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Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called “hotspots.” In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.

BACKGROUND: Definitive concomitant cisplatin-based chemoradiotherapy (CRT) is the current gold standard for most patients with advanced stage head and neck squamous cell carcinoma (HNSCC) of the pharynx and larynx. Since previous meta-analysis on CRT outcomes in HNSCC have been reported, advances have been made in radiotherapy techniques and clinical management, while HPV-status has been identified as a strong confounding prognostic factor in oropharyngeal cancer. Here, we present real-world outcome data from a large multicenter cohort of HPV-negative advanced stage HNSCC treated with CRT using contemporary IMRT-based techniques.METHOD: Retrospective data were collected from a multicenter cohort of 513 patients treated with definitive concurrent platinum-based CRT with curative intent between January 2009 and August 2017. Only patients with HPV-negative advanced stage (III-IV) HNSCC were included. A prognostic model for outcome was developed based on clinical parameters and compared to TNM.RESULTS: Nearly half of the 513 patients (49%) had an oropharyngeal tumor, often locally advanced (73.3% T3-T4b) and with involvement of the regional lymph nodes (84%). Most patients (84%) received cisplatin as single agent. 66% received the planned number of cycles and 75% reached a cumulative cisplatin dose of ≥200 mg/m2. Locoregional control was achieved in 324 (63%) patients during follow-up, and no association with tumor sites was observed (p = 0.48). Overall survival at 5 year follow-up was 47%, with a better survival for laryngeal cancer (p = 0.02) compared to other sites. A model with clinical variables (gender, high pre-treatment weight loss, N2c/N3-stage and CONCLUSION: Despite advances in clinical management, more than a third of patients with HPV-negative HNSCC do not complete CRT treatment protocols due to cisplatin toxicity. A model that consists of clinical variables and treatment parameters including cisplatin dose provided the strongest association with overall survival. Since cisplatin toxicity is a major obstacle in completing definitive CRT, the development of alternative and less toxic radiosensitizers is therefore warranted to improve treatment results. The association of RT-boost technique with distant metastasis is an important finding and requires further study.

Psychoneurological symptoms are commonly reported by newly diagnosed head and neck cancer (HNC) patients, yet there is limited research on the associations of these symptoms with biomarkers of stress and inflammation. In this article, pre-treatment data of a multi-center cohort of HNC patients were analyzed using a network analysis to examine connections between symptoms (poor sleep quality, anxiety, depression, fatigue, and oral pain), biomarkers of stress (diurnal cortisol slope), inflammation markers (c-reactive protein [CRP], interleukin [IL]-6, IL-10, and tumor necrosis factor alpha [TNF-α]), and covariates (age and body mass index [BMI]). Three centrality indices were calculated: degree (number of connections), closeness (proximity of a variable to other variables), and betweenness (based on the number of times a variable is located on the shortest path between any pair of other variables). In a sample of 264 patients, poor sleep quality and fatigue had the highest degree index; fatigue and CRP had the highest closeness index; and IL-6 had the highest betweenness index. The model yielded two clusters: a symptoms—cortisol slope—CRP cluster and a IL-6—IL-10—TNF-α—age—BMI cluster. Both clusters were connected most prominently via IL-6. Our findings provide evidence that poor sleep quality, fatigue, CRP, and IL-6 play an important role in the interconnections between psychoneurological symptoms and biomarkers of stress and inflammation in newly diagnosed HNC patients.

This study aimed to assess the prognostic value of intratumoral CD57+ cells in head and neck squamous cell carcinoma (HNSCC) and to examine the reproducibility of these analyses using QuPath. Pretreatment biopsies of 159 patients with HPV-negative, stage III/IV HNSCC treated with chemoradiotherapy were immunohistochemically stained for CD57. The number of CD57+ cells per mm2 tumor epithelium was quantified by two independent observers and by QuPath, software for digital pathology image analysis. Concordance between the observers and QuPath was assessed by intraclass correlation coefficients (ICC). The correlation between CD57 and clinicopathological characteristics was assessed; associations with clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan-Meier curves. The patient cohort had a 3-year OS of 65.8% with a median follow-up of 54 months. The number of CD57+ cells/mm2 tumor tissue did not correlate to OS, DFS, or LRC. N stage predicted prognosis (OS: HR 0.43, p = 0.008; DFS: HR 0.41, p = 0.003; LRC: HR 0.24, p = 0.007), as did WHO performance state (OS: HR 0.48, p = 0.028; LRC: 0.33, p = 0.039). Quantification by QuPath showed moderate to good concordance with two human observers (ICCs 0.836, CI 0.805–0.863, and 0.741, CI 0.692–0.783, respectively). In conclusion, the presence of CD57+ TILs did not correlate to prognosis in advanced stage, HPV-negative HNSCC patients treated with chemoradiotherapy. Substantial concordance between human observers and QuPath was found, confirming a promising future role for digital, algorithm driven image analysis.

Supplementary Figure 4. Growth of shMASTL cells and the representation of BUB1 knockdown.

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers. (Mol Cancer Res 2006;4(2):71–7)

Supplementary Table S5. Characteristics of the patient groups extracted from Wichmann et al.(64) and used in Figure 4. Patients were selected on basis of a negative HPV test result as HPV will confound the prognostic associations. All patients were treated with multimodality therapy regimens with curative intent.

Gene list: bleo comparison

Supplementary figures S1-S4

Supplementary Table 5. Top 10 enriched clusters from phosphoproteomics analyses.

Supplementary Figure 1. PRKDC knockdown induces radiosensitization.

Supplementary Figure Legends. Legends to the Supplementary Figures

Supplementary Figure 3. MASTL knockdown in A549 cells.

Frequency plots of CNAs detected in tumor and precancer samples. (A) A frequency plot of the 22 tumor cell lines. Note that almost all samples contained a loss at chromosome 3p and a gain at 3q. (B) The same plot was made for 12 precancer cultures originating from the margins, including VU-preSCC-M3 (HN439-M1 was excluded). Although the number of samples is low, there is a clear peak visible at chromosome 9p, where CDKN2A is located. (C) A similar plot was made for the five (erythro)leukoplakia cultures. Again the numbers are low, but a trend towards HNSCC-associated CNAs is clearly visible. All graphs represent the frequency of losses and gains in the group of samples, where blue represent gains and red losses.

Supplementary Table 2. Proteins phosporylated to a lower extent in SW1573 MASTL knockdown cells in comparison to SW1573 control cells.

Patients with advanced stage head and neck squamous cell carcinoma (HNSCC) are often treated with cisplatin-containing chemoradiation protocols. Although cisplatin is an effective radiation sensitizer, it causes severe toxicity and not all patients benefit from the combination treatment. HNSCCs expectedly not responding to cisplatin may better be treated with surgery and postoperative radiation or cetuximab and radiation, but biomarkers to personalize chemoradiotherapy are not available. We performed an unbiased genome-wide functional genetic screen in vitro to identify genes that influence the response to cisplatin in HNSCC cells. By siRNA-mediated knockdown, we identified the Fanconi anemia/BRCA pathway as the predominant pathway for cisplatin response in HNSCC cells. We also identified the involvement of the SHFM1 gene in the process of DNA cross-link repair. Furthermore, expression profiles based on these genes predict the prognosis of radiation- and chemoradiation-treated head and neck cancer patients. This genome-wide functional analysis designated the genes that are important in the response of HNSCC to cisplatin and may guide further biomarker validation. Cisplatin imaging as well as biomarkers that indicate the activity of the Fanconi anemia/BRCA pathway in the tumors are the prime candidates. Mol Cancer Ther; 16(3); 540–50. ©2016 AACR.

Figure S3. Visible lesions in non-transplanted FA patients. Visible lesions were found in 27 of 110 patients (25%) with follow-up. Five patients with visible lesions (4 with LOH, 1 without LOH) developed HNSCC.

LOH at chromosome arms 3p, 9p, 11q, and 17p are well-established oncogenetic aberrations in oral precancerous lesions and promising biomarkers to monitor the development of oral cancer. Noninvasive LOH screening of brushed oral cells is a preferable method for precancer detection in patients at increased risk for head and neck squamous cell carcinoma (HNSCC), such as patients with Fanconi anemia. We determined the prevalence of LOH in brushed samples of the oral epithelium of 141 patients with Fanconi anemia and 144 aged subjects, and studied the association between LOH and HNSCC. LOH was present in 14 (9.9%) nontransplanted patients with Fanconi anemia, whereas LOH was not detected in a low-risk group (n = 50, >58 years, nonsmoking/nonalcohol history) and a group with somewhat increased HNSCC risk (n = 94, >58 years, heavy smoking/excessive alcohol use); Fisher exact test, P = 0.023 and P = 0.001, respectively. Most frequent genetic alteration was LOH at 9p. Age was a significant predictor of LOH (OR, 1.13, P = 0.001). Five patients with Fanconi anemia developed HNSCC during the study at a median age of 39.6 years (range, 24.8–53.7). LOH was significantly associated with HNSCC (Fisher exact test, P = 0.000). Unexpectedly, the LOH assay could not be used for transplanted patients with Fanconi anemia because donor DNA in brushed oral epithelium, most likely from donor leukocytes present in the oral cavity, disturbed the analysis. Noninvasive screening using a LOH assay on brushed samples of the oral epithelium has a promising outlook in patients with Fanconi anemia. However, assays need to be adapted in case of stem cell transplantation, because of contaminating donor DNA. Cancer Prev Res; 8(11); 1102–11. ©2015 AACR.

Supplementary Table 4. DNA damage cluster obtained from DAVID analysis on proteins upregulated upon exposure to radiation.

Genetic characterization of the five established tumor cultures. The five tumor cultures we generated contained multiple CNAs, three samples showed a loss of CDKN2A and all contained a mutation in TP53.

Supplementary Table 6. Cell cycle cluster obtained from DAVID analysis on proteins that are less phosphorylated in SW1573 shMASTL cells upon radiation.

Head and neck squamous cell carcinomas (HNSCC) develop in fields of genetically altered cells. These fields are often dysplastic and a subset can be recognized as (erythro)leukoplakia, but most are macroscopically invisible. There is a lack of adequate treatment options to eradicate these fields, whereas they underlie the development of primary tumors as well as part of the local relapses. Unfortunately, there are almost no representative cellular models available to identify suitable treatment options. To this end, clinical biopsy specimens (n = 98) were cultured from normal appearing mucosa of the surgical margins of patients with primary HNSCCs (n = 32) to generate precancer cell culture models. This collection was extended with six previously established precancer cell cultures. Genetic analysis was performed on cultures with an extended life span (≥20 population doublings), the previously established cultures, and some randomly selected cultures. In total, cancer-associated changes were detected in 18 out of 34 (53%) cultures analyzed, which appeared to be independent of life span. A variety of genetic changes were identified, including somatic mutations as well as chromosomal copy-number aberrations (CNA). Loss of CDKN2A/p16Ink4A and mutations in TP53/p53 were most prominent. Remarkably, in some of these precancer cell cultures only chromosomal CNAs were detected, and none of the frequently occurring driver mutations.Implications:The precancer cell cultures, characterized herein, form a representative collection of field models that can be exploited to identify and validate new therapeutic strategies to prevent primary HNSCCs and local relapses.

Supplementary Figure 2. Knockdowns of FOXM1 and MASTL achieved by siRNAs or shRNA constructs.

Supplementary Methods. Supplementary methods describing the materials and methods for the phosphoproteomics.

Supplementary Table 3. Proteins phosporylated to a lower extent after 2Gy of ionizing radiation in SW1573 MASTL knockdown cells in comparison to SW1573 control cells.

Supplementary Table 1. Hit list genome wide siRNA screen

Supplementary tables and figure legends

Legends to supplementary figures S1-S4

Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non–small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle–related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2–M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy. Mol Cancer Ther; 14(6); 1434–44. ©2015 AACR.

Overview of type of changes found in 12 target genes in 89 OL samples. Top: In the left graph the distribution of the classes of mutations is depicted. In the middle graph the type of variants is shown. In the right graph the nucleotide changes are indicated. Bottom: In the left graph the number of mutations per sample is indicated. In the middle graph the number of variant classes found per sample is shown. In the right graph the top 10 mutated genes are depicted. Del = deletion. Ins = insert. SNP = single nucleotide polymorphism. SNV = Single nucleotide variant.

Purpose:Oral leukoplakia is the most common oral potentially malignant disorder with an annual malignant transformation rate of 1% to 5%. Consequently, oral leukoplakia patients have a 30% to 50% lifetime risk to develop oral squamous cell carcinoma. Although risk factors for malignant transformation of oral leukoplakia have been investigated, no definitive risk stratification model has been proposed. Next-generation sequencing can elucidate the genetic landscape of oral leukoplakia, which may be used to predict the risk for malignant transformation.Experimental Design:We investigated a retrospective cohort of 89 oral leukoplakia patients, and analyzed their oral leukoplakia lesions for the presence of genomic copy-number alterations and mutations in genes associated with oral squamous cell carcinoma.Results:In 25 of 89 (28%) patients, oral squamous cell carcinoma developed during follow-up. Seventy-nine of 89 (89%) oral leukoplakias harbored at least one genetic event. Copy-number alterations were present in 61 of 89 (69%) oral leukoplakias, most commonly gains of chromosome regions 8q24 (46%) and 20p11 (20%) and loss of 13q12 (19%). Mutations were present in 59 of 89 (66%) oral leukoplakias, most commonly in TP53 (28%), FAT1 (20%), and NOTCH1 (13%). Genetic data were combined with the presence of dysplasia to generate a prediction model, identifying three groups with a distinct risk for malignant transformation.Conclusions:We provide an extensive description of genetic alterations in oral leukoplakia and its relation to malignant transformation. On the basis of our data we provide a model for the prediction of malignant transformation of oral leukoplakia using dysplasia and genetic markers.

Supplementary Table S1: Per sample sequencing characteristics; Supplementary Table S2: Co-occurrence rates and odds ratios for presence of dysplasia and investigated genomic markers.

Patient characteristics

PDF file - 24K, The genome-wide siRNA screen in SW1573 revealed 293 siRNAs that caused a significant decrease in cell viability.

Purpose: Despite continuous improvement of treatment regimes, the mortality rates for non–small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) remain disappointingly high and novel anticancer agents are urgently awaited.Experimental Design: We combined the data from genome-wide siRNA screens on tumor cell lethality in a lung and a head and neck cancer cell line.Results: We identified 71 target genes that seem essential for the survival of both cancer types. We identified a cluster of 20 genes that play an important role during G2–M phase transition, underlining the importance of this cell-cycle checkpoint for tumor cell survival. Five genes from this cluster (CKAP5, KPNB1, RAN, TPX2, and KIF11) were evaluated in more detail and have been shown to be essential for tumor cell survival in both tumor types, but most particularly in HNSCC. Phenotypes that were observed following siRNA-mediated knockdown of KIF11 (kinesin family member 11) were reproduced by inhibition of KIF11 using the small-molecule inhibitor ispinesib (SB-715992). We showed that ispinesib induces a G2 arrest, causes aberrant chromosome segregation, and induces cell death in HNSCC in vitro, whereas primary keratinocytes are less sensitive. Furthermore, growth of HNSCC cells engrafted in immunodeficient mice was significantly inhibited after ispinesib treatment.Conclusion: This study identified a wide array of druggable genes for both lung and head and neck cancer. In particular, multiple genes involved in the G2–M checkpoint were shown to be essential for tumor cell survival, indicating their potential as anticancer targets. Clin Cancer Res; 19(8); 1994–2003. ©2013 AACR.

PDF file - 3.65 MB

Supplementary Table S4 from Genomic Profiles Associated with Early Micrometastasis in Lung Cancer: Relevance of 4q Deletion

PDF file - 43K, KIF11 protein expression in NSCLC and HNSCC cell lines.

Table with detailed follow-up data of the complete CRT treated patient cohort.

PDF file, 96K, Supplementary Figure S1 First validation of the identified 19 microRNAs that seemed to elicit a tumor-selective lethal effect. Supplementary Figure S2 Effect of the six tumor-selective microRNAs on non-HNSCC cell lines. Supplementary Figure S3 Phenotypic effect of miR-181a overexpression in time, either when introduced by retroviral transduction or transient transfection in HNSCC cell line VU-SCC-120. Supplementary Figure S4 ATM expression after transient transfection of expression constructs with ATM and a kinase-dead ATM mutant lacking the 3'UTR of ATM.