuploid and 13 euploid tumors. SPF ranged from 4 to 14.5% (median 10.5%). Neither SPF nor ploidy had a significant influence on outcome. Patients were divided according to PCNA index in higher (n = 10) and lower (n = 10) than the median. Survival and DFS were 13 and 6 months for the group >9% and 20 and 15 months for the group <9%. The difference in DFS was significant (p = 0.03, log rank test). These results fall in line with other studies showing the influence of pretherapeutic proliferation on outcome after radiotherapy. Although the moderately accelerated therapy regimen certainly reduces the influence of proliferation on outcome, patients with faster proliferating tumors still have a worse outcome. DFS is the more relevant endpoint in this study because of effective salvage therapies, which influence survival.

Correlation between p53 status, DNA ploidy, proliferation rate and nuclear morphology in breast cancer. An image cytometric study.

The study was designed to detect differences in the nuclear morphology of tumours and tumour cell populations with different p53 expression in correlation with DNA ploidy and proliferation rate. The paraffin sections from routinely processed samples of 88 breast cancers were immunostained with the monoclonal p53‐antibody DO‐1. After localization and evaluation with a scoring system the sections were destained and stained by the Feulgen method. The nuclei were relocated automatically and measured by means of the image cytometry workstation. Significant differences between the tumours and tumour cell populations with different p53 expression were found in the euploid tumours as well as in the aneuploid tumours and in the breast cancers with a high proliferation rate. The breast cancers with a low immunoreactive score (IRS 1–4) differ from the negative cancers as well as from the cancers with a higher immunoreactive score (IRS 5–12). Evaluating the nuclear populations of the p53 positive cancers, there were differences in the features of the chromatin amount and distribution in the groups of the euploid breast cancers and in cancer with a high proliferation rate. In contrast, the nuclear populations of the aneuploid cancers did not show any differences in their nuclear morphology.The results showed the different impacts of the p53 expression, DNA ploidy and the proliferation rate on the nuclear morphology in breast cancer.

Lectin histochemistry, ploidy level, and proliferative activity in rhabdomyosarcoma subtypes

The glycohistochemical expression of binding sites for eight lectins is characterized in a series of 8 embryonal, 4 alveolar and 4 pleomorphic rhabdomyosarcomas. The correlation between lectin staining and either the proliferation index or the ploidy level was also investigated. The data show that rhabdomyosarcomas exhibit heterogeneous lectin binding expressions. A comparable level of lectin labeling is observed in euploid and aneuploid tumours. In contrast to other neoplasms, lectin staining has proved to be of doubtful value in distinguishing between different RMS subtypes. The data also reveal that a significantly lower level of proliferative activity was observed in the pleomorphic group as compared to the alveolar one.

Prognostic impact of DNA content and AUER classification in primary fallopian tube carcinoma.

DNA ploidy has been studied in 61 primary fallopian tube carcinomas using image-cytometry. The investigation also included survival analysis, and ploidy classification according to AUER was performed in order to evaluate its prognostic impact for fallopian tube carcinoma. A high number of aneuploid cases were observed (79% aneuploid vs. 21% euploid tumors). The high incidence of aneuploid tumors was consistently observed among all FIGO-stages as well as all groups of histologic grading. There was no correlation between ploidy and FIGO-stage or histologic grading. Patients with euploid DNA content showed a median survival of 34 months compared to 24 months for aneuploid cases (log-rank, P = 0.83). No correlation between the AUER classification and FIGO-stage or histologic grading could be observed. Tumors with an AUER type I and II (75th quantile 41 months) showed a better outcome than tumors with AUER III and IV (75th quantile 19 months). Although these results did not reach statistical significance (P = 0.07), a trend could be observed. Therefore AUER classification may be useful as an objective prognostic parameter. The high incidence of aneuploid tumors could be an expression of the high biologic aggressiveness of primary fallopian tube cancer which has been repeatedly mentioned in the past.

Granulosa Cell Tumor of Ovary: A Clinicopathologic and Flow Cytometric DNA Analysis

Flow cytometric DNA ploidy and S-phase fraction (SPF) analysis was performed on 18 granulosa cell tumors of the ovary. Clinical and pathologic data from patients followed for an average of 10 years were compared to flow cytometry ploidy to determine its prognostic usefulness. Eleven (62%) tumors were diploid and seven (38%) were aneuploid. Aneuploidy was significantly associated with high stage, vascular or capsular invasion, and nuclear atypia but not with SPF or necrosis. Of the patients on whom follow-up was available, only 1 of 10 (10%) with euploid tumors died of disease, while 4 of 5 (80%) with aneuploid tumors died of their disease; this, however, did not reach a significant statistical correlation. SPF was not correlated with any clinical or histologic parameter. Aneuploidy in granulosa cell tumors is associated with other adverse histopathologic parameters and an apparent trend toward aggressive behavior.

Evaluation of deoxyribonucleic acid ploidy and S-phase fraction as prognostic parameters in advanced epithelial ovarian carcinoma: A prospective study

OBJECTIVE: Our goal was to study the prognostic value of deoxyribonucleic acid ploidy and S-phase fraction in advanced ovarian carcinoma. STUDY DESIGN: Prognostic factors for corrected survival were evaluated in a prospective study including 169 patients with stage III and IV ovarian cancer treated between 1985 and 1990. RESULTS: A total of 79% of the tumors were deoxyribonucleic acid aneuploid. Deoxyribonucleic acid aneuploidy was associated with grade of differentiation. S-phase fraction could be calculated in all deoxyribonucleic acid euploid tumors and 76% of the deoxyribonucleic acid aneuploid tumors. By multivariate analysis deoxyribonucleic acid ploidy, histologic type and grade, age, International Federation of Gynecology and Obstetrics stage, and amount of residual tumor were independent prognostic variables for corrected survival. On the basis of Cox regression a relative risk for the individual patient could be calculated. CONCLUSION: Deoxyribonucleic acid ploidy gives additive prognostic information and is a useful parameter for dividing patients with advanced ovarian cancer into risk groups for treatment decisions. (AM J OBSTET GYNECOL 1994;170:479-87.)

Combined analysis of flow cytometry and morphometry of ovarian granulosa cell tumor

It is difficult to determine the prognosis of granulosa cell tumors (GCT) at the time of diagnosis. The nuclear DNA content of 17 patients with ovarian GCT was investigated by flow cytometry using paraffin-embedded tissue. Nuclear area (NA), nuclear perimeter (NP), and nuclear shape factor (NSF) were measured by an image analyzer using hematoxylin- and-eosin-stained sections. The follow-up period of the patients ranged from 2 months to 11 years. Thirteen tumors were diploid or near diploid, whereas one was tetraploid, and three were aneuploid. Two tumors had varying degrees of DNA content heterogeneity. Crude survival of the patients with an euploid tumor (13 diploid, 1 tetraploid) was more favorable than that of the patients with an aneuploid tumor. Patients with S-phase fraction (SPF) greater than 10% or DNA content heterogeneity experienced disease recurrence or metastasis. A significant difference was observed in NA and NP between those with and without metastasis. Our results indicate that DNA aneuploidy, large SPF, DNA content heterogeneity, and large NA and NP are adverse prognostic factors in GCT. Thus, flow cytometric and morphometric measurement may provide a rapid and valuable method to predict the biologic behavior of GCT.

Lack of intratumoral heterogeneity in DNA ploidy pattern of hepatocellular carcinoma

Background : From biological and clinical perspectives, it is important to clarify tumor heterogeneity. This study was aimed to investigate whether or not intratumoral heterogeneity of DNA ploidy pattern exists in hepatocellular carcinoma (HCC). Methods : Using fresh materials resected from 31 untreated patients, DNA ploidy was analyzed at different sites of the same HCC by means of flow cytometry. The tumor size ranged from 1.7 to 25.0 cm. Results : There was no case in which euploid and aneuploid HCCs coexisted in the same tumor. The DNA ploidy pattern was euploid in 15 and aneuploid in 16 instances. Of 15 euploid tumors, the areas analyzed were all diploid in 12 and tetraploid in 2 but diploid/tetraploid in 1. Among 16 aneuploid tumors, the DNA indices (DI) at different sites were similar in 9, but apparently, different aneuploid subclones coexisted in 7 cases. The incidence of DI heterogeneity in aneuploid HCCs was similar between small (<5 cm) and large (≧5 cm) tumors; 3 of 7 (42.9%) versus 4 of 9 (44.4%). Conclusions : It is assumed that euploid and aneuploid HCCs develop in their own ploidy pattern and that the evolution of aneuploid subpopulations from euploid HCC is rare, but new aneuploid subclones can evolve from aneuploid HCC due to increased instability of its karyotype.

Flow cytometric DNA analysis does not predict the radiochemoresponsiveness of esophageal cancer

The relationship between the DNA pattern and the responsiveness to chemotherapy or chemoradiotherapy has been evaluated in 30 patients with squamous cell carcinoma of the esophagus. In 24 patients polychemotherapy with cisplatin (100 mg/m2 on day 1) and 5-fluorouracil (1,000 mg/m2/24 h, continuous infusion of 120 h) every 3 weeks, was performed. Six other patients received chemoradiotherapy with cisplatin 80 mg/m2 on day 1 and 18.5 Gy (split course). Before treatment, at least three endoscopic biopsies were taken from each tumor and frozen at -85 degrees C. Five patients were excluded from the evaluation, three because of interrupted treatment and two due to unsuitable biopsy material obtained endoscopically. The response rate to the cytoreductive treatment was 40% (10/25). DNA content was analyzed with flow cytometry. Out of 25 evaluable patients, a diploid and aneuploid tumor was present in 8 (32.0%) and 17 (68.0%) patients, respectively. According to the DNA pattern, a major response was observed in 4 of 8 patients with a diploid tumor and in 6 of 17 patients with an aneuploid tumor (P = 0.5). No relationship between the percentage of cells in the S-phase and the response to the cytoreductive treatment was evident. Although a slightly higher percentage of major responses was found in euploid tumors, there is no evidence that flow-cytometric DNA analysis can be helpful in the selection of patients for chemotherapy or chemoradiotherapy.