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BACKGROUND: In the Netherlands, lower high-risk human papillomavirus (hrHPV) positivity but higher cervical intraepithelial neoplasia (CIN) 2+ detection were found in self-collected compared with clinician-collected samples. To investigate the possible reason for these differences, we compared sociodemographic and screening characteristics of women and related these to screening outcomes. METHODS: We extracted data from PALGA on all primary hrHPV screens and associated follow-up tests for 857,866 screened women, invited in 2017 and 2018. We linked these data with sociodemographic data from Statistics Netherlands. Logistic regression was performed for hrHPV positivity and CIN 2+/3+ detection. RESULTS: Out of the 857,866 women, 6.8% chose to use a self-sampling device. A higher proportion of self-sampling users was ages 30 to 35 years, was not previously screened, was living in a one-person household, or was the breadwinner in the household. After adjustment for these factors self-sampling had lower hrHPV positivity (aOR, 0.65; 95% CI, 0.63-0.68)) as compared with clinician-collected sampling, as well as lower odds of CIN 2+ (aOR, 0.76; 95% CI, 0.70-0.82) and CIN 3+ (aOR, 0.86; 95% CI, 0.78-0.95) detection. CONCLUSIONS: It is likely that the observed differences between the two sampling methods are not only related to sociodemographic differences, but related to differences in screening test accuracy and/or background risk. IMPACT: Self-sampling can be used for targeting underscreened women, as a more convenient screening tool. Further investigation is required to evaluate how to implement self-sampling, when it is used as a primary instrument in routine screening. See related commentary by Arbyn et al., p. 159.

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BACKGROUND: High-risk human papillomavirus (hrHPV) testing on self-collected samples has potential as a primary screening tool in cervical screening, but real-world evidence on its accuracy in hrHPV-based screening programmes is lacking. METHODS: In the Netherlands, women aged 30-60 years invited for cervical screening can choose between sampling at the clinician's office (Cervex Brush) or self-sampling at home (Evalyn Brush). HrHPV testing is performed using Roche Cobas 4800. We collected screening test results between January 2017 and March 2018 and histological follow-up until August 2019. The main outcome measures were mean cycle threshold (Ct) value, cervical intraepithelial neoplasia (CIN) grade 3 or cancer (CIN3+) and CIN grade 2 or worse (CIN2+). FINDINGS: 30,808 women had a self-collected and 456,207 had a clinician-collected sample. In hrHPV-positive women with adequate cytology, Ct values were higher for self-collection than clinician-collection with a mean Ct difference of 1·25 (95% CI 0·98-1·52) in women without CIN2+, 2·73 (1·75-3·72) in CIN2 and 3·59 (3·03-4·15) in CIN3+. The relative sensitivity for detecting CIN3+ was 0·94 (0·90-0·97) for self-collection versus clinician-collection and the relative specificity was 1·02 (1·02-1·02). INTERPRETATION: The clinical accuracy of hrHPV testing on a self-collected sample for detection of CIN3+ is high and supports its use as a primary screening test for all invited women. Because of the slightly lower sensitivity of hrHPV testing on a self-collected compared to a clinician-collected sample, an evaluation of the workflow procedure to optimise clinical performance seems warranted. FUNDING: National Institute for Public Health and the Environment (the Netherlands) and the European Commission.

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BACKGROUND: In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme. METHODS: We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018 (maximum follow-up of almost 21 months) and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral (screen positivity; referral rate) and detection (cervical intraepithelial neoplasia (CIN) detection; number of referrals per detected CIN lesion). RESULTS: Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme (positivity rate: 5% vs 9%; referral rate: 1% vs 3%). CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme. CONCLUSIONS: This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening.

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BACKGROUND: The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results.During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. RESULTS: In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. CONCLUSION: The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research.

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The diagnostic landscape of non-small cell lung cancer (NSCLC) is changing rapidly with the availability of novel treatments. Despite high-level healthcare in the Netherlands, not all patients with NSCLC are tested with the currently relevant predictive tumor markers that are necessary for optimal decision-making for today's available targeted or immunotherapy. An expert workshop on the molecular diagnosis of NSCLC involving pulmonary oncologists, clinical chemists, pathologists, and clinical scientists in molecular pathology was held in the Netherlands on December 10, 2018. The aims of the workshop were to facilitate cross-disciplinary discussions regarding standards of practice, and address recent developments and associated challenges that impact future practice. This paper presents a summary of the discussions and consensus opinions of the workshop participants on the initial challenges of harmonization of the detection and clinical use of predictive markers of NSCLC. A key theme identified was the need for broader and active participation of all stakeholders involved in molecular diagnostic services for NSCLC, including healthcare professionals across all disciplines, the hospitals and clinics involved in service delivery, healthcare insurers, and industry groups involved in diagnostic and treatment innovations. Such collaboration is essential to integrate different technologies into molecular diagnostics practice, to increase nationwide patient access to novel technologies, and to ensure consensus-preferred biomarkers are tested.

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Cellular activation in trans by interferons, cytokines, and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and/or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling, and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation.

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BACKGROUND: In order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required. METHODS: An active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to -50 °C then 10 °C/min to -120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time. RESULTS: Repeat testing revealed that tissue viability between fresh and freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage.

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Recent thymic emigrants (RTEs) represent a source of antigen-naïve T cells that enter the periphery throughout life. However, whether RTEs contribute to the control of chronic parasitic infection and how their potential might be harnessed by therapeutic intervention is currently unclear. Here, we show that CD4+ recent thymic emigrants emerging into the periphery of mice with ongoing Leishmania donovani infection undergo partial activation and are recruited to sites of granulomatous inflammation. However, CD4+ RTEs displayed severely restricted differentiation either into IFNγ+ or IFNγ+TNFα+ effectors, or into IL-10-producing regulatory T cells. Effector cell differentiation in the chronically infected host was not promoted by adoptive transfer of activated dendritic cells or by allowing extended periods of post-thymic differentiation in the periphery. Nevertheless, CD4+ RTEs from infected mice retained the capacity to transfer protection into lymphopenic RAG2-/- mice. Taken together, our data indicate that RTEs emerging into a chronically inflamed environment are not recruited into the effector pool, but retain the capacity for subsequent differentiation into host protective T cells when placed in a disease-free environment.

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BACKGROUND & AIMS: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. METHODS: We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types. RESULTS: Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. CONCLUSIONS: Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology. LAY SUMMARY: Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist. DATA REPOSITORY: Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).

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BACKGROUND: Unintentional weight loss occurs among advanced non-small-cell lung cancer (NSCLC) patients and is associated with worse survival. Small studies have suggested that weight gain during treatment is associated with superior survival. PATIENTS AND METHODS: A retrospective analysis analyzed data from three international phase III studies comprising 2301 advanced, non-squamous NSCLC patients who received a platinum-based, first-line doublet, with or without bevacizumab and maintenance therapy. Body weight was recorded before and after treatment by each study's schedule. The relationship between weight gain and overall survival (OS) and progression-free survival (PFS) was assessed using log-rank test and adjusted Cox modeling. Logistic regression assessed the association between baseline covariates and post-baseline weight gain. RESULTS: Four hundred and twenty-one (18.3%) patients had >5% weight gain after baseline. More than half of the weight gain cohort exhibited initial weight gain by 3 weeks. The median OS was 16.7 months versus 10.7 months for the >5% versus ≤5% weight gain subgroup (n = 1880) (P < 0.001). PFS was 6.9 versus 4.8 months, respectively (P < 0.001). Differences in overall tumor response rate (50.8% versus 25.4%, respectively) and disease control rate (tumor response or stable disease) (91.5% versus 63.6%, respectively) were also significant (P < 0.001). The Cox modeling revealed the >5% subgroup had longer survival [hazard ratio (HR) = 0.54, 95% confidence interval (CI) 0.47-0.62; P < 0.001] than the ≤5% subgroup after adjusting for baseline factors. Similar significant results were found for PFS (HR = 0.59, 95% CI 0.52-0.67; P < 0.001). Unadjusted logistic regression indicated a significant association between weight gain (>5% versus ≤5%) and age, and BMI. CONCLUSIONS: Weight gain during treatment may be an early indicator of clinical benefit. If confirmed in prospective studies, monitoring weight change may provide important information regarding survival outcomes in NSCLC and may provide ideas for new therapeutic strategies.

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BACKGROUND: Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. METHOD: We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. RESULTS: Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. CONCLUSION: Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

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Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.

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Type 17 helper T-cell cytokines have been implicated in the pathogenesis of inflammatory bowel disease, a chronic condition affecting the gastrointestinal tract, but information regarding their contribution to pathology in different regions of the gut is lacking. By using a murine model of bacteria-induced typhlocolitis, we investigated the role of IL-17A, IL-17F, and IL-22 in cecal versus colonic inflammation. Cecal, but not colonic, pathology in C57BL/6 mice inoculated with Helicobacter hepaticus plus anti-IL-10 receptor (IL-10R) monoclonal antibody was exacerbated by co-administration of anti-IL-17A monoclonal antibody, suggesting a disease-protective role for IL-17A in the cecum. In contrast, anti-IL-17F had no effect on H. hepaticus-induced intestinal pathology. Neutralization of IL-22 prevented the development of colonic, but not cecal, inflammation in H. hepaticus-infected anti-IL-10R-treated mice, demonstrating a pathogenic role for IL-22 in the colon. Analysis of transcript levels revealed differential expression of IL-22R, IL-22 binding protein, and IL-23R between cecum and colon, a finding that may help explain why these tissues respond differently after anti-IL-22 treatment. Analysis of microarray data from healthy human intestine further revealed significant differences in cytokine receptor transcript levels (including IL-22RA1 and IL-23R) in distinct parts of the human gut. Together, our findings demonstrate that individual type 17 helper T-cell cytokines can have proinflammatory or anti-inflammatory effects in different regions of the intestine, an observation that may have implications for interventions against human inflammatory bowel disease.

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OBJECTIVES: To compare next-generation sequencing (NGS) platforms with mutation-specific analysis platforms in a clinical setting, in terms of sensitivity, mutation specificity, costs, capacity, and ease of use. METHODS: We analyzed 25 formalin-fixed, paraffin-embedded lung cancer samples of different size and tumor percentage for known KRAS and EGFR hotspot mutations with two dedicated genotyping platforms (cobas [Roche Diagnostics, Almere, The Netherlands] and Rotor-Gene [QIAGEN, Venlo, The Netherlands]) and two NGS platforms (454 Genome Sequencer [GS] junior [Roche Diagnostics] and Ion Torrent Personal Genome Machine [Life Technologies, Bleiswijk, The Netherlands]). RESULTS: All platforms, except the 454 GS junior, detected the mutations originally detected by Sanger sequencing and high-resolution melting prescreening and detected an additional KRAS mutation. The dedicated genotyping platforms outperformed the NGS platforms in speed and ease of use. The large sequencing capacity of the NGS platforms enabled them to deliver all mutation information for all samples at once. CONCLUSIONS: Sensitivity for detecting mutations was highly comparable among all platforms. The choice for either a dedicated genotyping platform or an NGS platform is basically a trade-off between speed and genetic information.

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BACKGROUND: Aggressive fibromatosis (AF) comprises tumors with a varying biological behavior. Genetic tumor characteristics may be predictive of recurrence; hence, the prognostic value of three specific mutations on the CTNNB1 gene was evaluated in relation to known clinicopathologic risk factors in patients with primary, sporadic AF. METHODS: In a multi-institutional retrospective cohort study of patients with primary extra-abdominal and abdominal wall AF who underwent surgical treatment, the original pathology specimens were reviewed for the presence of a T41A, S45F, and 45P mutations on the CTNNB1 gene. For these mutations, the risk of recurrence was analyzed by the Kaplan-Meier method with log-rank test. Univariable and multivariable Cox regression was performed to calculate hazard ratios. RESULTS: A total of 101 patients were analyzed. During a median follow-up of 41 months, 17 recurrences were detected; the cumulative 5-year recurrence rate was 22.8 %. A specific CTNNB1 mutation was found in 76 patients, with the majority of patients having a T41A mutation (n = 49). CTNNB1 mutations were associated with the risk of recurrence: the presence of a S45F mutation was associated with a 5-year cumulative risk of recurrence of 63.8 % (P < 0.001). Multivariable analysis showed that young age and S45F mutation were independent risk factors (P = 0.011 and P < 0.001). CONCLUSIONS: The presence of specific CTNNB1 mutations was predictive for recurrence in patients after surgical treatment for primary, sporadic extra-abdominal and abdominal AF. A S45F mutation increased the risk of recurrence significantly.

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In routine cancer molecular pathology, various independent experiments are required to determine mutation and amplification status of clinically relevant genes. Most of these tests are designed to identify a limited number of genetic aberrations, most likely in a given tumor type. We present a modified version of a multiplexed PCR and IonTorrent-based sequencing approach that can replace a large number of existing assays. The test allows for the simultaneous detection of point mutations and gene amplifications in 40 genes, including known hotspot regions in oncogenes (KRAS, BRAF), inactivating mutations in tumor suppressors (TP53, PTEN), and oncogene amplifications (ERBB2, EGFR). All point mutations were confirmed using certified diagnostic assays, and a sensitivity and specificity of 100% (95% CI, 0.875-1.0) and 99% (95% CI, 0.960-0.999), respectively, were determined for amplifications in FFPE material. Implementation of a single assay to effectively detect mutations and amplifications in clinically relevant genes not only improves the efficiency of the workflow within diagnostic laboratories but also increases the chance of detecting (rare) actionable variants for a given tumor type that are typically missed in routine pathology. The ability to obtain comprehensive and rapid mutational overviews is key for improving the efficiency of cancer patient care through tailoring treatments based on the genetic characteristics of individual tumors.

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BCR-ABL1-specific tyrosine kinase inhibitors prolong the life of patients with chronic myeloid leukemia (CML) but cannot completely eradicate CML progenitors. The BH3 mimetic, ABT-263, targets prosurvival BCL2 family members, and has activity against CML progenitors. However, the inhibitory effect of ABT-263 on BCL-XL, which mediates platelet survival, produces dose-limiting thrombocytopenia. A second-generation BH3 mimetic, ABT-199, has been developed to specifically bind BCL2 but not BCL-XL. We determined the activity of ABT-199 against CML cell lines, as well as primary CML and normal cord blood (NCB) progenitors. We find that BCL2 expression levels predict sensitivity to ABT-199 in CML and NCB progenitors, and that high NCB BCL2 levels may explain the reported hematologic toxicities in ABT-199-treated patients. Also, while single agent ABT-199 has modest activity against CML progenitors, when combined with imatinib, ABT-199 significantly enhances imatinib activity against CML progenitors at concentrations predicted to avoid hematologic toxicities.

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A broad range of anti-cancer agents, including glucocorticoids (GCs) and tyrosine kinase inhibitors (TKIs), kill cells by upregulating the pro-apoptotic BCL2 family member, BIM. A common germline deletion in the BIM gene was recently shown to favor the production of non-apoptotic BIM isoforms, and to predict inferior responses in TKI-treated chronic myeloid leukemia (CML) and EGFR-driven lung cancer patients. Given that both in vitro and in vivo GC resistance are predictive of adverse outcomes in acute lymphoblastic leukemia (ALL), we hypothesized that this polymorphism would mediate GC resistance, and serve as a biomarker of poor response in ALL. Accordingly, we used zinc finger nucleases to generate ALL cell lines with the BIM deletion, and confirmed the ability of the deletion to mediate GC resistance in vitro. In contrast to CML and lung cancer, the BIM deletion did not predict for poorer clinical outcome in a retrospective analysis of 411 pediatric ALL patients who were uniformly treated with GCs and chemotherapy. Underlying the lack of prognostic significance, we found that the chemotherapy agents used in our cohort (vincristine, L-asparaginase, and methotrexate) were each able to induce ALL cell death in a BIM-independent fashion, and resensitize BIM deletion-containing cells to GCs. Together, our work demonstrates how effective therapy can overcome intrinsic resistance in ALL patients, and suggests the potential of using combinations of drugs that work via divergent mechanisms of cell killing to surmount BIM deletion-mediated drug resistance in other cancers.

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BACKGROUND: HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH). METHODS: As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases). RESULTS: The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining. CONCLUSIONS: MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.

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