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40 Gene Miseq primers.

Supplementary Methods - PDF file 148K, Contains supplementary methods used in data analysis

Supplementary Tables 1-12 - XLSX file 156K, This file contains Supplementary Tables 1-12. Supplementary Table 1. Clinical information of 12 patients in discovery cohort. Supplementary Table 2. Whole genome sequnecing coverage of uveal melanoma samples. Supplementary Table 3. Ploidy and tumor fraction in uveal melanoma samples. Supplementary Table 4. Predicted somatic structural variants identified by WGS. Supplementary Table 5. List of nonsynonymous mutations in uveal melanoma samples. Supplementary Table 6.. Recurrent mutations / events in uveal melanoma Supplementary Table 7. Clinical features of extension cohort patients. Supplementary Table 8. Effects of SF3B1 mutations on gene expression. Supplementary Table 9. Effects of SF3B1 mutations on exon usage. Supplementary Table 10. The effects of SF3B1 mutations on alternative splicing 1: DEXSeq analysis. Supplementary Table 11. The effects of SF3B1 mutations on alternative splicing 2: MATS analysis. Supplementary Table 12. Primers for qRT-PCR validations

Supplementary Figures S1-S7 - PDF file 4131K, This file contains Supplementary Figures S1-S7. S1 Summary of somatic copy number alterations in uveal melanoma S2 Circos plots of uveal melanoma genomes S3 Distribution of missense mutations in SF3B1 S4 Kaplan-Meier curves for uveal melanoma patients S5 Alternative splicing in Harbour et al. SF3B1 mutant uveal melanomas S6 qRT-PCR of alternative gene splicing in SF3B1 mutant uveal melanoma S7 SF3B1 mutations Harbour et al. uveal melanomas

Supplementary Methods

Supplementary Figure S1. IGV visualization for whole exome sequencing of codon Q61 of NRAS in the pre-treatment tumor vs. the relapsed tumor in patient 1. The red color indicates the A>G for the p.Q61R mutation in the tumor. Supplementary Figure S2. A. IGV visualization for the targeted sequencing of BRAF and PI3KCA in patient 6. B. IGV visualization for the targeted sequencing of BRAF and NRAS in patient 7. Supplementary Figure S3. Response of patient 5 PDX to PLX4720.

Purpose:Cancer susceptibility and mortality are higher in males, and the mutational and transcriptomic landscape of cancer differs by sex. The current assumption is that men are at higher risk of epithelial cancers as they expose more to carcinogens and accumulate more damage than women. We present data showing women present with less aggressive primary cutaneous squamous cell carcinoma (cSCC) and early strong immune activation.Experimental Design:We explored clinical and molecular sexual disparity in immunocompetent and immunosuppressed patients with primary cSCC (N = 738, N = 160), advanced-stage cSCC (N = 63, N = 20) and FVB/N mice exposed to equal doses of DMBA, as well as in human keratinocytes by whole-exome, bulk, and single-cell RNA sequencing.Results:We show cSCC is more aggressive in men, and immunocompetent women develop mild cSCC, later in life. To test whether sex drives disparity, we exposed male and female mice to equal doses of carcinogen, and found males present with more aggressive, metastatic cSCC than females. Critically, females activate cancer immune-related expression pathways and CD4 and CD8 T-cell infiltration independently of mutations, a response that is absent in prednisolone-treated animals. In contrast, males increase the rate of mitosis and proliferation in response to carcinogen. Women's skin and keratinocytes also activate immune-cancer fighting pathways and immune cells at UV radiation–damaged sites. Critically, a compromised immune system leads to high-risk, aggressive cSCC specifically in women.Conclusions:This work shows the immune response is sex biased in cSCC and highlights female immunity offers greater protection than male immunity.

Supplementary Tables 1-6

SF1, SF2 Supp Methods Supp Table Legends

Background: De novo and acquired resistance to HER2 directed therapy is frequently encountered. Upregulation of the phosphatidylinositol-3-kinase (PI3K) pathway is an important mediator of treatment resistance. This can occur through an activating mutation of the PIK3CA gene or PTEN loss. PIK3CA mutations are present in approximately 20% of HER2 positive breast cancers and as such, the PI3K pathway has emerged as an attractive target for restoring sensitivity to HER2 directed therapy. Methods: We performed a single arm, multicentre, open label Phase Ib/II trial. Patients (pts) with advanced HER2-positive breast cancer whose disease had progressed on at least 1 line of Trastuzumab/T-DM1 based treatment in the metastatic setting were eligible if they met following criteria: ECOG PS ≤ 2 and adequate organ function. Pts with treated, controlled brain metastases were permitted to enrol. Exclusion criteria included uncontrolled hypertension or diabetes mellitus. Pts on Phase IB were treated according to a 6+6 study design with a dose escalation schedule of Copanlisib IV (level 1 = 45mg, level 2 = 60mg) on Day 1, 8 and 15 of a 28-day cycle along with a fixed dose of Trastuzumab 2mg/kg weekly. Phase II treatment was the MTD (maximum tolerated dose) of Copanlisib in combination with Trastuzumab. Archival tumour tissue, voluntary biopsies and serial plasma samples were collected for genomic sequencing. Primary endpoints were MTD (Phase I) and clinical benefit rate (CBR) which was defined as complete response (CR) or partial response (PR) at any time point; or stable disease (SD) lasting at least 24 weeks (Phase II). Secondary endpoints included safety and tolerability, tumor response rate, duration of response, time to treatment failure (TTF) and progression free (PFS) and overall survival (OS). Results: Twelve pts were enrolled in Phase IB. No dose limiting toxicity was observed. The MTD was established as Copanlisib 60mg and Trastuzumab 2mg/kg. Fourteen pts were enrolled in Phase II (6 pts treated at the MTD in Phase IB were included in the final Phase II analysis resulting in a total of 20 pts). The median number of lines of prior treatment in the metastatic setting was 3 (1-8). The most common grade 3-4 toxicities encountered in the Phase Ib/II cohorts included hypertension (n=7, 27%), hyperglycaemia (n=2, 8%) and vomiting (n=2, 8%). Three pts discontinued treatment due to toxicity. The median follow-up for the Phase II cohort was 7.5 months (95% CI 6.0-14.5). PR was observed in 4 pts (20%) and SD (at any time point) was seen in 8 pts (40%). The CBR was 30% (n=6). The duration of response was 15.0 weeks (95% CI 4.9 - 16.1). The median TTF was 11.9 weeks (95% CI 7.5 - 21.1). The median PFS was 3.0 mo (95% CI 0.2 - 5.8) and OS was 14.0 mo (95% CI 5.2-22.8). At the time of analysis, 9 of 20 patients were alive. PIK3CA mutations were detectable in the archival tissue of 11 of 26 pts (42%). PIK3CA hotspot mutations (H1047R, E542K and E545K) were detectable in the plasma of all 26 pts at various points throughout treatment. Pre and post treatment biopsies of 2 pts in the Phase IB trial revealed somatic mutations in DNAH3 and TRRAP, the latter of which encodes a PI3K-like protein kinase. Targeted next generation sequencing was performed on the circulating tumour DNA of 20 pts in the Phase II cohort taken before, during and after treatment to further validate these findings and to assess for other mechanisms of response or resistance. The final translational results will be presented at the meeting. Conclusions: The combination of Copanlisib and Trastuzumab is a safe and tolerable regimen and is associated with clinical efficacy in a heavily pre-treated metastatic HER2-positive breast cancer population. Translational studies may have identified novel resistance biomarkers in this pt cohort. Citation Format: Lisa Prior, Niamh M Keegan, Simon J Furney, Janice M Walshe, Giuseppe Gullo, John Crown, M John Kennedy, Diarmuid Smith, John McCaffrey, Catherine M Kelly, Keith Egan, Jennifer Kerr, Mark Given, Niall Sheehy, Peter O'Donovan, Andres Hernando, Ausra Teiserskiene, Imelda Parker, Elaine Kay, Ray McDermott, Maccon M Keane, Seamus O'Reilly, Liam Grogan, Oscar Breathnach, Patrick G Morris, Sinead Toomey, Bryan T Hennessy. Phase Ib/II trial evaluating safety and efficacy of copanlisib (PI3K inhibitor) and trastuzumab in pre-treated advanced HER2-positive breast cancer: Results from the PantHER study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-08.

Background Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. Methods We performed TIL analysis and T-cell analysis by IHC on the pretreatment and ‘On-treatment’ samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. Results In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10–3) but not TILs (p = 0.1) in their ‘On-treatment’ tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p Conclusions The immune system may be ‘primed’ prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Trastuzumab has significantly improved the overall survival of patients with HER2+ metastatic breast cancer (MBC). However, outcomes can vary, with patients progressing within 1 year of treatment or exceptional cases of complete response to trastuzumab for ≥10 years. Identification of the underlying genomic aberrations of “exceptional responders (ExRs)” compared to “rapid non-responders (NRs)” increases our understanding of the mechanisms involved in MBC progression and identification of biomarkers of trastuzumab response and resistance. Whole-exome sequencing was performed on six ExRs compared to five NR. The overall fraction of genome copy number alteration (CNA) burden was higher in NR patients (P = 0.07), while more significantly pronounced in copy number gains (P = 0.03) in NR compared to ExRs. Delineation of the distribution of CNA burden across the genome identified a greater degree of CNA burden in NR within Chr8 (P = 0.02) and in Chr17 (P = 0.06) and conferred a statistically significant benefit in overall survival. Clinical trial number: NCT01722890 [ICORG 12/09].

Mucinous colorectal cancer is a unique histological subtype that is known to respond poorly to cytotoxic chemotherapy and radiotherapy. There are a number of genes known to be associated with resistance to 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The aim of this study was to compare the somatic mutation frequency and copy number variation (CNV) in these genes between mucinous and non-mucinous colorectal cancer. A systematic search of PubMed was performed to identify papers investigating drug resistance in colorectal cancer. From this review, a list of 26 drug-resistance-associated genes was compiled. Using patient data from The Cancer Genome Atlas (TCGA), the somatic mutation rate and CNV was compared between patients with mucinous and non-mucinous colorectal cancer. Statistical analysis was carried out using GraphPad PRISM® version 5.00. Data were available on 531 patients (464 non-mucinous, 67 mucinous). A statistically significant difference in the somatic mutation rate between the two cohorts was identified in the TYMP (p = 0.0179), ATP7B (p = 0.0465), SRPK1 (p = 0.0135), ABCB1 (p = 0.0423), and ABCG2 (p = 0.0102) genes. A statistically significant difference in CNV was identified between the two cohorts in the GSTP1 (p = 0.0405), CCS (p = 0.0063), and TOP1 (p = 0.0048) genes. Differences in somatic mutation rate and CNV in genes associated with resistance to 5-FU, oxaliplatin, and irinotecan may partly account for the pattern of resistance observed in mucinous colorectal cancers. These genetic alterations may prove useful when deciding on a personalized approach to chemotherapy and may also represent potential therapeutic targets going forward.

Background and objectives Mucinous adenocarcinoma is a distinct subtype of colorectal cancer (CRC) with a worse prognosis when compared with non-mucinous adenocarcinoma. The aim of this study was to compare somatic mutations and copy number alteration (CNA) between mucinous and non-mucinous CRC. Methods Data from The Cancer Genome Atlas-colon adenocarcinoma and rectum adenocarcinoma projects were utilized. Mucinous and non-mucinous CRC were compared with regard to microsatellite status, overall mutation rate, the most frequently mutated genes, mutations in genes coding for mismatch repair (MMR) proteins and genes coding for mucin glycoproteins. CNA analysis and pathway analysis was undertaken. Results Mucinous CRC was more likely to be microsatellite instability-high (MSI-H) and hypermutated. When corrected for microsatellite status the single-nucleotide variation and insertion-deletion rate was similar between the two cohorts. Mucinous adenocarcinoma was more likely to have mutations in genes coding for MMR proteins and mucin glycoproteins. Pathway analysis revealed further differences between the two histological subtypes in the cell cycle, RTK-RAS, transforming growth factor-β, and TP53 pathways. Conclusions Mucinous CRC has some distinct genomic aberrations when compared with non-mucinous adenocarcinoma, many of which are driven by the increased frequency of MSI-H tumors. These genomic aberrations may play an important part in the difference seen in response to treatment and prognosis in mucinous adenocarcinoma.

BackgroundMucinous differentiation occurs in 5–15 per cent of colorectal adenocarcinomas. This subtype of colorectal cancer responds poorly to chemoradiotherapy and has a worse prognosis. The genetic aetiology underpinning this cancer subtype lacks consensus. The aim of this study was to use meta-analytical techniques to clarify the molecular associations of mucinous colorectal cancer.MethodsThis study adhered to MOOSE guidelines. Databases were searched for studies comparing KRAS, BRAF, microsatellite instability (MSI), CpG island methylator phenotype (CIMP), p53 and p27 status between patients with mucinous and non-mucinous colorectal adenocarcinoma. A random-effects model was used for analysis.ResultsData from 46 studies describing 17 746 patients were included. Mucinous colorectal adenocarcinoma was associated positively with KRAS (odds ratio (OR) 1·46, 95 per cent c.i. 1·08 to 2·00, P = 0·014) and BRAF (OR 3·49, 2·50 to 4·87; P < 0·001) mutation, MSI (OR 3·98, 3·30 to 4·79; P < 0·001) and CIMP (OR 3·56, 2·85 to 4·43; P < 0·001), and negatively with altered p53 expression (OR 0·46, 0·31 to 0·67; P < 0·001).ConclusionThe genetic origins of mucinous colorectal adenocarcinoma are predominantly associated with BRAF, MSI and CIMP pathways. This pattern of molecular alterations may in part explain the resistance to standard chemotherapy regimens seen in mucinous adenocarcinoma.

Pulmonary enteric adenocarcinoma (PEAC) is a rare variant of lung adenocarcinoma first described in the early 1990s in a lung tumour with overlapping lung and small intestine features. It is a rare tumour with fewer than 300 cases described in the published literature and was only formally classified in 2011. Given these characteristics the diagnosis is challenging, but even more so in a patient with prior gastrointestinal malignancy. A 68-year-old Caucasian female presented with a cough and was found to have a right upper lobe mass. Her history was significant for a pT3N1 colon adenocarcinoma. The resected lung tumour showed invasive lung adenocarcinoma but also features of colorectal origin. Immuno-stains were strongly and diffusely positive for lung and enteric markers. Multi-region, whole-exome sequencing of the mass and archival tissue from the prior colorectal cancer showed distinct genomic signatures with higher mutational burden in the PEAC and very minimal overlap in mutations between the two tumours. This case highlights the challenge of diagnosing rare lung tumours, but more specifically PEAC in a patient with prior gastro-intestinal cancer. Our use of multi-region, next-generation sequencing revealed distinct genomic signatures between the two tumours further supporting our diagnosis, and evidence of PEAC intra-tumour heterogeneity.

Background KIT mutations are primarily associated with acral and mucosal melanoma, and have been reported to show higher prevalence in chronic sun-damaged (CSD) than non-CSD melanomas. Objectives To investigate the prevalence of KIT mutations in melanoma according to subtype, and determine the clinical role of such mutations. Material & methods We present results from a study of a Spanish population of 492 melanomas, classified according to the latest World Health Organization (WHO) guidelines. We analysed the mutational status of KIT and correlated with different clinical variables related to sun exposure and family history. Results KIT mutations were significantly more frequent in acral (3/36; 8.3%) and mucosal (4/8; 50%) melanomas than non-acral cutaneous melanomas. No significant difference was observed in KIT mutational status between CSD and non-CSD melanomas. Conclusion Our results suggest that KIT mutations in melanoma tumours are unrelated to the development of nevi or chronic sun damage, but their presence is associated with aggressive melanomas which show ulceration, vascular invasiveness, and increased Breslow thickness. These findings are consistent with those reported by The Cancer Genome Atlas network.

Introduction Mucinous adenocarcinoma (MA) of the rectum accounts for 5-15% of rectal cancers. This histological subtype has been shown to have a worse response to chemoradiotherapy and worse long term outcomes when compared to non-mucinous adenocarcinoma (NMA). The aim of this study was to compare the genomic landscape of rectal MA to rectal NMA. Method Using the BGISEQ PE100 platform, paired end whole genome sequencing was carried out on tumour and matched normal tissue from cases of MA. The Cancer Genome Atlas was interrogated to identify further cases of rectal MA and cases of rectal NMA with sequencing data that could be used as a control group. In-depth bioinformatic analysis was undertaken and the genomic landscapes of the two subtypes were compared. Result A total of 14 cases of MA were compared to 74 cases of NMA. The microsatellite instability-high rate in the MA group was 14.29% vs 4.05% in the NMA group. POLE mutations were found in 5.41% of the NMA group compared to 0.00% of the MA group. KRAS, BRAF and PIK3CA mutations were more frequent in the MA group whereas TP53 and APC mutations were more common in the NMA group. Significant differences between the groups were identified in the most frequently mutated genes. Copy number alteration, mutational signatures, structural variation and microbiome findings will also be described. Conclusion MA of the rectum is not just phenotypically distinct, it is a distinct genotype. This could have implications when considering neoadjuvant and adjuvant treatment strategies in the future. Take-home message Mucinous adenocarcinoma differs from a genomic perspective from non-mucinous adenocarcinoma and this may have implications for treatment strategies in the future

Introduction The incidence of brain metastases is increasing despite longer survival rates for patients with advanced breast cancer. The identification of novel therapeutic targets for these patients is an urgent unmet clinical need. Sequencing of metastatic tumours have largely focused on mutations however gene fusions have an important, yet underappreciated role in tumorigenesis and disease progression. In this study, we investigate the role of gene fusions in brain metastatic disease and their impact on altered therapeutic responses. Method RNA sequencing was performed on the largest reported cohort of patient matched primary and resected brain metastatic tumours (45 patients n=90 samples). Expressed gene fusions were detected computationally using STAR-Fusion and Arriba. Result We identified differential gene fusion burden in brain metastatic tumours (medium of 58) vs. primary breast tumours (medium of 38) (p Conclusion These results highlight the significant role of gene fusions in breast cancer brain metastases. Abbreviations MAPK Mitogen Activated Protein Kinase, HER Human Epidermal Receptor, CDK12 Cyclin Dependent Kinase 12 Take-home message We highlight the significant role of gene fusions in breast cancer brain metastases and offer specific actionable genomic alterations to be exploited.

Simple Summary Patients with HER-2 positive breast cancer who progress through available HER2-targeted therapy, at present, have few effective treatment options. PIK3CA is mutated in approximately 20% of HER2 positive breast cancers, contributes to HER-2 therapy resistance and may be predictive of response to PI3K inhibitors, including copanlisib. PIK3CA gene mutations were assessed in archival tumour tissue and serially in plasma circulating tumour DNA over the course of treatment with copanlisib. Disease stabilisation (stable disease ≥16 weeks) was seen with copanlisib and trastuzumab in a proportion of participants (n = 6, 50%). PIK3CA mutation detected in archival tumour tissue did not appear to predict tumour response to copanlisib and trastuzumab in this small, heavily pre-treated cohort. Notably, PIK3CA circulating tumour DNA mutations were detected in the plasma of all trial participants, including those who tested negative for the mutation in tissue. This study established a dosing strategy for the novel combination of the PI3K inhibitor copanlisib with trastuzumab and suggested clinical activity for the combination in heavily pre-treated HER-2 positive advanced breast cancer. Further evaluation in a phase 2 study in patients with HER2 therapy resistant tumours is ongoing (NCT02705859). Abstract Background: Activation of the phosphoinositide-3 kinase (PI3K) pathway is a resistance mechanism to anti-human epidermal growth factor receptor 2 (HER2) therapy. This phase Ib trial was conducted to determine the maximum tolerated dose (MTD) of copanlisib, an intravenous (IV) pan-class I PI3K inhibitor, combined with trastuzumab. Methods: Patients with advanced HER2-positive breast cancer and disease progression following at least one prior line of HER2 therapy in the metastatic setting were treated with copanlisib (45 or 60 mg) IV on days 1, 8 and 15 of a 28-day cycle with a fixed dose of trastuzumab 2 mg/kg weekly. Results: Twelve patients were enrolled. The MTD was determined as copanlisib 60 mg plus trastuzumab 2 mg/kg weekly. The most common adverse events of any grade occurring in more than two patients were hyperglycaemia (58%), fatigue (58%), nausea (58%) and hypertension (50%). Stable disease was confirmed at 16 weeks in six participants (50%). PIK3CA mutations were detected in archival tumour of six participants (50%). PIK3CA hotspot mutations, were detectable in pre- and on-treatment plasma of all participants. Pre- and post-treatment tumour biopsies for two patients identified temporal genomic heterogeneity, somatic mutations in the TRRAP gene, which encodes a PI3K-like protein kinase, and emergent somatic mutations related to protein kinase signalling. Conclusion: Copanlisib and trastuzumab can be safely administered with fair overall tolerability. Preliminary evidence of tumour stability was observed in patients with heavily pre-treated, metastatic HER2 positive breast cancer. Several potential biomarkers were identified for further study in the current phase 2 clinical trial. NCT: 02705859.

BackgroundMany cancer types display sex and age disparity in incidence and outcome. The mutational load of tumours, including melanoma, varies according to sex and age. However, there are no tools to systematically explore if clinical variables such as age and sex determine the genomic landscape of cancer.ObjectivesTo establish a mathematical approach using melanoma mutational data to analyze how sex and age shape the tumour genome.MethodsWe model how age‐related (clock‐like) somatic mutations that arise during cell division, and extrinsic (environmental ultraviolet light) mutations accumulate in cancer genomes.ResultsMelanoma is primarily driven by cell‐intrinsic age‐related mutations and extrinsic ultraviolet light‐induced mutations, and we show these mutation types differ in magnitude, chronology and by sex in the distinct molecular melanoma subtypes. Our model confirms age and sex are determinants of cellular mutation rate, shaping the final mutation composition. We show mathematically for the first time how, similar to non‐cancer tissues, melanoma genomes reflect a decline in cell division during ageing. We find clock‐like mutations strongly correlate with the acquisition of ultraviolet light‐induced mutations, but critically, males present a higher number and rate of cell division‐linked mutations.ConclusionsThese data indicate the contribution of environmental damage to melanoma likely extends beyond genetic damage to affect cell division. Sex and age determine the final mutational composition of melanoma.

Purpose Cancer susceptibility and mortality are higher in males, and the mutational and transcriptomic landscape of cancer differs by sex. The current assumption is that men are at higher risk of epithelial cancers as they expose more to carcinogens and accumulate more damage than women. We present data showing women are more protected from aggressive cutaneous squamous cell carcinoma (cSCC) due to strong immune activation. Methods We explored clinical and molecular sexual disparity in immunocompetent and immunosuppressed patients (N= 738, N=160) with carcinoma cSCC, in FVB/N mice exposed to equal doses of DMBA, and in human keratinocytes by whole exome sequencing, bulk and single cell RNA sequencing. Results We show cSCC is more aggressive in men, and immunocompetent women develop mild cSCC, later in life. To test if sex drives disparity, we exposed male and female mice to equal doses of carcinogen, and found males present more aggressive, metastatic cSCC than females. Critically, females activate cancer immune-related expression pathways and CD4 and CD8 T cell infiltration independently of mutations. In contrast, males increase the rate of mitoses and proliferation in response to carcinogen. Human female skin and keratinocytes also activate immune-cancer fighting pathways and immune cells at ultraviolet radiation-damaged sites. Critically, a compromised immune system leads to high-risk, aggressive cSCC specifically in women. Conclusions This work shows the immune response is sex biased in cSCC, and highlights female immunity offers greater protection than male immunity. Translational relevance Sex bias affects cancer incidence, mortality and therapy response; and the molecular landscape of cancer differs by sex. However, it is not known if the sex discrepancy is due to a difference in behaviour and exposure to carcinogens, or due to sex-linked susceptibility. This work reveals men are inherently more susceptible to cutaneous aggressive squamous cell carcinoma, in contrast to women who activate stronger immune responses when challenged with the same carcinogens. The loss of immunity particularly affects women. Personalised medicine approaches stratify cancer patients by genotype; however, to date, the potential for cancer stratification, prevention strategies and therapy by sex and immune competency has not been explored. These data indicate men require targeted prevention programs and increased monitoring. Furthermore, we provide a rationale to prioritise men and immunosuppressed women for adjuvant therapy and immunotherapy.

Synchronous colorectal cancers (syCRCs) are two or more primary tumours identified simultaneously in a patient. Previous studies report high inter-tumour heterogeneity between syCRCs, suggesting independent origin and different treatment response, making their management particularly challenging, with no specific guidelines currently in place. Here, we performed in-depth bioinformatic analyses of genomic and transcriptomic data of a total of eleven syCRCs and one metachronous CRC collected from three patients. We found mixed microsatellite status between and within patients. Overlap of mutations between synchronous tumours was consistently low (Fusobacterium nucleatum species in patients with MSI tumours, while quantification of tumour immune infiltration showed varying immune responses between syCRCs. Our results suggest high heterogeneity of syCRCs within patients but find clinically actionable biomarkers that help predict responses to currently available targeted therapies. Our study highlights the importance of personalised genome and transcriptome sequencing of all synchronous lesions to aid therapy decision and improve management of syCRC patients.

Background: Trastuzumab (T) has shown clinical efficacy in early-stage and MBC. However, within 1-year 40-50% develop resistance to T. The exact mechanism of the development of T resistance is not completely understood. Anecdotal observations suggest that a small fraction of patients with HER2+ MBC may be "extraordinary durable complete responders (ExdCR)". Indeed, we previously reported that 9% of MBC achieve dCR following T and chemotherapy. Understanding the genomic mechanisms underlying exceptional dCR to T may improve patient selection and treatment rationale to identify HER2+ MBC pts who are more likely to achieve dCR following T treatment. Methods: Genomic DNA was extracted from paraffin embedded formalin fixed (FFPE) tissue. Whole exome sequencing (WES) on primary tumours from 9 MBC ExdCR > 60 mo (5 matched T:N) and 6 non-responders (NR) or PR < 6 mo (3 matched T:N). Tumours were analysed for single nucleotide variants (SNVs) point mutations, insertions/deletions (indels), copy number alterations (CNA), and tumour mutational burden. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of matched tumour:normal samples revealed differences in SNVs and indels between the ExdCR pts compared to NR. Mutations in TP53 were found in 2/5 ExdCR pts and in 0/3 NR. Initial analysis of CNA revealed that HER2 is significantly more amplified in ExdCR pts compared to NR, and this was also shown by IHC and FISH. Conclusions: We present a genomic landscape of extraordinary durable complete responders compared to non-responders using WES. High variability exists in mutation profile of ExdCR pts with few overlapping genes. Further analysis into clinically relevant genomic and molecular alterations will be performed to potential aid in patient selection and choice of therapy, and novel drug targets. Citation Format: Walsh N, Gullo G, Quinn C, Furney SJ, Crown J. Whole exome sequencing of HER2+ metastatic breast cancer (MBC) patients (pts) with extraordinary durable complete responses (ExdCR) to trastuzumab (T) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-13.

Standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (NACRT), followed by surgical resection. However, &gt, 70% of patients do not achieve a complete pathological response and have higher rates of relapse and death. There are no validated pre- or on-treatment factors that predict response to NACRT besides tumour stage and size. We characterised the response of 33 LARC patients to NACRT, collected tumour samples from patients prior to, during and after NACRT, and performed whole exome, transcriptome and high-depth targeted sequencing. The pre-treatment LARC genome was not predictive of response to NACRT. However, in line with the increasing recognition of microbial influence in cancer, RNA analysis of pre-treatment tumours suggested a greater abundance of Fusobacteria in intermediate and poor responders. In addition, we investigated tumour heterogeneity and evolution in response to NACRT. While matched pre-treatment, on-treatment and post-treatment tumours revealed minimal genome evolution overall, we identified cases in which microsatellite instability developed or was selected for during NACRT. Recent research has suggested a role for adaptive mutability to targeted therapy in colorectal cancer cells. We provide preliminary evidence of selection for mismatch repair deficiency in response to NACRT. Furthermore, pre-NACRT genomic landscapes do not predict treatment response but pre-NACRT microbiome characteristics may be informative.

Many cancer types display sex and age disparity in incidence and outcome. Here, we establish a mathematical approach using cancer mutational data to analyze how sex and age shape the tumour genome. We model how age-related (clock-like) somatic mutations that arise during cell division, and extrinsic (environmental) mutations accumulate in cancer genomes. As a proof-of-concept, we apply our approach to melanoma, a cancer driven by cell-intrinsic age-related mutations and extrinsic ultraviolet light-induced mutations, and show these mutation types differ in magnitude, chronology and by sex in the distinct molecular melanoma subtypes.Our model confirms age and sex are determinants of cellular mutation rate, shaping the final mutation composition. We show mathematically for the first time how, similar to non-cancer tissues, melanoma genomes reflect a decline in cell division during ageing. We find clock-like mutations strongly correlate with the acquisition of ultraviolet light-induced mutations, but critically, males present a higher number and rate of cell division-linked mutations. These data indicate the contribution of environmental damage to melanoma likely extends beyond genetic damage to affect cell division.

Background & Aims: KH-type splicing regulatory protein (KHSRP) is a multifunctional nucleic acid binding protein. KHSRP regulates transcription, mRNA decay and translation, and miRNA biogenesis. These distinct functions are associated with key aspects of cancer cell biology: inflammation and cell-fate determination. However, the role KHSRP plays in colorectal cancer (CRC) tumorigenesis remains largely unknown. Methods: We investigated the oncogenic role of KHSRP in CRC using a combination of in silico analysis of large datasets, ex vivo analysis of protein expression in patient samples, and mechanistic studies using in vitro models of CRC. Results: KHSRP was expressed in the epithelial and stromal compartments of both primary and metastatic tumors. Elevated KHSRP expression was found in tumor versus matched normal tissue in a cohort of 62 patients. This finding was validated in larger independent cohorts in silico. KHSRP expression was a prognostic indicator of worse overall survival (HR=3.74, 95% CI = 1.43-22.97, p=0.0138). Mechanistic data in CRC cell line models supported a role of KHSRP in driving epithelial cell proliferation in both a primary and metastatic setting, through control of the G1/S transition. Additionally, epithelial KHSRP was involved in promoting a pro-angiogenic extracellular environment, as well as regulating the secretion of oncogenic proteins involved in diverse cellular processes such as migration and response to cellular stress. Conclusion: Our study has uncovered novel mechanistic-based data on the tumor- promoting effects of KHSRP in CRC.

Mucinous colorectal cancer is a unique histological subtype that is known to respond poorly to cytotoxic chemotherapy and radiotherapy. There are a number of genes known to be associated with resistance to 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The aim of this study was to compare the somatic mutation frequency and copy number variation (CNV) in these genes between mucinous and non-mucinous colorectal cancer. A systematic search of PubMed was performed to identify papers investigating drug resistance in colorectal cancer. From this review, a list of 26 drug-resistance-associated genes was compiled. Using patient data from The Cancer Genome Atlas (TCGA), the somatic mutation rate and CNV was compared between patients with mucinous and non-mucinous colorectal cancer. Statistical analysis was carried out using GraphPad PRISM® version 5.00. Data were available on 531 patients (464 non-mucinous, 67 mucinous). A statistically significant difference in the somatic mutation rate between the two cohorts was identified in the TYMP (p = 0.0179), ATP7B (p = 0.0465), SRPK1 (p = 0.0135), ABCB1 (p = 0.0423), and ABCG2 (p = 0.0102) genes. A statistically significant difference in CNV was identified between the two cohorts in the GSTP1 (p = 0.0405), CCS (p = 0.0063), and TOP1 (p = 0.0048) genes. Differences in somatic mutation rate and CNV in genes associated with resistance to 5-FU, oxaliplatin, and irinotecan may partly account for the pattern of resistance observed in mucinous colorectal cancers. These genetic alterations may prove useful when deciding on a personalized approach to chemotherapy and may also represent potential therapeutic targets going forward.

The treatment landscape for metastatic melanoma has been revolutionised by the introduction of immunotherapy and targeted therapies. Despite these advances, some patients exhibit primary or acquired resistance to treatment. We present the case of a resected mucosal melanoma that on relapse underwent transformation to a dedifferentiated state. The relapsed tumour was phenotypically disparate and demonstrated loss of all typical melanoma-associated immunohistochemical markers. Furthermore, it demonstrated aggressive biological behaviour and immunotherapy resistance. We performed genomic profiling of the original and relapsed tumour to further elucidate the mechanisms underlying this rare phenomenon. Mass spectrometry-based single-nucleotide polymorphism genotyping technology was used to screen for mutations in the original and recurrent tumour. Whole-exome sequencing was performed on the original tumour, recurrent tumour and blood. Both the original and recurrent tumour shared a NRAS mutation, a similar aneuploidy profile and proportion of somatic single-nucleotide variants. However, in contrast to the original tumour, the recurrent tumour demonstrated a lower mutational burden and deletions in the CDKN2A/CDKN2B and CHEK2 genes. The genomic similarity between the original and recurrent tumour attests to a common ancestry and the possible existence of nongenomic drivers inciting phenotype plasticity. In contrast, the low mutational load and potential inactivation of tumour suppressor genes in the recurrent tumour may underlie its rapid proliferative rate and immunoresistance. Dynamic treatment models are desired in the future to track the genomic and epigenetic evolution of a tumour to guide optimal therapy choice and sequencing.

Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell–derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. Significance: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments. Cancer Discov; 6(3); 286–99. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 217

BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains < 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR < 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p < 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.

Background: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. Methods: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. Results: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status. Conclusions: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.

The comprehensive identification of mutations contributing to the development of cancer is a priority of large cancer sequencing projects. To date, most studies have scrutinized mutations in coding regions of the genome, but several recent discoveries, including the identification of recurrent somatic mutations in the TERT promoter in multiple cancer types, support the idea that mutations in non-coding regions are also important in tumour development. Furthermore, analysis of whole-genome sequencing data from tumours has elucidated novel mutational patterns and processes etched into cancer genomes. Here, we present an overview of insights gleaned from the analysis of mutations from sequenced cancer genomes. We then review the mechanisms by which non-coding mutations can play a role in cancer. Finally, we discuss recent efforts aimed at identifying non-coding driver mutations, as well as the unique challenges that the analysis of non-coding mutations present in contrast to the identification of driver mutations in coding regions.

Ongoing advances in massively parallel sequencing technologies have changed the analysis of cancer genomes dramatically. Tumor sequencing studies have created an unprecedented amount of oncogenomic data and the identification of driver mutations and genes contributing to tumor development in sequenced samples is pivotal. Here we present an overview of the methods used to identify driver genes and mutations in coding and noncoding regions of the genome, and we consider the mutational processes acting in tumor genomes that confound these approaches.

We used whole-exome sequencing to identify variants other than APOE associated with the rate of hippocampal atrophy in amnestic mild cognitive impairment. An in-silico predicted missense variant in REST (rs3796529) was found exclusively in subjects with slow hippocampal volume loss and validated using unbiased whole-brain analysis and meta-analysis across 5 independent cohorts. REST is a master regulator of neurogenesis and neuronal differentiation that has not been previously implicated in Alzheimer's disease. These findings nominate REST and its functional pathways as protective and illustrate the potential of combining next-generation sequencing with neuroimaging to discover novel disease mechanisms and potential therapeutic targets.

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear. The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event. To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a BRAF(V600E) mouse model. In mice expressing BRAF(V600E) in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden. Here we show that sunscreen (UVA superior, UVB sun protection factor (SPF) 50) delayed the onset of UVR-driven melanoma, but only provided partial protection. The UVR-exposed tumours showed increased numbers of single nucleotide variants and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in approximately 40% of cases. TP53 is an accepted UVR target in human non-melanoma skin cancer, but is not thought to have a major role in melanoma. However, we show that, in mice, mutant Trp53 accelerated BRAF(V600E)-driven melanomagenesis, and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans. Furthermore, we identify TP53/Trp53 as a UVR-target gene that cooperates with BRAF(V600E) to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis. Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

We used a combination of whole-genome sequencing and in vitro validation to show that mutations that activated at least two pro-growth/survival pathways mediated intrinsic resistance to BRAF inhibition in a melanoma patient. These data demonstrate how in-depth analysis can reveal intrinsic resistance to standard of care, providing an opportunity for alternative therapeutic strategies for patients who are likely to fail first-line treat-575 ment., Background BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAFV600 mutations. Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond. Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance. Methods We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient. We validated mechanisms of resistance in a cell line derived from the patient's tumour. Results We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites. We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours. A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway. Inhibition of both pathways synergised to block the growth of the cells. Conclusions Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination. We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient. These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered. Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.