Spatial genomics maps the structure, nature and evolution of cancer clones.

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour1-3. Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive4,5. Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology.

High-throughput molecular assays for inclusion in personalised oncology trials - State-of-the-art and beyond.

In the last decades, the development of high-throughput molecular assays has revolutionised cancer diagnostics, paving the way for the concept of personalised cancer medicine. This progress has been driven by the introduction of such technologies through biomarker-driven oncology trials. In this review, strengths and limitations of various state-of-the-art sequencing technologies, including gene panel sequencing (DNA and RNA), whole-exome/whole-genome sequencing and whole-transcriptome sequencing, are explored, focusing on their ability to identify clinically relevant biomarkers with diagnostic, prognostic and/or predictive impact. This includes the need to assess complex biomarkers, for example microsatellite instability, tumour mutation burden and homologous recombination deficiency, to identify patients suitable for specific therapies, including immunotherapy. Furthermore, the crucial role of biomarker analysis and multidisciplinary molecular tumour boards in selecting patients for trial inclusion is discussed in relation to various trial concepts, including drug repurposing. Recognising that today's exploratory techniques will evolve into tomorrow's routine diagnostics and clinical study inclusion assays, the importance of emerging technologies for multimodal diagnostics, such as proteomics and in vivo drug sensitivity testing, is also discussed. In addition, key regulatory aspects and the importance of patient engagement in all phases of a clinical trial are described. Finally, we propose a set of recommendations for consideration when planning a new precision cancer medicine trial.

IMPRESS-Norway: improving public cancer care by implementing precision medicine in Norway; inclusion rates and preliminary results.

BACKGROUND AND PURPOSE: In Norway, comprehensive molecular tumour profiling is implemented as part of the public healthcare system. A substantial number of tumours harbour potentially targetable molecular alterations. Therapy outcomes may improve if targeted treatments are matched with actionable genomic alterations. In the IMPRESS-Norway trial (NCT04817956), patients are treated with drugs outside the labelled indication based on their tumours molecular profile. PATIENTS AND METHODS: IMPRESS-Norway is a national, prospective, non-randomised, precision cancer medicine trial, offering treatment to patients with advanced-stage disease, progressing on standard treatment. Comprehensive next-generation sequencing, TruSight Oncology 500, is used for screening. Patients with tumours harbouring molecular alterations with matched targeted therapies available in IMPRESS-Norway, are offered treatment. Currently, 24 drugs are available in the study. Primary study endpoints are percentage of patients offered treatment in the trial, and disease control rate (DCR) defined as complete or partial response or stable disease in evaluable patients at 16 weeks (W16) of treatment. Secondary endpoint presented is DCR in all treated patients. RESULTS: Between April 2021 and October 2023, 1,167 patients were screened, and an actionable mutation with matching drug was identified for 358 patients. By the data cut off 186 patients have initiated treatment, 170 had a minimum follow-up time of 16 weeks, and 145 also had evaluable disease. In patients with evaluable disease, the DCR was 40% (58/145). Secondary endpoint analysis of DCR in all treated patients, showed DCR of 34% (58/170). INTERPRETATION: Precision cancer medicine demonstrates encouraging clinical effect in a subset of patients included in the IMPRESS-Norway trial.

PCM4EU and PRIME-ROSE: Collaboration for implementation of precision cancer medicine in Europe.

BACKGROUND: In the two European Union (EU)-funded projects, PCM4EU (Personalized Cancer Medicine for all EU citizens) and PRIME-ROSE (Precision Cancer Medicine Repurposing System Using Pragmatic Clinical Trials), we aim to facilitate implementation of precision cancer medicine (PCM) in Europe by leveraging the experience from ongoing national initiatives that have already been particularly successful. PATIENTS AND METHODS: PCM4EU and PRIME-ROSE gather 17 and 24 partners, respectively, from 19 European countries. The projects are based on a network of Drug Rediscovery Protocol (DRUP)-like clinical trials that are currently ongoing or soon to start in 11 different countries, and with more trials expected to be established soon. The main aims of both the projects are to improve implementation pathways from molecular diagnostics to treatment, and reimbursement of diagnostics and tumour-tailored therapies to provide examples of best practices for PCM in Europe. RESULTS: PCM4EU and PRIME-ROSE were launched in January and July 2023, respectively. Educational materials, including a podcast series, are already available from the PCM4EU website (http://www.pcm4eu.eu). The first reports, including an overview of requirements for the reimbursement systems in participating countries and a guide on patient involvement, are expected to be published in 2024. CONCLUSION: PCM4EU and PRIME-ROSE were launched in January and July 2023, respectively. Educational materials, including a podcast series, are already available from the PCM4EU website (http://www.pcm4eu.eu). The first reports, including an overview of requirements for the reimbursement systems in participating countries and a guide on patient involvement, are expected to be published in 2024. CONCLUSION: European collaboration can facilitate the implementation of PCM and thereby provide affordable and equitable access to precision diagnostics and matched therapies for more patients.

Incidence of breast cancer subtypes in immigrant and non-immigrant women in Norway.

BACKGROUND: Breast cancer incidence differs between non-immigrants and immigrants from low- and middle-income countries. This study investigates whether immigrants also have different subtype-specific incidences. METHODS: We used national health registries in Norway and calculated subtype-specific incidence rate ratios (IRRs) for invasive breast cancer among women aged 20-75 and 20-49 years between 2005 and 2015. Immigrant groups were classified by country of birth broadly defined based on WHO regional groupings. Subtype was defined using estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) status as luminal A-like (ER+ PR+ HER2-), luminal B-like/HER2- (ER+ PR- HER2-), luminal B-like/HER2+ (ER+ PR any HER2+), HER2+ (ER-PR-HER2+) and triple-negative breast cancer (TNBC) (ER-PR-HER2-). RESULTS: Compared to non-immigrants, incidence of the luminal A-like subtype was lower in immigrants from Sub-Saharan Africa (IRR 0.43 95% CI 0.28-0.66), South East Asia (IRR 0.63 95% CI 0.51-0.79), South Asia (IRR 0.67 95% CI 0.52-0.86) and Eastern Europe (IRR 0.86 95% CI 0.76-0.99). Immigrants from South Asia had higher rates of HER2 + tumors (IRR 2.02 95% CI 1.26-3.23). The rates of TNBC tended to be similar regardless of region of birth, except that women from South East Asia had an IRR of 0.54 (95% CI 0.32-0.91). CONCLUSIONS: Women from Eastern Europe, Sub-Saharan Africa and Asia had different subtype-specific incidences compared to women from high-income countries (including non-immigrants). These differences in tumor characteristics between immigrant groups should be taken into consideration when planning preventive or screening strategies.

Ipilimumab in a real-world population: A prospective Phase IV trial with long-term follow-up.

Ipilimumab was the first treatment that improved survival in advanced melanoma. Efficacy and toxicity in a real-world setting may differ from clinical trials, due to more liberal eligibility criteria and less intensive monitoring. Moreover, high costs and lack of biomarkers have raised cost-benefit concerns about ipilimumab in national healthcare systems and limited its use. Here, we report the prospective, interventional study, Ipi4 (NCT02068196), which aimed to investigate the toxicity and efficacy of ipilimumab in a real-world population with advanced melanoma. This national, multicentre, phase IV trial included 151 patients. Patients received ipilimumab 3 mg/kg intravenously and were followed for at least 5 years or until death. Treatment interruption or cessation occurred in 38%, most frequently due to disease progression (19%). Treatment-associated grade 3 to 4 toxicity was observed in 28% of patients, and immune-related toxicity in 56%. The overall response rate was 9%. Median overall survival was 12.1 months (95% CI: 8.3-15.9); and progression-free survival 2.7 months (95% CI: 2.6-2.8). After 5 years, 20% of patients were alive. In a landmark analysis from 6 months, improved survival was associated with objective response (HR 0.16, P = .001) and stable disease (HR 0.49, P = .005) compared to progressive disease. Poor performance status, elevated lactate dehydrogenase and C-reactive protein were identified as biomarkers. This prospective trial represents the longest reported follow-up of a real-world melanoma population treated with ipilimumab. Results indicate safety and efficacy comparable to phase III trials and suggest that the use of ipilimumab can be based on current cost-benefit estimates.

Improving public cancer care by implementing precision medicine in Norway: IMPRESS-Norway.

BACKGROUND: Matching treatment based on tumour molecular characteristics has revolutionized the treatment of some cancers and has given hope to many patients. Although personalized cancer care is an old concept, renewed attention has arisen due to recent advancements in cancer diagnostics including access to high-throughput sequencing of tumour tissue. Targeted therapies interfering with cancer specific pathways have been developed and approved for subgroups of patients. These drugs might just as well be efficient in other diagnostic subgroups, not investigated in pharma-led clinical studies, but their potential use on new indications is never explored due to limited number of patients. METHODS: In this national, investigator-initiated, prospective, open-label, non-randomized combined basket- and umbrella-trial, patients are enrolled in multiple parallel cohorts. Each cohort is defined by the patient's tumour type, molecular profile of the tumour, and study drug. Treatment outcome in each cohort is monitored by using a Simon two-stage-like 'admissible' monitoring plan to identify evidence of clinical activity. All drugs available in IMPRESS-Norway have regulatory approval and are funded by pharmaceutical companies. Molecular diagnostics are funded by the public health care system. DISCUSSION: Precision oncology means to stratify treatment based on specific patient characteristics and the molecular profile of the tumor. Use of targeted drugs is currently restricted to specific biomarker-defined subgroups of patients according to their market authorization. However, other cancer patients might also benefit of treatment with these drugs if the same biomarker is present. The emerging technologies in molecular diagnostics are now being implemented in Norway and it is publicly reimbursed, thus more cancer patients will have a more comprehensive genomic profiling of their tumour. Patients with actionable genomic alterations in their tumour may have the possibility to try precision cancer drugs through IMPRESS-Norway, if standard treatment is no longer an option, and the drugs are available in the study. This might benefit some patients. In addition, it is a good example of a public-private collaboration to establish a national infrastructure for precision oncology. Trial registrations EudraCT: 2020-004414-35, registered 02/19/2021; ClinicalTrial.gov: NCT04817956, registered 03/26/2021.

Cost-effectiveness of molecularly matched off-label therapies for end-stage cancer - the MetAction precision medicine study.

BACKGROUND: Precision cancer medicine (PCM), frequently used for the expensive and often modestly efficacious off-label treatment with medications matched to the tumour genome of end-stage cancer, challenges healthcare resources. We compared the health effects, costs and cost-effectiveness of our MetAction PCM study with corresponding data from comparator populations given best supportive care (BSC) in two external randomised controlled trials. METHODS: We designed three partitioned survival models to evaluate the healthcare costs and quality-adjusted life years (QALYs) as the main outcomes. Cost-effectiveness was calculated as the incremental cost-effectiveness ratio (ICER) of PCM relative to BSC with an annual willingness-to-pay (WTP) threshold of EUR 56,384 (NOK 605,000). One-way and probabilistic sensitivity analyses addressed uncertainty. RESULTS: We estimated total healthcare costs (relating to next-generation sequencing (NGS) equipment and personnel wages, molecularly matched medications to the patients with an actionable tumour target and follow-up of the responding patients) and the health outcomes for the MetAction patients versus costs (relating to estimated hospital admission) and outcomes for the BSC cases. The ICERs for incremental QALYs were twice or more as high as the WTP threshold and relatively insensitive to cost decrease of the NGS procedures, while reduction of medication prices would contribute significantly towards a cost-effective PCM strategy. CONCLUSIONS: The models suggested that the high ICERs of PCM were driven by costs of the NGS diagnostics and molecularly matched medications, with a likelihood for the strategy to be cost-effective defying WTP constraints. Reducing drug expenses to half the list price would likely result in an ICER at the WTP threshold. This can be an incentive for a public-private partnership for sharing drug costs in PCM, exemplified by ongoing European initiatives. CLINICALTRIALS.GOV, IDENTIFIER: NCT02142036.

In modern times, how important are breast cancer stage, grade and receptor subtype for survival: a population-based cohort study.

BACKGROUND: In breast cancer, immunohistochemistry (IHC) subtypes, together with grade and stage, are well-known independent predictors of breast cancer death. Given the immense changes in breast cancer treatment and survival over time, we used recent population-based data to test the combined influence of IHC subtypes, grade and stage on breast cancer death. METHODS: We identified 24,137 women with invasive breast cancer aged 20 to 74 between 2005 and 2015 in the database of the Cancer Registry of Norway. Kaplan-Meier curves, mortality rates and adjusted hazard ratios for breast cancer death were estimated by IHC subtypes, grade, tumour size and nodal status during 13 years of follow-up. RESULTS: Within all IHC subtypes, grade, tumour size and nodal status were independent predictors of breast cancer death. When combining all prognostic factors, the risk of death was 20- to 40-fold higher in the worst groups compared to the group with the smallest size, low grade and ER+PR+HER2- status. Among node-negative ER+HER2- tumours, larger size conferred a significantly increased breast cancer mortality. ER+PR-HER2- tumours of high grade and advanced stage showed particularly high breast cancer mortality similar to TNBC. When examining early versus late mortality, grade, size and nodal status explained most of the late (> 5 years) mortality among ER+ subtypes. CONCLUSIONS: There is a wide range of risks of dying from breast cancer, also across small breast tumours of low/intermediate grade, and among node-negative tumours. Thus, even with modern breast cancer treatment, stage, grade and molecular subtype (reflected by IHC subtypes) matter for prognosis.

Whole genome copy number analyses reveal a highly aberrant genome in TP53 mutant lung adenocarcinoma tumors.

BACKGROUND: Genetic alterations are common in non-small cell lung cancer (NSCLC), and DNA mutations and translocations are targets for therapy. Copy number aberrations occur frequently in NSCLC tumors and may influence gene expression and further alter signaling pathways. In this study we aimed to characterize the genomic architecture of NSCLC tumors and to identify genomic differences between tumors stratified by histology and mutation status. Furthermore, we sought to integrate DNA copy number data with mRNA expression to find genes with expression putatively regulated by copy number aberrations and the oncogenic pathways associated with these affected genes. METHODS: Copy number data were obtained from 190 resected early-stage NSCLC tumors and gene expression data were available from 113 of the adenocarcinomas. Clinical and histopathological data were known, and EGFR-, KRAS- and TP53 mutation status was determined. Allele-specific copy number profiles were calculated using ASCAT, and regional copy number aberration were subsequently obtained and analyzed jointly with the gene expression data. RESULTS: The NSCLC tumors tissue displayed overall complex DNA copy number profiles with numerous recurrent aberrations. Despite histological differences, tissue samples from squamous cell carcinomas and adenocarcinomas had remarkably similar copy number patterns. The TP53-mutated lung adenocarcinomas displayed a highly aberrant genome, with significantly altered copy number profiles including gains, losses and focal complex events. The EGFR-mutant lung adenocarcinomas had specific arm-wise aberrations particularly at chromosome7p and 9q. A large number of genes displayed correlation between copy number and expression level, and the PI(3)K-mTOR pathway was highly enriched for such genes. CONCLUSIONS: The genomic architecture in NSCLC tumors is complex, and particularly TP53-mutated lung adenocarcinomas displayed highly aberrant copy number profiles. We suggest to always include TP53-mutation status when studying copy number aberrations in NSCLC tumors. Copy number may further impact gene expression and alter cellular signaling pathways.

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Molecularly matched therapy in the context of sensitivity, resistance, and safety; patient outcomes in end-stage cancer - the MetAction study.

Background: In precision cancer medicine, the challenge is to prioritize DNA driver events, account for resistance markers, and procure sufficient information for treatment that maintains patient safety. The MetAction project, exploring how tumor molecular vulnerabilities predict therapy response, first established the required workflow for DNA sequencing and data interpretation (2014-2015). Here, we employed it to identify molecularly matched therapy and recorded outcome in end-stage cancer (2016-2019).Material and methods: Metastatic tissue from 26 patients (16 colorectal cancer cases) was sequenced by the Oncomine assay. The study tumor boards interpreted called variants with respect to sensitivity or resistance to matched therapy and recommended single-agent or combination treatment if considered tolerable. The primary endpoint was the rate of progression-free survival 1.3-fold longer than for the most recent systemic therapy. The objective response rate and overall survival were secondary endpoints.Results: Both common and rare actionable alterations were identified. Thirteen patients were found eligible for therapy following review of tumor sensitivity and resistance variants and patient tolerability. The interventions were inhibitors of ALK/ROS1-, BRAF-, EGFR-, FGFR-, mTOR-, PARP-, or PD-1-mediated signaling for 2-3 cases each. Among 10 patients who received treatment until radiologic evaluation, 6 (46% of the eligible cases) met the primary endpoint. Four colorectal cancer patients (15% of the total study cohort) had objective response. The only serious adverse event was a transient colitis, which appeared in 1 of the 2 patients given PD-1 inhibitor with complete response. Apart from those two, overall survival was similar for patients who did and did not receive study treatment.Conclusions: The systematic MetAction approach may point forward to a refined framework for how to interpret the complexity of sensitivity versus resistance and patient safety that resides in tumor sequence data, for the possibly improved outcome of precision cancer medicine in future studies. ClinicalTrials.gov, identifier: NCT02142036.

Risk of breast cancer by prior screening results among women participating in BreastScreen Norway.

BACKGROUND: A premalignant lesion in the breast is associated with an increased risk of breast cancer. The aim of this article was to identify women with an increased risk of breast cancer based on prior screening results (PSRs). METHODS: This registry-based cohort study followed women who participated in the organized breast cancer screening program in Norway, BreastScreen Norway, in 1995-2016. Incidence rates and incidence rate ratios were used to estimate absolute and relative risks of breast cancer associated with PSRs. Histopathological characteristics of subsequent breast cancers were presented by PSRs. RESULTS: This study included 762,643 women with up to 21 years of follow-up. In comparison with negatively screened women, increased incidence rate ratios of 1.8, 2.0, 2.9, and 3.8 were observed after negative additional imaging, for benign biopsy, for hyperplasia with atypia, and for carcinoma in situ, respectively. Subsequent breast cancers did not differ in tumor diameter or histological grade, whereas the proportion of lymph node-positive breast cancers decreased as the presumed malignancy potential of PSRs increased. CONCLUSIONS: The risk of subsequent breast cancer increased with the presumed malignancy potential of PSRs, whereas the tumor characteristics of subsequent cancers did not differ except for the lymph node status. Women with screen-detected benign lesions or hyperplasia with atypia might benefit from more frequent screening.

Breast Cancer Molecular Stratification: From Intrinsic Subtypes to Integrative Clusters.

Breast carcinomas can be stratified into different entities based on clinical behavior, histologic features, and/or by biological properties. A classification of breast cancer should be based on underlying biology, which we know must be determined by the somatic genomic landscape of mutations. Moreover, because the latest generations of anticancer agents are founded on biological mechanisms, a detailed molecular stratification is a requirement for appropriate clinical management. Such stratification, based on genomic drivers, will be important for selecting patients for clinical trials. It will also facilitate the discovery of novel drivers, the study of tumor evolution, and the identification of mechanisms of treatment resistance. Assays for risk stratification have focused mainly on response prediction to existing treatment regimens. Molecular stratification based on gene expression profiling revealed that breast cancers could be classified in so-called intrinsic subtypes (luminal A and B, HER2-enriched, basal-like, and normal-like), which mostly corresponded to hormone receptor and HER2 status, and further stratified luminal tumors based on proliferation. The realization that a significant proportion of the gene expression landscape is determined by the somatic copy number alterations that drive expression in cis led to the newer classification of breast cancers into integrative clusters. This stratification of breast cancers into integrative clusters reveals prototypical patterns of single-nucleotide variants and is associated with distinct clinical courses and response to therapy.

Prognostic value of PAM50 and risk of recurrence score in patients with early-stage breast cancer with long-term follow-up.

BACKGROUND: The aim of this study was to investigate the prognostic value of the PAM50 intrinsic subtypes and risk of recurrence (ROR) score in patients with early breast cancer and long-term follow-up. A special focus was placed on hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) pN0 patients not treated with chemotherapy. METHODS: Patients with early breast cancer (n = 653) enrolled in the observational Oslo1 study (1995-1998) were followed for distant recurrence and breast cancer death. Clinicopathological parameters were collected from hospital records. The primary tumors were analyzed using the Prosigna® PAM50 assay to determine the prognostic value of the intrinsic subtypes and ROR score in comparison with pathological characteristics. The primary endpoints were distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS). RESULTS: Of 653 tumors, 52.2% were classified as luminal A, 26.5% as luminal B, 10.6% as HER2-enriched, and 10.7% as basal-like. Among the HR+/HER2- patients (n = 476), 37.8% were categorized as low risk by ROR score, 22.7% as intermediate risk, and 39.5% as high risk. Median follow-up durations for BCSS and DDFS were 16.6 and 7.1 years, respectively. Multivariate analysis showed that intrinsic subtypes (all patients) and ROR risk classification (HR+/HER2- patients) yielded strong prognostic information. Among the HR+/HER2- pN0 patients with no adjuvant treatment (n = 231), 53.7% of patients had a low ROR, and their prognosis at 15 years was excellent (15-year BCSS 96.3%). Patients with intermediate risk had reduced survival compared with those with low risk (p = 0.005). In contrast, no difference in survival between the low- and intermediate-risk groups was seen for HR+/HER2- pN0 patients who received tamoxifen only. Ki-67 protein, grade, and ROR score were analyzed in the unselected, untreated pT1pN0 HR+/HER2- population (n = 171). In multivariate analysis, ROR score outperformed both Ki-67 and grade. Furthermore, 55% of patients who according to the PREDICT tool ( http://www.predict.nhs.uk/ ) would be considered chemotherapy candidates were ROR low risk (33%) or luminal A ROR intermediate risk (22%). CONCLUSIONS: The PAM50 intrinsic subtype classification and ROR score improve classification of patients with breast cancer into prognostic groups, allowing for a more precise identification of future recurrence risk and providing an improved basis for adjuvant treatment decisions. Node-negative patients with low ROR scores had an excellent outcome at 15 years even in the absence of adjuvant therapy.

Quantitative multigene FISH on breast carcinomas identifies der(1;16)(q10;p10) as an early event in luminal A tumors.

In situ detection of genomic alterations in cancer provides information at the single cell level, making it possible to investigate genomic changes in cells in a tissue context. Such topological information is important when studying intratumor heterogeneity as well as alterations related to different steps in tumor progression. We developed a quantitative multigene fluorescence in situ hybridization (QM FISH) method to detect multiple genomic regions in single cells in complex tissues. As a "proof of principle" we applied the method to breast cancer samples to identify partners in whole arm (WA) translocations. WA gain of chromosome arm 1q and loss of chromosome arm 16q are among the most frequent genomic events in breast cancer. By designing five specific FISH probes based on breakpoint information from comparative genomic hybridization array (aCGH) profiles, we visualized chromosomal translocations in clinical samples at the single cell level. By analyzing aCGH data from 295 patients with breast carcinoma with known molecular subtype, we found concurrent WA gain of 1q and loss of 16q to be more frequent in luminal A tumors compared to other molecular subtypes. QM FISH applied to a subset of samples (n = 26) identified a derivative chromosome der(1;16)(q10;p10), a result of a centromere-close translocation between chromosome arms 1q and 16p. In addition, we observed that the distribution of cells with the translocation varied from sample to sample, some had a homogenous cell population while others displayed intratumor heterogeneity with cell-to-cell variation. Finally, for one tumor with both preinvasive and invasive components, the fraction of cells with translocation was lower and more heterogeneous in the preinvasive tumor cells compared to the cells in the invasive component.