The Drug Rediscovery protocol facilitates the expanded use of existing anticancer drugs.

The large-scale genetic profiling of tumours can identify potentially actionable molecular variants for which approved anticancer drugs are available1-3. However, when patients with such variants are treated with drugs outside of their approved label, successes and failures of targeted therapy are not systematically collected or shared. We therefore initiated the Drug Rediscovery protocol, an adaptive, precision-oncology trial that aims to identify signals of activity in cohorts of patients, with defined tumour types and molecular variants, who are being treated with anticancer drugs outside of their approved label. To be eligible for the trial, patients have to have exhausted or declined standard therapies, and have malignancies with potentially actionable variants for which no approved anticancer drugs are available. Here we show an overall rate of clinical benefit-defined as complete or partial response, or as stable disease beyond 16 weeks-of 34% in 215 treated patients, comprising 136 patients who received targeted therapies and 79 patients who received immunotherapy. The overall median duration of clinical benefit was 9 months (95% confidence interval of 8-11 months), including 26 patients who were experiencing ongoing clinical benefit at data cut-off. The potential of the Drug Rediscovery protocol is illustrated by the identification of a successful cohort of patients with microsatellite instable tumours who received nivolumab (clinical benefit rate of 63%), and a cohort of patients with colorectal cancer with relatively low mutational load who experienced only limited clinical benefit from immunotherapy. The Drug Rediscovery protocol facilitates the defined use of approved drugs beyond their labels in rare subgroups of cancer, identifies early signals of activity in these subgroups, accelerates the clinical translation of new insights into the use of anticancer drugs outside of their approved label, and creates a publicly available repository of knowledge for future decision-making.

Molecular Tumor Boards: current practice and future needs.

BACKGROUND: Due to rapid technical advances, steeply declining sequencing costs, and the ever-increasing number of targeted therapies, it can be expected that extensive tumor sequencing such as whole-exome and whole-genome sequencing will soon be applied in standard care. Clinicians will thus be confronted with increasingly complex genetic information and multiple test-platforms to choose from. General medical training, meanwhile, can hardly keep up with the pace of innovation. Consequently, there is a rapidly growing gap between clinical knowledge and genetic potential in cancer care. Multidisciplinary Molecular Tumor Boards (MTBs) have been suggested as a means to address this disparity, but shared experiences are scarce in literature and no quality requirements or guidelines have been published to date. METHODS: Based on literature review, a survey among hospitals in The Netherlands, and our own experience with the establishment of a nationally operating MTB, this article evaluates current knowledge and unmet needs and lays out a strategy for successful MTB implementation. RESULTS: Having access to an MTB can improve and increase the application of genetics-guided cancer care. In our survey, however, <50% of hospitals and only 5% of nonacademic hospitals had access to an MTB. In addition, current MTBs vary widely in terms of composition, tasks, tools, and workflow. This may not only lead to variation in quality of care but also hinders data sharing and thus creation of an effective learning community. CONCLUSIONS: This article acknowledges a leading role for MTBs to govern (extensive) tumor sequencing into daily practice and proposes three basic necessities for successful MTB implementation: (i) global harmonization in cancer sequencing practices and procedures, (ii) minimal member and operational requirements, and (iii) an appropriate unsolicited findings policy. Meeting these prerequisites would not only optimize MTB functioning but also improve general interpretation and application of genomics-guided cancer care.

Triple-negative breast cancer: BRCAness and concordance of clinical features with BRCA1-mutation carriers.

BACKGROUND: BRCAness is defined as shared tumour characteristics between sporadic and BRCA-mutated cancers. However, how to exactly measure BRCAness and its frequency in breast cancer is not known. Assays to establish BRCAness would be extremely valuable for the clinical management of these tumours. We assessed BRCAness characteristics frequencies in a large cohort of triple-negative breast cancers (TNBCs). METHODS: As a measure of BRCAness, we determined a specific BRCA1-like pattern by array Comparative Genomic Hybridisation (aCGH), and BRCA1 promoter methylation in 377 TNBCs, obtained from 3 different patient cohorts. Clinicopathological data were available for all tumours, BRCA1-germline mutation status and chemotherapy response data were available for a subset. RESULTS: Of the tumours, 66-69% had a BRCA1-like aCGH profile and 27-37% showed BRCA1 promoter methylation. BRCA1-germline mutations and BRCA1 promoter methylation were mutually exclusive events (P=1 × 10(-5)). BRCAness was associated with younger age and grade 3 tumours. Chemotherapy response was significantly higher in BRCA1-mutated tumours, but not in tumours with BRCAness (63% (12 out of 19) vs 35% (18 out of 52) pathological complete remission rate, respectively). CONCLUSION: The majority of the TNBCs show BRCAness, and those tumours share clinicopathological characteristics with BRCA1-mutated tumours. A better characterisation of TNBC and the presence of BRCAness could have consequences for both hereditary breast cancer screening and the treatment of these tumours.

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers.

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.

Neoadjuvant chemotherapy in ER+ HER2- breast cancer: response prediction based on immunohistochemical and molecular characteristics.

A pathological complete remission (pCR) is rarely achieved by neoadjuvant chemotherapy in estrogen receptor-positive (ER+) HER2-negative (HER2-) tumors. Therefore, its use might be questionable in specific groups of this tumor type. To select which patients benefit and which could be spared neoadjuvant chemotherapy, we tested standard pathology and molecular markers in ER+ HER2- breast tumors. Pretreatment biopsies were available from 211 ER+ HER2- tumors, who had been treated with neoadjuvant chemotherapy (adriamycin/cyclophosphamide). mRNA expression data were available for 132 tumors. We determined progesterone receptor expression (PR), endocrine sensitivity, HER2 expression, histology, proliferation, and molecular subtypes. We correlated these data to chemotherapy response using pCR rates and the previously published neoadjuvant response index (NRI). PR-negative tumors (n = 65, 30.8%) and luminal B type tumors (n = 43, 20.4%) responded significantly better to chemotherapy than other tumors. These associations remained significant in multivariate analysis. However, even in the subgroup of patients with the lowest response rate, comprising tumors that had both a positive-PR expression and the luminal A subtype (n = 58, 44%), the majority of the patients had downstaging because of chemotherapy. For histology (lobular vs. ductal), endocrine sensitivity, and proliferation, no associations with chemotherapy response were observed. Gene expression array analysis resulted in 28 significant genes (FDR < 0.1). PR expression and luminal B status are associated with a better response to neoadjuvant chemotherapy. However, both markers had only weak response predictive power, and it was not possible to identify a subgroup with no or only minimal chemotherapy benefit. Therefore, the decision to refrain from neoadjuvant chemotherapy to ER+ HER2- breast tumors should not be based on predictive markers, but exclusively on estimates of prognosis.

Indicators of homologous recombination deficiency in breast cancer and association with response to neoadjuvant chemotherapy.

BACKGROUND: Tumors with homologous recombination deficiency (HRD), such as BRCA1-associated breast cancers, are not able to reliably repair DNA double-strand breaks (DSBs) and are therefore highly sensitive to both DSB-inducing chemotherapy and poly (ADP-ribose) polymerase inhibitors. We have studied markers that may indicate the presence of HRD in HER2-negative breast cancers and related them to neoadjuvant chemotherapy response. PATIENTS AND METHODS: Array comparative genomic hybridization (aCGH), BRCA1 promoter methylation, BRCA1 messenger RNA (mRNA) expression and EMSY amplification were assessed in 163 HER2-negative pretreatment biopsies from patients scheduled for neoadjuvant chemotherapy. RESULTS: Features of BRCA1 dysfunction were frequent in triple-negative (TN) tumors: a BRCA1-like aCGH pattern, promoter methylation and reduced mRNA expression were observed in, respectively, 57%, 25% and 36% of the TN tumors. In ER+ tumors, a BRCA2-like aCGH pattern and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13%, respectively) and this BRCA2-like profile was associated with a better response to neoadjuvant chemotherapy. CONCLUSIONS: Abnormalities associated with BRCA1 inactivation are present in about half of the TN breast cancers but were not predictive of chemotherapy response. In ER+/HER2- tumors, a BRCA2-like aCGH pattern was predictive of chemotherapy response. These findings should be confirmed in independent series.

An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients.

BACKGROUND: Breast cancer cells deficient for BRCA1 are hypersensitive to agents inducing DNA double-strand breaks (DSB), such as bifunctional alkylators and platinum agents. Earlier, we had developed a comparative genomic hybridisation (CGH) classifier based on BRCA1-mutated breast cancers. We hypothesised that this BRCA1-like(CGH) classifier could also detect loss of function of BRCA1 due to other causes besides mutations and, consequently, might predict sensitivity to DSB-inducing agents. PATIENTS AND METHODS: We evaluated this classifier in stage III breast cancer patients, who had been randomly assigned between adjuvant high-dose platinum-based (HD-PB) chemotherapy, a DSB-inducing regimen, and conventional anthracycline-based chemotherapy. Additionally, we assessed BRCA1 loss through mutation or promoter methylation and immunohistochemical basal-like status in the triple-negative subgroup (TN subgroup). RESULTS: We observed greater benefit from HD-PB chemotherapy versus conventional chemotherapy among patients with BRCA1-like(CGH) tumours [41/230 = 18%, multivariate hazard ratio (HR) = 0.12, 95% confidence interval (CI) 0.04-0.43] compared with patients with non-BRCA1-like(CGH) tumours (189/230 = 82%, HR = 0.78, 95% CI 0.50-1.20), with a significant difference (test for interaction P = 0.006). Similar results were obtained for overall survival (P interaction = 0.04) and when analyses were restricted to the TN subgroup. Sixty-three percent (20/32) of assessable BRCA1-like(CGH) tumours harboured either a BRCA1 mutation (n = 8) or BRCA1 methylation (n = 12). CONCLUSION: BRCA1 loss as assessed by CGH analysis can identify patients with substantially improved outcome after adjuvant DSB-inducing chemotherapy when compared with standard anthracycline-based chemotherapy in our series.

A non-BRCA1/2 hereditary breast cancer sub-group defined by aCGH profiling of genetically related patients.

Germline mutations in BRCA1 and BRCA2 explain approximately 25% of all familial breast cancers. Despite intense efforts to find additional high-risk breast cancer genes (BRCAx) using linkage analysis, none have been reported thus far. Here we explore the hypothesis that BRCAx breast tumors from genetically related patients share a somatic genetic etiology that might be revealed by array comparative genomic hybridization (aCGH) profiling. As BRCA1 and BRCA2 tumors can be identified on the basis of specific genomic profiles, the same may be true for a subset of BRCAx families. Analyses used aCGH to compare 58 non-BRCA1/2 familial breast tumors (designated BRCAx) to sporadic (non-familiar) controls, BRCA1 and BRCA2 tumors. The selection criteria for BRCAx families included at least three cases of breast cancer diagnosed before the age of 60 in the family, and the absence of ovarian or male breast cancer. Hierarchical cluster analysis was performed to determine sub-groups within the BRCAx tumor class and family heterogeneity. Analysis of aCGH profiles of BRCAx tumors indicated that they constitute a heterogeneous class, but are distinct from both sporadic and BRCA1/2 tumors. The BRCAx class could be divided into sub-groups. One subgroup was characterized by a gain of chromosome 22. Tumors from family members were classified within the same sub-group in agreement with the hypothesis that tumors from the same family would harbor a similar genetic background. This approach provides a method to target a sub-group of BRCAx families for further linkage analysis studies.

Proteasome: from structure to function.

During the past two years, significant progress has been made in understanding the structure and function of the proteasome. Recent work has revealed the three-dimensional structure of the 700 kDa proteolytic complex at atomic resolution and elucidated its novel catalytic mechanism. Close relationships to a number of other amino-terminal hydrolases have emerged, making the proteasomal subunits the prototype of this newly discovered structural superfamily.

Import of human and Thermoplasma 20S proteasomes into nuclei of HeLa cells requires functional NLS sequences.

Proteasomes are present both in the nucleus and cytoplasm of eukaryotic cells. Their localization is regulated and changes during the cell cycle. Nuclear localization signal (NLS) type sequences were identified in proteasomes from various organisms. In addition, acidic complementary sequences were identified (cNLS) which could interact with the positively charged NLS, masking or unmasking them and thereby modulating nuclear import. In this paper we show that fluorescently labeled human erythrocyte 20S proteasomes accumulate in the nucleus of digitonin-permeabilized cells. This translocation is ATP-dependent and occurs through the nuclear pore complex as is shown by blocking of the nuclear pores with wheat germ agglutinin. In addition, we used 20S proteasomes from Thermoplasma acidophilum as a model system. Recombinant 20S proteasomes from the archaebacterium Thermoplasma acidophilum are imported into nuclei of HeLa and 3T3 cells similar to their eukaryotic counterpart. We constructed mutants in the putative NLS and cNLS region to study their effect on import. The NLS mutant was not imported into nuclei and showed cytoplasmic staining only. This indicates that this sequence is indeed responsible for nuclear targeting. Mutational studies of the cNLS do not support the involvement of this sequence in regulation of nuclear transport.

A tumour with a neuroendocrine and papillary serous component: two or a pair?

AIMS: To examine the clonal origin of a tumour, made up of a neuroendocrine component and a papillary serous component by comparing the pattern of loss of heterozygosity (LOH) and the immunohistochemical protein expression of both components. METHODS/RESULTS: A 70 year old woman, known to have a metastasised neuroendocrine carcinoma, underwent resection of the distal part of the ileum because of obstruction by a mesenterial mass. The macroscopically homogeneous mesenterial mass consisted histologically of an admixture of a neuroendocrine component and a papillary serous carcinoma. Loss of heterozygosity (LOH) analysis of both components with a panel of 15 polymorphic microsatellite markers showed a distinctive pattern of LOH, and both components showed LOH on chromosome 4q and 17, but involving different alleles at the same locus. Moreover, both components showed different immunohistochemical staining patterns for neuroendocrine markers, cytokeratin 7, carcinoembryonic antigen, and CA125. CONCLUSION: Both LOH analysis of the neuroendocrine and papillary serous components of this tumour and the immunohistochemical profile of both components are consistent with a different clonal origin. The tumour is probably a collision tumour, in which the papillary serous carcinoma must have been of peritoneal origin because necropsy revealed a normal uterus and normal ovaries.

Quality control in diagnostic molecular pathology in the Netherlands; proficiency testing for patient identification in tissue samples.

AIMS: To describe the evolution of proficiency testing for molecular diagnostic pathology with respect to determining unambiguously the patient identity of tissue samples by microsatellite analysis. METHOD: Four rounds of quality control exchanges of samples from different patients were sent with the purpose of identifying the correct origin of these samples. The samples were either paraffin wax embedded sections on glass, sections in tubes, or isolated DNA. Blinded samples were distributed to all participating laboratories. No restrictions to the method and short tandem repeat markers used for identification were imposed. RESULTS: In four subsequent rounds the number of participating laboratories increased from three to 10. The numbers of samples tested increased in time from five to 12. The microsatellite markers used by the different laboratories showed little overlap. In the first three rounds, in which isolated DNA was provided, all samples were accurately classified irrespective of the microsatellite markers used. In the last round, which also included paraffin wax embedded sections, a small number of laboratories experienced problems, either with amplification or incorrect classification of a few samples. CONCLUSION: Proficiency testing was useful, and showed country wide high quality and correct identification of (patient) samples with molecular techniques for diagnostic purposes.

Detection of chromosome aberrations in interphase tumor nuclei by nonradioactive in situ hybridization.

In a blind study, chromosome aberrations in tumor cells were analyzed by conventional cytogenetic techniques (G banding) and nonradioactive in situ hybridization with chromosome-specific probes. The material was obtained directly from patients with hematologic diseases and from colon tumor derived cell lines. The cytogenetic data obtained with G banding were in accord with those obtained by in situ hybridization to metaphase chromosomes. Most importantly, in situ hybridization to interphase nuclei gave reliable results and even allowed detection of cell subpopulations that were not detected by analyzing metaphase chromosomes. Furthermore, in retrospect, even structural aberrations could be detected in interphase nuclei; abnormal cells with either an i(1q) or a translocation der(1)t(1;7) could be identified. Our results show that the application of in situ hybridization in combination with routine cytogenetic techniques offers significant advantages for cytogenetic analysis of solid tumors and hematologic malignancies.

Goblet cell carcinoid of the appendix: a specific type of carcinoma.

AIMS: Goblet cell carcinoid is a poorly understood tumour of the appendix. The aim of this study was to determine whether it should be regarded as a separate entity or as a variant of classical carcinoid. METHODS AND RESULTS: The immunohistochemical expression pattern of 21 markers and the mutation status of KRas codon 12 were determined in 16 goblet cell carcinoids and compared with 14 classical carcinoids, 19 colonic adenocarcinomas and 10 appendiceal mucinous cystadeno (carcino)mas. The results were subjected to a stepwise linear discriminant analysis. Goblet cell carcinoids were significantly different from the control groups. The most important markers for discriminating between the groups were CEA (classical carcinoid versus all others), KRas mutation (present in all mucinous cystadeno (carcino)mas), beta-catenin (goblet cell carcinoid versus left sided colonic adenocarcinoma) and chromogranin (goblet cell carcinoid versus right sided colonic adenocarcinoma). Expression of Math1 and HD5 was similar in goblet cell carcinoid and colonic adenocarcinoma but absent in classical carcinoid. CONCLUSION: The results suggest that goblet cell carcinoids should be regarded as a separate entity. The formerly used term 'crypt cell carcinoma' may be more appropriate because it reflects the more aggressive clinical behaviour of these tumours as well as their greater similarity to adenocarcinomas rather than to carcinoids.

A multiplex PCR predictor for aCGH success of FFPE samples.

Formalin-fixed, paraffin-embedded (FFPE) tissue archives are the largest and longest time-spanning collections of patient material in pathology archives. Methods to disclose information with molecular techniques, such as array comparative genomic hybridisation (aCGH) have rapidly developed but are still not optimal. Array comparative genomic hybridisation is one efficient method for finding tumour suppressors and oncogenes in solid tumours, and also for classification of tumours. The fastest way of analysing large numbers of tumours is through the use of archival tissue samples with first, the huge advantage of larger median follow-up time of patients studied and second, the advantage of being able to locate and analyse multiple tumours, even across generations, from related individuals (families). Unfortunately, DNA from archival tissues is not always suitable for molecular analysis due to insufficient quality. Until now, this quality remained undefined. We report the optimisation of a genomic-DNA isolation procedure from FFPE pathology archives in combination with a subsequent multiplex PCR-based quality-control that simply identified all samples refractory to further DNA-based analyses.

Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro.

Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits. We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (Hsc iota, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct non-nuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both cell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence.

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Thick-section fluorescence in situ hybridization on formalin-fixed, paraffin-embedded archival tissue provides a histogenetic profile.

Fluorescence in situ hybridization has become a major tool for analysis of gene and chromosome copy number in normal and malignant tissue. The technique has been applied widely to fresh tissue and dispersed formalin-fixed, paraffin-embedded archival tissue, but its use on sections of archival tissue has largely been limited to sections < 6 mu thick. This does not provide intact, uncut nuclei for accurate analysis of gene or chromosome copy number. We report here a method of hybridization to sections > 20 microns thick that overcomes these difficulties. Key developments were the use of DNA probes directly labeled with fluorochromes and optical sectioning using laser-scanning confocal microscopy.

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.

Gene amplification in human cells may involve interchromosomal transposition and persistence of the original DNA region.

In tumor cells in vivo and in vitro the amplification of large DNA sequences is a spontaneous and frequently occurring genetic event. We have used human cells to study independent events leading to a low level of amplification of a single copy of an integrated plasmid. Fluorescence in situ hybridization, chromosome banding, and chromosome painting revealed that the new amplified DNA sequences can become located on chromosomes that are totally unrelated to the chromosome that harbors the original DNA sequences, indicating that the transposition of amplified DNA sequences is interchromosomal. In cells containing amplified DNA sequences the integrated single-copy plasmid remained at its original location. The unit of amplification contained a DNA fragment of at least a 800 kb and the same fragment was also present in the parental single-copy cell clone. The data suggest that a doubling of the DNA region at the original location precedes or is coupled to gene amplification.