At last, cancer reproducibility project releases some results — and they’re mixed

Nearly five years ago, researchers suggested that the vast majority of preclinical cancer research wouldn’t hold up to follow-up experiments, delaying much needed treatments for patients. In a series of articles publishing tomorrow morning, eLife has released the results of the first five attempts to replicate experiments in cancer biology — and the results are decidedly mixed.

As our co-founders Adam Marcus and Ivan Oransky write in STAT, the overall take-home message was that two studies generated findings similar to the original, one did not replicate the original, and two others were inconclusive.

They quote Brian Nosek, a psychologist at the University of Virginia, in Charlottesville, who runs the Center for Open Science, who has been leading the replication effort in his own field:

Reproducibility is hard, and once you fail to reproduce something it isn’t always obvious why.

The new articles, added Nosek,

are additional evidence that we don’t quite understand this as well as we thought we did.

(Full disclosure: Retraction Watch is partnering with the Center on a database of retractions.)

This is just the first wave of results: The authors plan on performing and publishing more than 20 replication efforts. And they had hoped for more, Marcus and Oransky write:

The effort isn’t cheap: Each replication took an average of nearly seven months to complete, at an average cost of about $27,000, according to data from the investigators.  (Budget concerns had earlier forced the group to cut from 50 to 37 the number of trials they could re-run.)

The effort would be easier if researchers cooperated, they add:

Although many researchers the group contacted were gracious and eager to help – providing additional information about their methods – others were less forthcoming. One group took 111 days to respond; another ignored the request.

In an accompanying editorial publishing tomorrow, Nosek and Timothy Errington, the “metascience manager” at the Center for Open Science, caution that being able to replicate a result doesn’t automatically mean the result is correct — and vice versa:

Scientific claims gain credibility by accumulating evidence from multiple experiments, and a single study cannot provide conclusive evidence for or against a claim. Equally, a single replication cannot make a definitive statement about the original finding. However, the new evidence provided by a replication can increase or decrease confidence in the reproducibility of the original finding.

Indeed, as an accompanying editorial notes, one of the studies whose replication efforts produced somewhat inconclusive results has led to a clinical trial for an antibody therapy. Whether or not it is effective in patients remains to be seen.

For Adam and Ivan’s whole column, click here.

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Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy

Teresa Davoli, Hajime Uno, Eric C. Wooten, Stephen J. Elledge

Structured Abstract

INTRODUCTION

Aneuploidy, also known as somatic copy number alterations (SCNAs), is widespread in human cancers and has been proposed to drive tumorigenesis. The relationship between SCNAs and the characteristic functional features or “hallmarks” of cancer is not well understood. Among these cancer hallmarks is immune evasion, which is accomplished by neoantigen editing, defects in antigen presentation and inhibition of tumor infiltration, and/or cytotoxic activities of immune cells. Whether and how tumor SCNA levels influence immune evasion is of particular interest as this information could potentially be used to improve the efficacy of immune checkpoint blockade, a therapy that has produced durable responses in a subset of cancer patients.

RATIONALE

Understanding how SCNAs and mutation load affect tumor evolution, and through what mechanisms, is a key objective in cancer research. To explore the relationships between SCNA levels, tumor mutations, and cancer hallmarks, we examined data from 5255 tumor/normal samples representing 12 cancer types from The Cancer Genome Atlas project. We assigned each tumor an SCNA score and looked for correlations with the number and types of tumor mutations. We also compared the gene expression profiles of tumors with high versus low SCNA levels to identify differences in cellular signaling pathways.

RESULTS

First, we found that, for most tumors, there was a positive correlation between SCNA levels and the total number of mutations. Second, tumors harboring activating oncogenic mutations in the receptor tyrosine kinase–RAS–phosphatidylinositol 3-kinase pathway showed fewer SCNAs, a finding at odds with the hypothesis of oncogene-driven genomic instability. Third, we found that tumors with high levels of SCNAs showed elevated expression of cell cycle and cell proliferation markers (cell cycle signature) and reduced expression of markers for cytotoxic immune cell infiltrates (immune signature). The increased expression level of the cell cycle signature was primarily predicted by focal SCNAs, with a lesser contribution of arm and whole-chromosome SCNAs. In contrast, the lower expression level of the immune signature was primarily predicted by high levels of arm and whole-chromosome SCNAs. SCNA levels were a stronger predictor of markers of cytotoxic immune cell infiltration than tumor mutational load. Finally, through analysis of data from two published clinical trials of immunotherapy in melanoma patients, we found that high SCNA levels in tumors correlated with poorer survival of patients. The combination of the tumor SCNA score and the tumor mutational load was a better predictor of survival after immunotherapy than either biomarker alone.

CONCLUSION

We found that two hallmarks of cancer, cell proliferation and immune evasion, are predicted by distinct types of aneuploidy that likely act through distinct mechanisms. Proliferation markers mainly correlated with focal SCNAs, implying a mechanism related to the action of specific genes targeted by these SCNAs. Immune evasion markers mainly correlated with arm- and chromosome-level SCNAs, consistent with a mechanism related to general gene dosage imbalance rather than the action of specific genes. A retrospective analysis of melanoma patients treated with immune checkpoint blockade anti–CTLA-4 (cytotoxic T lymphocyte–associated protein 4) therapy revealed that high SCNA levels were associated with a poorer response, suggesting that tumor aneuploidy might be a useful biomarker for predicting which patients are most likely to benefit from this therapy.

Genetic events associated with two cancer hallmarks: cell proliferation and immune evasion.

Across several human tumor types, high SCNA levels correlate with increased expression of cell cycle markers and decreased expression of markers of cytotoxic immune cell infiltrates. A high load of tumor neoantigens (reflecting a high level of point mutations) promotes the detection of tumors by the immune system, limiting immune evasion. The relative contribution of focal, arm/chromosome, and neoantigen load to the prediction of proliferation and immune evasion is shown.

Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination

Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination

 

Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ~60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

Determinants of HIV-1 broadly neutralizing antibody induction

Determinants of HIV-1 broadly neutralizing antibody induction

Broadly neutralizing antibodies (bnAbs) are a focal component of HIV-1 vaccine design, yet basic aspects of their induction remain poorly understood. Here we report on viral, host and disease factors that steer bnAb evolution using the results of a systematic survey in 4,484 HIV-1-infected individuals that identified 239 bnAb inducers. We show that three parameters that reflect the exposure to antigen—viral load, length of untreated infection and viral diversity—independently drive bnAb evolution. Notably, black participants showed significantly (P = 0.0086–0.038) higher rates of bnAb induction than white participants. Neutralization fingerprint analysis, which was used to delineate plasma specificity, identified strong virus subtype dependencies, with higher frequencies of CD4-binding-site bnAbs in infection with subtype B viruses (P = 0.02) and higher frequencies of V2-glycan-specific bnAbs in infection with non–subtype B viruses (P = 1 × 10−5). Thus, key host, disease and viral determinants, including subtype-specific envelope features that determine bnAb specificity, remain to be unraveled and harnessed for bnAb-based vaccine design.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes

Accepted
Published online

Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing

http://www.pnas.org/content/early/2016/08/10/1607794113.full.pdf

Margaret L. Hoanga,b,1, Isaac Kindea,b,2, Cristian Tomasettic,d, K. Wyatt McMahona,b, Thomas A. Rosenquiste , Arthur P. Grollmane,f, Kenneth W. Kinzlera,3, Bert Vogelsteina,b,g,3, and Nickolas Papadopoulos

We present the bottleneck sequencing system (BotSeqS), a nextgeneration sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. We use BotSeqS to show age- and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal human tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues.

 

 

 

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children

Michelle M. Stein, B.S., Cara L. Hrusch, Ph.D., Justyna Gozdz, B.A., Catherine Igartua, B.S., Vadim Pivniouk, Ph.D., Sean E. Murray, B.S., Julie G. Ledford, Ph.D., Mauricius Marques dos Santos, B.S., Rebecca L. Anderson, M.S., Nervana Metwali, Ph.D., Julia W. Neilson, Ph.D., Raina M. Maier, Ph.D., Jack A. Gilbert, Ph.D., Mark Holbreich, M.D., Peter S. Thorne, Ph.D., Fernando D. Martinez, M.D., Erika von Mutius, M.D., Donata Vercelli, M.D., Carole Ober, Ph.D., and Anne I. Sperling, Ph.D.

N Engl J Med 2016; 375:411-421August 4, 2016DOI: 10.1056/NEJMoa1508749

BACKGROUND

The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities.

METHODS

We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma.

RESULTS

Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling.

CONCLUSIONS

The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.)

NIH sets new postdoc stipend levels

In response to a new rule governing overtime pay in the United States, the National Institutes of Health yesterday announced its new, increased postdoc stipend levels, which will go into effect 1 December 2017. The starting base salary will be $47,484, up approximately 9% from the 2016 level of $43,692 and just above the threshold of $47,476, below which employers are required to pay employees for overtime. The new rates apply specifically to those supported by Ruth L. Kirschstein National Research Service Awards (NRSAs), but they are likely to be relevant to postdocs funded by other mechanisms as well because many institutions use the NRSA levels as guidelines for setting postdoctoral salaries.

“It’s not as much as we hoped,” says Future of Research Executive Director Gary McDowell, noting that the National Academies and other groups have recommended that postdocs be paid a minimum $50,000, “but it’s certainly a step in the right direction.”

Under the new stipend schedule, the annual increases for the first 2 years will be significantly decreased: just 0.8%, as compared to the current increase of approximately 4%. At the third year, the new stipends begin to increase at about the 4% rate. “It’ll be interesting to see if there’s a loss of postdocs around year 3 because that’s when they start becoming progressively more expensive for labs,” says Rescuing Biomedical Research Director Chris Pickett. The pressure of supporting a higher salary at that point may increase the pressure for a postdoc to move on, which isn’t necessarily a bad situation, he says. “It’s definitely important to reduce the postdoc period to get people moving on to their next career spot, especially if they’re doing a postdoc as a default. There’s no reason to stay in a [default] postdoc terribly long.”

The next step, Pickett adds, will be monitoring how institutions implement the new regulation, such as determining whether postdocs will be required to pay more for their health insurance. In addition, McDowell says, more uniformity in how postdocs are identified would help ensure that institutions comply with the new law.

Dense transcript profiling in single cells by image correlation decoding

Nat Methods. 2016 Aug;13(8):657-60. doi: 10.1038/nmeth.3895. Epub 2016 Jun 6.
Dense transcript profiling in single cells by image correlation decoding.
Coskun AF1,2, Cai L1,2.
Author information
Abstract
Sequential barcoded fluorescent in situ hybridization (seqFISH) allows large numbers of molecular species to be accurately detected in single cells, but multiplexing is limited by the density of barcoded objects. We present correlation FISH (corrFISH), a method to resolve dense temporal barcodes in sequential hybridization experiments. Using corrFISH, we quantified highly expressed ribosomal protein genes in single cultured cells and mouse thymus sections, revealing cell-type-specific gene expression.

PMID: 27271198 PMCID: PMC4965285 [Available on 2017-02-01] DOI: 10.1038/nmeth.3895

Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease

For those following the C9ORF72 story, this is a very interesting twist!

Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease

Aaron Burberry1,2, Naoki Suzuki1,2, Jin-Yuan Wang1,2, Rob Moccia1,2, Daniel A. Mordes1,2,3, Morag H. Stewart1,4, Satomi Suzuki-Uematsu1,2, Sulagna Ghosh1,2, Ajay Singh1,2, Florian T. Merkle1,2, Kathryn Koszka1,2, Quan-Zhen Li5, Leonard Zon1,6, Derrick J. Rossi1,4,6, Jennifer J. Trowbridge7, Luigi D. Notarangelo6,8 and Kevin Eggan1,2,*

Abstract

C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.