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In the past decade, major advances have been made in the understanding of melanoma. New predisposition genes have been reported and key somatic events, such as BRAF mutation, directly translated into therapeutic management. Surgery for localised melanoma and regional lymph node metastases is the standard of care. Sentinel-node biopsy provides precise staging, but has not been reported to affect survival. The effect of lymph-node dissection on survival is a topic of investigation. Two distinct approaches have emerged to try to extend survival in patients with metastatic melanoma: immunomodulation with anti-CTLA4 monoclonal antibodies, and targeted therapy with BRAF inhibitors or MEK inhibitors for BRAF-mutated melanoma. The combination of BRAF inhibitors and MEK inhibitors might improve progression-free survival further and, possibly, increase overall survival. Response patterns differ substantially—anti-CTLA4 immunotherapy can induce long-term responses, but only in a few patients, whereas targeted drugs induce responses in most patients, but nearly all of them relapse because of pre-existing or acquired resistance. Thus, the long-term prognosis of metastatic melanoma remains poor. Anti-PD1 and anti-PDL1 antibodies have emerged as breakthrough drugs for melanoma that have high response rates and long durability. Biomarkers that have predictive value remain elusive in melanoma, although emerging data for adjuvant therapy indicate that interferon sensitivity is associated with ulceration of the primary melanoma. Intense investigation continues for clinical and biological markers that predict clinical benefit of immunotherapeutic drugs, such as interferon alfa or anti-CTLA4 antibodies, and the mechanisms that lead to resistance of targeted drugs.

Respiratory screening assays lacking Sample Adequacy Controls (SAC) may result in inadequate sample quality and thus false negative results. The non-adequate samples might represent a significant proportion of the total performed tests, thus resulting in sub-optimal infection control measures with implications that may be critical during pandemic times. The quantitative sample adequacy threshold can be established empirically, measuring the change in the frequency of positive results, as a function of the numerical value of “sample adequacy”. Establishing a quantitative threshold for SAC requires a big number/volume of tests to be analyzed in order to have a statistically valid result. Herein, we are offering for the first time clear clinical evidence that a subset of results, which did not pass minimal sample adequacy criteria, have a significantly lower frequency of positivity compared with the “adequate” samples. Flagging these results and/or re-sampling them is a mitigation strategy, which can dramatically improve infection control measures.

BackgroundCheckpoint blockade with ipilimumab provides long-term survival to a significant proportion of patients with metastatic melanoma. New approaches to increase survival and to predict which patients will benefit from treatment are needed. This phase II trial combined ipilimumab with carboplatin/paclitaxel (CP) to assess its safety, efficacy, and to search for peripheral and tumor-based predictive biomarkers.MethodsThirty patients with untreated unresectable/metastatic melanoma were treated with ipilimumab and CP. Adverse events (AEs) were monitored and response to treatment was evaluated. Tumor tissue and peripheral blood were collected at specified time points to characterize tumor immune markers by immunohistochemistry and systemic immune activity by multiplex assays and flow cytometry.ResultsEighty three percent of patients received all 5 cycles of CP and 93% completed ipilimumab induction. Serious AEs occurred in 13% of patients, and no treatment-related deaths were observed. Best Overall Response Rate (BORR) and Disease Control Rate (DCR) were 27 and 57%, respectively. Median overall survival was 16.2 months. Response to treatment was positively correlated with a higher tumor CD3+ infiltrate (immune score) at baseline. NRAS and BRAF mutations were less frequent in patients who experienced clinical benefit. Assessment of peripheral blood revealed that non-responders had elevated baseline levels of CXCL8 and CCL4, and a higher proportion of circulating late differentiated B cells. Pre-existing high levels of chemokines (CCL3, CCL4 and CXCL8) and advanced B cell differentiation were strongly associated with worse patient overall survival. Elevated proportions of circulating CD8+/PD-1+ T cells during treatment were associated with worse survival.ConclusionsThe combination of ipilimumab and CP was well tolerated and revealed novel characteristics associated with patients likely to benefit from treatment. A pre-existing systemic inflammatory state characterized by elevation of selected chemokines and advanced B cell differentiation, was strongly associated with poor patient outcomes, revealing potential predictive circulating biomarkers.Trial registration Clinicaltrials.gov, NCT01676649, registered on August 29, 2012.

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor’s biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

Therapeutic resistance remains a major cause of cancer-related deaths. Resistance can occur from the outset of treatment or as an acquired phenomenon after an initial clinical response. Therapeutic resistance is an almost universal phenomenon in the treatment of metastatic cancers. The advent of molecularly targeted treatments brought greater efficacy in patients whose tumors express a particular target or molecular signature. However, resistance remains a predictable challenge. This article provides an overview of somatic genomic events that confer resistance to cancer therapies. Some examples, including BCR-Abl, EML4-ALK, and the androgen receptor, contain mutations in the target itself, which hamper binding and inhibitory functions of therapeutic agents. There are also examples of somatic genetic changes in other genes or pathways that result in resistance by circumventing the inhibitor, as in resistance to trastuzumab and BRAF inhibitors. Yet other examples results in activation of cytoprotective genes. The fact that all of these mechanisms of resistance are due to somatic changes in the tumor's genome makes targeting them selectively a feasible goal. To identify and validate these changes, it is important to obtain biopsies of clinically resistant tumors. A rational consequence of this evolving knowledge is the growing appreciation that combinations of inhibitors will be needed to anticipate and overcome therapeutic resistance.

Stemness and chromosomal instability (CIN) are two common contributors to intratumor heterogeneity and therapy relapse in advanced cancer, but their interplays are poorly defined. Here, in anaplastic thyroid cancer (ATC), we show that ALDH+ stem-like cancer cells possess increased CIN-tolerance owing to transcriptional upregulation of the scaffolding protein NEDD9. Thyroid patient tissues and transcriptomic data reveals NEDD9/ALDH1A3 to be co-expressed and co-upregulated in ATC. Compared to bulk ALDH− cells, ALDH+ cells were highly efficient at propagating CIN due to their intrinsic tolerance of both centrosome amplification and micronuclei. ALDH+ cells mitigated the fitness-impairing effects of centrosome amplification by partially inactivating supernumerary centrosomes. Meanwhile, ALDH+ cells also mitigated cell death caused by micronuclei-mediated type 1 interferon secretion by suppressing the expression of the DNA-sensor protein STING. Both mechanisms of CIN-tolerance were lost upon RNAi-mediated NEDD9 silencing. Both in vitro and in vivo, NEDD9-depletion attenuated stemness, CIN, cell/tumor growth, while enhancing paclitaxel effectiveness. Collectively, these findings reveal that ATC progression can involve an ALDH1A3/NEDD9-regulated program linking their stemness to CIN-tolerance that could be leveraged for ATC treatment.

Insights into tumour biology require biospecimens from the primary tumour and those that reflect the patient's disease in specific contexts. Tumour samples must be representative, viable, and adequate both in quantity and quality for subsequent molecular applications. This Review article outlines the existing procedures for sample procurement and processing of next-generation biospecimens, and highlights the issues involved in this endeavour. The majority of samples in existing tumour biobanks are surgical specimens of primary tumours. Insights into tumour biology, such as intratumoural heterogeneity, tumour–host crosstalk, and the evolution of the disease during therapy, require biospecimens from the primary tumour and those that reflect the patient's disease in specific contexts. Next-generation 'omics' technologies facilitate deep interrogation of tumours, but the characteristics of the samples can determine the ultimate accuracy of the results. The challenge is to biopsy tumours, in some cases serially over time, ensuring that the samples are representative, viable, and adequate both in quantity and quality for subsequent molecular applications. The collection of next-generation biospecimens, tumours, and blood samples at defined time points during the disease trajectory—either for discovery research or to guide clinical decisions—presents additional challenges and opportunities. From an organizational perspective, it also requires new additions to the multidisciplinary therapeutic team, notably interventional radiologists, molecular pathologists, and bioinformaticians. In this Review, we describe the existing procedures for sample procurement and processing of next-generation biospecimens, and highlight the issues involved in this endeavour, including the ethical, logistical, scientific, informational, and financial challenges accompanying next-generation biobanking. Key Points Next-generation biospecimens are biopsy-type clinical specimens collected from patients at distinct time points and in a prespecified clinical context of treatment, made available for multidimensional high-throughput technologies Biopsies of recurrent primary or metastatic tumours are highly sought after next-generation biospecimens for both research purposes and the clinical management of patients Controlling preanalytical variables is critical to ensure that the results of multidimensional high-throughput profiling are accurate and reproducible Standard operating procedures for biospecimen collection and processing, with quality assurance of every specimen, must be developed and adhered to, with particular emphasis placed on the training of personnel Collection of next-generation biospecimens requires increased resources and a multidisciplinary team consisting of interventional radiologists and molecular pathologists

Tumour budding is defined as the presence of a cluster of fewer than 5 cells along the invasive margin. It may confer a worse prognosis in colorectal cancer, but its importance in pT2N0 colorectal cancer is unknown. This study aimed to determine the prognostic value of tumour budding in pT2N0 colorectal cancer. This was a retrospective cohort study with prospective assessment of tumour budding by 2 pathologists. We included all patients who underwent elective curative resection for pT2N0 colorectal cancer except those with hereditary colorectal cancer syndromes, inflammatory bowel disease or positive resection margins, those who received neoadjuvant or adjuvant therapy and those who died within 90 days of operation. Patients were classified as having high-grade tumour budding (≥ 10 budding foci per high-power field) or low-grade tumour budding (< 9 budding foci per high-power field). The main outcome measure was locoregional or distant recurrence. Of 85 patients, 36 had high-grade tumour budding and 49 had low-grade tumour budding. The overall recurrence rate was 11% (9/85) and median follow-up was 41.0 months (interquartile range 22.0–68.0). Interrater reliability for tumour budding assessment was excellent (κ = 0.86, 95% confidence interval [CI] 0.76–0.96). There were more recurrences in patients with high-grade tumour budding (7/36, 19.4% v. 2/49, 4.1%; p = 0.020). On multivariate analysis, after we adjusted for confounders, the presence of high-grade tumour budding was independently associated with recurrence (hazard ratio 5.11, 95% CI 1.01–25.9). Tumour budding was independently associated with increased recurrence after pT2N0 colorectal cancer resection. It offers additional prognostic information that may affect treatment strategy. Le bourgeonnement tumoral se définit par la présence d'un amas de 5 cellules ou moins le long de la marge invasive. Il pourrait conférer un pronostic plus sombre dans le cancer colorectal, mais on ignore sa portée dans le cancer colorectal de stade pT2N0. Cette étude visait à déterminer la valeur pronostique du bourgeonnement tumoral dans un cancer colorectal de stade pT2N0. Il s'agit d'une étude de cohorte rétrospective avec évaluation prospective du bourgeonnement tumoral par 2 pathologistes. Nous avons inclus tous les patients ayant subi une résection curative non urgente pour un cancer colorectal de stade pT2N0, sauf ceux qui présentaient un syndrome de cancer colorectal héréditaire, une maladie inflammatoire de l'intestin ou des marges de résection positives ceux qui avaient reçu des traitements néoadjuvants ou adjuvants et ceux qui étaient décédés dans les 90 jours suivant l'intervention. Les patients ont été classés selon qu'ils avaient un bourgeonnement tumoral de haut grade (≥ 10 foyers bourgeonnants par champ à fort grossissement) ou bourgeonnement tumoral de bas grade (< 9 foyers bourgeonnants par champ à fort grossissement). Le principal paramètre était la récurrence locorégionale ou distante. Sur 85 patients, 36 présentaient un bourgeonnement tumoral de haut grade, et 49, de bas grade. Le taux de récurrence global a été de 11 % (9/85) et le suivi médian a été de 41,0 mois (intervalle interquartile 22,0–68,0). La fiabilité interévaluateur de l’évaluation du bourgeonnement tumoral a été excellente (κ = 0,86, intervalle de confiance [IC] de 95 % 0,76–0,96). Les récurrences ont été plus nombreuses chez les patients qui avaient un bourgeonnement tumoral de haut grade (7/36, 19,4 % c. 2/49, 4,1 %; p = 0,020). À l'analyse multivariée, après ajustement pour tenir compte des variables de confusion, la présence de bourgeonnement tumoral de haut grade s'est révélée indépendamment liée à la récurrence (risque relatif 5,11, IC de 95 % 1,01–25,9). Le bourgeonnement tumoral s'est révélé indépendamment lié à l'augmentation des récurrences après la résection pour cancer colorectal de stade pT2N0. Il offre une information pronostique additionnelle qui pourrait influer sur la stratégie thérapeutique.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Background: Metastatic prostate cancers frequently harbour pathogenic aberrations in Homologous Recombination Repair (HRR) genes that confer sensitivity to PARP inhibitors (PARPi). Therefore, accurate identification of all eligible patients is needed. The development of a circulating tumour DNA (ctDNA) testing alternative is promising as genomic testing of archived tissue leads to a failure rate of up to 30–40% in prostate cancer. Methods: This was a bi-institutional retrospective cohort study of patients with metastatic prostate cancer treated at the Jewish General Hospital or the McGill University Health Center, Montreal, Canada, between 2021 and 2023. Molecular data and treatment information were abstracted from a chart review. Chi-square, Fisher’s exact test, and Mann–Whitney tests were used to assess differences between groups. Results: We identified 484 metastatic prostate cancer patients. Somatic and germline testing for HRR was performed in 55.4% (n = 268) and 20% (n = 97) patients, respectively. Somatic testing was performed on tissue (n = 192, 71.6%) or ctDNA from liquid biopsies (n = 18, 6.7%) or both (n = 58, 21.7%). Pathogenic somatic HRR alterations were detected in 48 patients (17.9%). BRCA2 was the most frequent (n = 17), followed by ATM (n = 11), then CHEK2 (n = 5). Amongst patients with germline testing, 13/97 (13.4%) had pathogenic alterations predicted to lead to deficient HRR, mostly BRCA2 (n = 9), and three had detectable BRCA2 in tissue. Dual testing modality (tissue+ctDNA) significantly enhanced the detection rate of HRR alterations 19/58 (32.7%) vs. 29/210 (13.8%) for single testing modality (tissue or ctDNA), p = 0.008. The rate of inconclusive results was significantly lower in dual testing modality 0/58 (0%) vs. 25/210 in single testing modality (11.9%), p = 0.003. Amongst the 14 patients who had discordant results between liquid and tissue tests, HRR abnormalities were more frequently identified in ctDNA (n = 11) vs. tissue (n = 3). Patients who had HRR deficiency detected only in ctDNA had older tissue samples (median 5.6 years) compared to those who had deficient HRR detected only in tissue (median 0.2 years; p = 0.14). Conclusions: These data highlight a potential role in implementing liquid biopsy—especially in patients who only have older archival tissue available or failed tissue testing—to improve the detection rate of deficient HRR. Our ongoing prospective study will further validate whether the addition of liquid biopsy can identify more patients who are eligible to receive precision therapies by increasing the rate of detection of HRR deficiency compared to routine tissue testing alone.