Dermatology-related quality-of-life outcomes in patients with RAS wild-type metastatic colorectal cancer treated with fluorouracil and folinic acid with or without panitumumab (Pmab) maintenance after FOLFOX + Pmab induction: a prespecified secondary analysis of the phase II randomized PanaMa (AIO KRK 0212) trial.

BACKGROUND: The key endpoints for the assessment of the effect of maintenance therapy for metastatic colorectal cancer (mCRC) are survival and quality-of-life outcomes. We aimed to compare dermatology-related quality of life (DRQOL) in patients with RAS wild-type (wt) mCRC treated with fluorouracil and folinic acid (FU/FA) + panitumumab (Pmab) versus FU/FA alone as maintenance therapy after folinic acid, fluorouracil and oxaliplatin + Pmab induction. PATIENTS AND METHODS: The phase II randomized PanaMa (AIO KRK 0212; NCT01991873) trial included 387 patients at 70 community/academic sites in Germany. For this prespecified secondary analysis, DRQOL outcomes were assessed using the Functional Assessment of Cancer Therapy-epidermal growth factor receptor inhibitor (FACT-EGFRI), Dermatology Life Quality Index (DLQI), and Skindex-16 questionnaires at every second cycle of therapy until disease progression/death. RESULTS: At least one DRQOL questionnaire was completed by a total of 310/377 (82%) patients who received induction therapy, and by 216/248 (87%) patients who were randomized and received maintenance therapy. Patients who experienced skin toxicity according to the National Cancer Institute (NCI)-Common Terminology Criteria for Adverse Events (CTCAE) during induction therapy had significantly worse DRQOL according to all three measures, compared to those who did not [i.e. Skindex-16, mean difference at cycle 2 -12.87; 95% confidence interval (CI) -20.01 to -5.73; P < 0.001]. During maintenance therapy, significantly improved recovery was observed in all DRQOL measures for patients receiving FU/FA, compared to those receiving additional Pmab (i.e. Skindex-16, mean difference at cycle 6 -16.53; 95% CI -22.68 to -10.38; P < 0.001). CONCLUSIONS: In this secondary analysis of a phase II randomized clinical trial, patient-reported DRQOL outcomes correlated with skin toxicity according to NCI-CTCAE during induction therapy. Maintenance therapy with FU/FA + Pmab was associated with deteriorated DRQOL versus FU/FA alone in patients with RAS wt mCRC.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. METHODS: We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. RESULTS: MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. CONCLUSION: These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

Health-related quality of life in patients with RAS wild-type metastatic colorectal cancer treated with fluorouracil and folinic acid with or without panitumumab as maintenance therapy: a prespecified secondary analysis of the PanaMa (AIO KRK 0212) trial.

BACKGROUND: The PanaMa trial demonstrated significant benefit in progression-free survival with the addition of panitumumab (Pmab) to fluorouracil and folinic acid (FU/FA) as maintenance therapy following first-line induction therapy with FOLFOX/Pmab in patients with RAS wild-type metastatic colorectal cancer. Here, we report health-related quality of life (HRQOL) analyses from the PanaMa trial. METHODS: HRQOL outcomes were evaluated using European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) at every cycle of therapy until disease progression/death. HRQOL outcomes were mean and individual changes in EORTC QLQ-C30 from baselines (before induction therapy and before maintenance therapy) to each cycle of treatment. Comparative analyses were performed by randomisation status and treatment arm for induction- and maintenance-therapy, respectively. The trial is registered with clinicaltrials.gov (NCT01991873). RESULTS: At least one HRQOL questionnaire was completed by a total of 349/377 (93%) patients who received induction therapy, and by 237/248 (96%) patients who were randomised and received maintenance therapy. During induction therapy, most HRQOL dimensions remained stable or showed improvement, while appetite loss and diarrhoea significantly deteriorated. During maintenance therapy, HRQOL dimensions remained stable, while those that deteriorated during induction therapy showed significant improvement, without significant differences between the treatment arms. CONCLUSION: Maintenance therapy improves HRQOL dimensions that initially deteriorated during induction therapy while stabilising HRQOL in other dimensions. The addition of Pmab to FU/FA as maintenance therapy in patients with RAS wild-type metastatic colorectal cancer prolongs progression-free survival without negative impact on HRQOL.

Is there a role for rechallenge and reintroduction of anti-EGFR plus chemotherapy in later lines of therapy for metastatic colorectal carcinoma? A retrospective analysis.

BACKGROUND: Mechanisms of resistance have been described during disease progression (PD) for patients under treatment with anti-EGFR plus chemotherapy (CT). The aim of our study was to evaluate efficacy of anti-EGFR rechallenge (ReCH) and reintroduction (ReIn) in metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: This is a retrospective analysis of patients with mCRC that previously received anti-EGFR + CT and interrupted therapy due to PD in the ReCH group and other reasons in the ReIn group. We aimed to describe progression-free survival (PFS), overall survival (OS) and response rate (RR) after re-exposure and to evaluate prognostic factors associated with PFS. RESULTS: Sixty-eight patients met the inclusion criteria. The median follow-up after re-exposure was 39.3 months. ReCH was adopted in 25% and ReIn in 75%. The median anti-EGFR free interval was at 10.5 months. At re-exposure, the main CT regimen was FOLFIRI in 58.8%. Cetuximab and Panitumumab were used in 59 and 9 patients, respectively. mPFS for ReCH and ReIn was 3.3 × 8.4 months, respectively (p 0.001). The objective response rate for ReCH and ReIn was 18% and 52%, respectively. In univariate analysis, adverse prognostic factors related to PFS were: stable disease or PD at first anti-EGFR exposure (HR: 2.12, CI:1.20-3.74; p = 0.009); ReCH (HR: 3.44, CI:1.88-6.29, p < 0.0001); rechallenge at fourth or later lines (HR: 2.51, CI:1.49-4.23, p = 0.001); panitumumab use (HR: 2.26 CI:1.18-5.54, p = 0.017). In the multivariate model, only ReCH remained statistically significant (HR = 2.63, CI: 1.14-6.03, p = 0.022). CONCLUSION: In our analysis, ReCH resulted in short PFS and low RR. However, reintroduction of anti-EGFR plus CT before complete resistance arose resulted in prolonged PFS. These data could be clinically useful to guide a treatment break due to side effects or patient decisions. Our data should be confirmed by larger and prospective trials.

Prevalence, prognosis and predictive status of HER2 amplification in anti-EGFR-resistant metastatic colorectal cancer.

Numerous inherent and acquired genetic alterations have been demonstrated with resistance to anti-epidermal growth factor receptor (anti-EGFR) therapy in metastatic colorectal cancer (mCRC) patients. Although the common oncogenic driver mutations identified include KRAS, NRAS, BRAF, and PI3K, recent studies report a vital role played by human epithelial growth factor receptor-2 (HER2) amplification in acquired resistance to anti-EGFR therapy. HER2 amplification has been associated with poor prognosis in many malignancies including breast and gastric cancer and is also a negative predictor of anti-EGFR therapy. Given the relevance of HER2 amplification in conferring an anti-EGFR resistance, this paper reviews the prevalence of HER2 amplification in mCRC while exploring the prognostic and predictive values of this biomarker. Further, we also discuss the results of the studies that explored the utilization of anti-HER2-targeted therapies in mCRC. HER2-directed therapies have the ability to change the treatment algorithm in clinically relevant small subset of patients with HER2-amplified mCRC.

Circulating Tumor DNA Detection in the Management of Anti-EGFR Therapy for Advanced Colorectal Cancer.

Background: Anti-EGFR antibodies are a standard care for advanced KRAS-wild type colorectal cancers. Circulating tumor DNA (ctDNA) monitoring during therapy can detect emergence of KRAS mutant clones and early resistance to therapy. Case Presentation: We describe a 61-years-old man presenting a metastatic and recurrent rectal cancer treated with different chemotherapy regimens. His tumor was KRAS wild-type based on tissue analysis and he was treated sequentially with cetuximab-based chemotherapy, chemotherapy alone and panitumumab-based chemotherapy. We performed sequential analysis of ctDNA using droplet digital PCR (ddPCR) and a commercial assay designed for the detection of frequent KRAS mutations during his clinical follow-up. Prior to the first cetuximab-based chemotherapy ctDNA analysis demonstrated an absence of KRAS mutations. Emergence of KRAS mutations in ctDNA occurred ~3 months after treatment initiation and preceded clinical and imaging progression in about 2 months. Fractional abundance of KRAS mutation rapidly increased to 70.7% immediately before a chemotherapy alone regimen was initiated. Interestingly, KRAS mutation abundance decreased significantly during the first two months of chemotherapy, reaching a fractional abundance of 3.0%, despite minimal clinical benefit with this therapy. Re-challenge with a different anti-EGFR antibody was attempted as later line, but high levels of KRAS mutations in ctDNA before therapy correlated with an absence of clinical benefit. Conclusions: The monitoring of resistance mutations in KRAS using ctDNA during the treatment of KRAS wild-type advanced colorectal cancers can detect the emergence of resistant clones prior to clinical progression. Dynamics of resistant clones may alter during periods on and off anti-EGFR antibodies, detecting window of opportunities for a re-challenge with these therapies.

Skeletal muscle loss during anti-EGFR combined chemotherapy regimens predicts poor prognosis in patients with RAS wild metastatic colorectal cancer.

PURPOSE: We aimed to assess whether anti-EGFR combined chemotherapy regimens are related with loss of skeletal muscle mass and to compare cetuximab and panitumumab therapies in the aspect of skeletal muscle area change as well as to assess whether skeletal muscle mass loss has prognostic significance in the RAS wild mCRC patients. MATERIALS AND METHODS: A total of 56 patients (30 patients in cetuximab arm and 26 patients in panitumumab) who had computed tomography images were retrospectively evaluated at the diagnosis and follow up during the treatment period before progression. RESULTS: During treatment period 24 patients (42.8%) had muscle loss. Of these, 7 (29.2%) patients were treated at first-line and 17 (70.8%) patients were treated at second-line setting. There was no significant difference in the aspect of skeletal muscle loss among cetuximab and panitumumab combined treatment regimens. Median PFS was 9.1 (8.6-9.6) months in muscle loss group and 13.9 (7.2-20.6) months in muscle stable group (p = 0.001). Median OS was 23.4 (95% CI 15.8-31.0) months in muscle stable group and 19.1 (95% CI 17.0-21.3) months in muscle loss group (p = 0.57) at first-line setting. For second-line, median OS was 21.2 (14.7-27.7) months in muscle stable group and 14.4 (6.0-22.4) months in muscle loss group (p = 0.003). CONCLUSIONS: Decrease in skeletal muscle mass before progression on CT imaging is an independent indicator for shorter PFS value in RAS WT mCRC patients who received anti-EGFR combined chemotherapy regimens at both the first and second-line settings. Beside that shorter overall survival values also were significantly seen in patients who had muscle loss during anti-EGFR therapy in the second-line setting.

Clinical management of cutaneous adverse events in patients on targeted anticancer therapies and immunotherapies: a national consensus statement by the Spanish Academy of Dermatology and Venereology and the Spanish Society of Medical Oncology.

Progress in the understanding of many tumors has enabled the development of new therapies, such as those targeted at specific molecules involved in cell growth (targeted therapies) or intended to modulate the immune system (immunotherapy). However, along with the clinical benefit provided by these new treatments, new adverse effects have also appeared. Dermatological toxicities such as papulopustular eruptions, xerosis, and pruritus are common with EGFR inhibitors. Other adverse effects have also been described with PDGFR, BCR-ABL, and MAPK tyrosine kinase inhibitors, antiangiogenic drugs, and inhibitors at immune checkpoints such as CTLA-4 and PD-1/PD-L1. Onset of these adverse effects often causes dose reductions and/or delays in administering the prescribed therapy, which can affect patient survival and quality of life. It is, therefore, important to prevent the occurrence of these adverse effects, or to treat unavoidable ones as soon as possible. This requires cooperation between medical oncologists and dermatologists. This article reviews the various dermatological toxicities associated with targeted therapies and immunotherapies, along with their diagnosis and therapeutic management.

AKT can modulate the in vitro response of HNSCC cells to irreversible EGFR inhibitors.

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of HNSCC patients. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib, represents a significant therapeutic promise. In this study, we intend to compare the potential cytotoxicity of two anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The higher AKT phosphorylation level was associated with resistance to anti-EGFR agents. Therefore, we further performed drug combinations with anti-AKT agent (MK2206) and AKT1 gene editing, which demonstrated afatinib and allitinib sensitivity restored. Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab. AKT pathway constitutes a predictive marker of allitinib response and combination with AKT inhibitors could restore response and increase treatment success.

High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.

PURPOSE: Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy. METHODS: Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients' tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression. RESULTS: Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38 months vs 6.79 months; HR 2.77 95% CI 1.2-19.4) and median OS (2.59 months vs 9.82 months; HR 3.0 95% CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression. CONCLUSION: High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy.

RAS mutations vary between lesions in synchronous primary colorectal cancer: testing only one lesion is not sufficient to guide anti-EGFR treatment decisions.

INTRODUCTION: Mutations in KRAS and NRAS genes are negative predictors of anti-EGFR therapies response in metastatic colorectal cancer. There are few reports on RAS testing in synchronous primary colorectal cancer (SP-CRC) and a lack of recommendations on which tissue should be tested for the mutation in this disease. This study analyzed the RAS status of both lesions in SP-CRC patients and in their metastasis. MATERIALS AND METHODS: DNA was obtained from formalin-fixed-paraffin-embedded tissue, and mutations were analyzed by pyrosequencing. RESULTS: RAS status was heterogeneous in 6 (75%) of 8 SP-CRC patients between primary lesions. Five showed heterogeneity regarding RAS mutational status, and from these, four presented with metastasis: 3 cases (75%) had WT metastatic tissue, and 1 case (25%) had mutated metastatic tissue. One patient showed divergence regarding RAS mutation type. DISCUSSION: RAS mutations vary significantly between SP-CRC lesions, and the status of the metastasis is unpredictable. Testing for RAS mutations in only 1 of the primary lesions can misguide clinical decisions and hind the predictive potential of anti-EGFR treatment. A more appropriate approach in metastatic SP-CRC is to test the metastatic tissue or both primary lesions for providing more accurate mutation scenery and support more assertive clinical decisions.

[Clinical-pathological features and gene profile in colorectal cancer]. / Caracteres clínico-patológicos y perfil genético en el carcinoma colorrectal.

Colorectal cancer is the third most frequent cancer in men and the second most frequent in women, with a worldwide incidence of approximately 1.2 million new cases per year. Our primary objective was to study the relationship between clinical and histological features of individuals with colorectal cancer and the mutational status of codons 12 and 13 of the KRAS gene (7 validated mutations), in order to find a histopathological marker to mutated tumors. The secondary objective was to determine how many patients had additional mutations in codons 15 and 61 of the KRAS gene, and codon 600 of the BRAF gene, which could modify the tumor phenotype. Sixty individuals with colorectal cancer (30 wild-type subjects and 30 with validated mutations in codons 12 and 13 of the KRAS gene) were selected. Exons 2 and 3 of the KRAS gene, and exon 15 of the BRAF gene were amplified and sequenced. The data collected were reviewed by a descriptive, univariate and/or multivariate analysis, as appropriate. In conclusion, no relation was found between clinical and histological features of individuals with colorectal cancer and their mutational status for codons 12 and 13 of the KRAS gene. This suggests that those easily available data do not allow predicting the response to anti-EGFR therapy. In patients with advanced colorectal adenocarcinomas and KRAS wild-type status, further study of codon 600 of the BRAF gene could be required.

Caracteres clínico-patológicos y perfil genético en el carcinoma colorrectal / Clinical-pathological features and gene profile in colorectal cancer

El cáncer colorrectal es el tercer cáncer más frecuente en hombres y el segundo más frecuente en mujeres, con una incidencia mundial aproximada de 1.2 millones de casos nuevos por año. Nuestro objetivo primario fue estudiar la relación existente entre las características clínico-histológicas en individuos con cáncer colorrectal y el estado mutacional de los codones 12 y 13 del gen KRAS (7 mutaciones validadas), con el fin de hallar un marcador histopatológico para los tumores mutados. El objetivo secundario fue determinar cuántos pacientes tenían mutaciones adicionales en los codones 15 y 61 del gen KRAS y 600 del gen BRAF que podrían modificar el fenotipo tumoral. Fueron seleccionados 60 individuos con cáncer colorrectal (30 wild-type y 30 con mutaciones validadas en los codones 12 y 13 del gen KRAS). Se amplificaron y secuenciaron del gen KRAS los exones 2 y 3, y del gen BRAF el exón 15. La información recolectada se examinó mediante un análisis descriptivo, análisis univariado y/o análisis multivariado, según correspondiese. En conclusión, no se encontró relación entre las características clínico-histológicas de los tumores de individuos con diagnóstico de cáncer colorrectal y el estado mutacional de los codones 12 y 13 del gen KRAS. No hallamos un marcador histopatológico para los tumores mutados. En pacientes con adenocarcinomas colorrectales avanzados y KRAS wild-type resulta de interés considerar el estudio del codón 600 del gen BRAF.(AU)

Colorectal cancer is the third most frequent cancer in men and the second most frequent in women, with a worldwide incidence of approximately 1.2 million new cases per year. Our primary objective was to study the relationship between clinical and histological features of individuals with colorectal cancer and the mutational status of codons 12 and 13 of the KRAS gene (7 validated mutations), in order to find a histopathological marker to mutated tumors. The secondary objective was to determine how many patients had additional mutations in codons 15 and 61 of the KRAS gene, and codon 600 of the BRAF gene, which could modify the tumor phenotype. Sixty individuals with colorectal cancer (30 wild-type subjects and 30 with validated mutations in codons 12 and 13 of the KRAS gene) were selected. Exons 2 and 3 of the KRAS gene, and exon 15 of the BRAF gene were amplified and sequenced. The data collected were reviewed by a descriptive, univariate and/or multivariate analysis, as appropriate. In conclusion, no relation was found between clinical and histological features of individuals with colorectal cancer and their mutational status for codons 12 and 13 of the KRAS gene. This suggests that those easily available data do not allow predicting the response to anti-EGFR therapy. In patients with advanced colorectal adenocarcinomas and KRAS wild-type status, further study of codon 600 of the BRAF gene could be required.(AU)

Caracteres clínico-patológicos y perfil genético en el carcinoma colorrectal / Clinical-pathological features and gene profile in colorectal cancer

El cáncer colorrectal es el tercer cáncer más frecuente en hombres y el segundo más frecuente en mujeres, con una incidencia mundial aproximada de 1.2 millones de casos nuevos por año. Nuestro objetivo primario fue estudiar la relación existente entre las características clínico-histológicas en individuos con cáncer colorrectal y el estado mutacional de los codones 12 y 13 del gen KRAS (7 mutaciones validadas), con el fin de hallar un marcador histopatológico para los tumores mutados. El objetivo secundario fue determinar cuántos pacientes tenían mutaciones adicionales en los codones 15 y 61 del gen KRAS y 600 del gen BRAF que podrían modificar el fenotipo tumoral. Fueron seleccionados 60 individuos con cáncer colorrectal (30 wild-type y 30 con mutaciones validadas en los codones 12 y 13 del gen KRAS). Se amplificaron y secuenciaron del gen KRAS los exones 2 y 3, y del gen BRAF el exón 15. La información recolectada se examinó mediante un análisis descriptivo, análisis univariado y/o análisis multivariado, según correspondiese. En conclusión, no se encontró relación entre las características clínico-histológicas de los tumores de individuos con diagnóstico de cáncer colorrectal y el estado mutacional de los codones 12 y 13 del gen KRAS. No hallamos un marcador histopatológico para los tumores mutados. En pacientes con adenocarcinomas colorrectales avanzados y KRAS wild-type resulta de interés considerar el estudio del codón 600 del gen BRAF.

Colorectal cancer is the third most frequent cancer in men and the second most frequent in women, with a worldwide incidence of approximately 1.2 million new cases per year. Our primary objective was to study the relationship between clinical and histological features of individuals with colorectal cancer and the mutational status of codons 12 and 13 of the KRAS gene (7 validated mutations), in order to find a histopathological marker to mutated tumors. The secondary objective was to determine how many patients had additional mutations in codons 15 and 61 of the KRAS gene, and codon 600 of the BRAF gene, which could modify the tumor phenotype. Sixty individuals with colorectal cancer (30 wild-type subjects and 30 with validated mutations in codons 12 and 13 of the KRAS gene) were selected. Exons 2 and 3 of the KRAS gene, and exon 15 of the BRAF gene were amplified and sequenced. The data collected were reviewed by a descriptive, univariate and/or multivariate analysis, as appropriate. In conclusion, no relation was found between clinical and histological features of individuals with colorectal cancer and their mutational status for codons 12 and 13 of the KRAS gene. This suggests that those easily available data do not allow predicting the response to anti-EGFR therapy. In patients with advanced colorectal adenocarcinomas and KRAS wild-type status, further study of codon 600 of the BRAF gene could be required.