THE NIEHS AND RAMAZZINI INSTITUTE COLLABORATION: LOOKING TO THE PAST AND FRAMING THE FUTURE

John Bucher, Ph.D. Associate Director, National Toxicology ProgramJohn Bucher, Ph.D. Associate Director, National Toxicology Program

NIEHS

Fiorella Belpoggi, Ph.D., FIATP

Director, CMCRC, Ramazzini Institute

• Drs. Ken Olden and Prof. Cesare Maltoni sign agreement June 22, 2000

– Rodent cancer bioassays

– Interactions of genetic susceptibility and environmental exposures

– Basic mechanisms of carcinogenesis

– Statistical methods for the evaluation of bioassay data

Establishment of Formal Collaboration

– Statistical methods for the evaluation of bioassay data

• Renewals approximately every 2-3 years

• Philosophy behind rodent cancer assay designs

– Explored in reciprocal staff visits beginning in Feb. 2001

– Subject of New York Academy of Sciences Symposium honoring Prof. Maltoni in April. 2002, many RI and NIEHS staff spoke including Drs Soffritti, Belpoggi, Huff, Melnick

• Comparisons of pathology diagnoses

Early Initiatives 2001-2004

– Dr. Belpoggi first of several visits to NIEHS with selected lesions from rat studies in June 2001, discussions over intercurrent infections begin

– General agreement in lesions, differences in some terminology, medical vs veterinary

– Installation of electronic data capture system (TDMSE) at the RI in 2003

• Exploring mechanisms of carcinogenesis

– Dr. Luca Lambertini spends 2 years with Dr Robert Sills and Teddy Devereux, 2002-4

– Publishes on involvement on P53 and H-ras in squamous cell carcinomas

• Harmonization of nomenclature

• Loading of pathology findings

– Aspartame (1)- later evaluated by EFSA

– TAME- utilized by US EPA

2004-2007

– ETBE- utilized by US EPA

– Methanol- later utilized by US EPA

• Available on RI website 2007

• Loading of formaldehyde and gasoline mixtures

• Dr. Belpoggi visits NIEHS with selected mouse lesions

• Decision reached to carry out formal PWGs- 2009 renewal of agreement

• Dr. Soffritti visits NIEHS in Mar. 2009 and presents aspartame mouse studies and preliminary EMF findings

2008-2009

mouse studies and preliminary EMF findings

• Methanol Institute disputes EPA reliance on RI studies

• NTP pathologists visit RI in April to conduct a limited pathology audit and determine feasibility for an NTP style Pathology Working Group (PWG)

– Audit found few discrepancies in records and pathology specimens-“typical for laboratory doing these types of studies”

2010

– Somewhat fewer neoplasms and more inflammatory lesions than diagnosed by the RI

– Fewer disagreements in samples from more recent studies

• Recommendation to proceed with a full PWG

• EPA identified a number of studies of particular interest

• NIEHS and RI agree to begin studies to establish clonality of lymphomas diagnosed in the lung

• Quality Assurance team from Experimental Pathology Laboratories (EPL) visits RI to prepare PWG

• PWG carried out April 4-8, coordinated by Dr. Robert Maronpot, former Chief of Pathology at NIEHS

• PWG participants selected by NIEHS based on

2011

• PWG participants selected by NIEHS based on experience in rodent pathology, rodent diseases, including an MD pathologist familiar with human pathology terminology

• Good diagnostic agreement- only minor issues in terminology for acrylonitrile, ETBE, and VC

• More frequent disagreements for lymphoid neoplasms and neoplasms of the nasal cavity and ear canal in MTBE and methanol studies

• Neoplasms considered hyperplasias or bone remodeling with osteoid formation secondary to chronic airway inflammation and otis media, some hyperplasias had neoplastic features

PWG Results

some hyperplasias had neoplastic features

• Some pulmonary lymphomas without evidence of involvement of other organs were confirmed

• The frequency and nature of the differences in diagnostic opinions between the PWG and the RI SP were not considered unusual for studies of this type

• Enormous amount of work, outstanding cooperation and accommodation

• PWGs represent a second opinion and do not alter the RI conclusions

• Dr. Alex Merrick (NIEHS) examines the potential utilization of the RI archival tissues for gene expression studies by “NextGen” RNA seqencing

• Simona Panzacchi (RI) visited laboratory of Dr. David Malarkey (NIEHS) from May to Nov. 2013 to refine immunohistochemicaltechniques for lymphoma diagnosis

2012-present

techniques for lymphoma diagnosis

• Both remain active areas of investigation

• Collaboration is a continuing success

�10

The future The future The future The future collaborationcollaborationcollaborationcollaboration

Continuation of existing projects

� Characterization of lymphomas

� Toxicology Data Managing System (TDMSE) on compound of

interest

� PWGs on compound of interest

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� Comparison of results for the long-term studies on ELFEMF

and RFEMF performed both at NTP and CMCRC (blue

book?..). This includes:

� TDMSE

� PWG on lesions of interest

� reports shared between the 2 institutions

48

hours

1-2

years

1

week

4-5

years

Example: CD68 on normal spleen fixed in alcohol

week years

1

month

5-10

years

DNA/RNA NanoDrop quantification (NTP Dr. Merrick )

�“As NextGen sequencing becomes less expensive and methods for fixed tissue become more robust, the Ramazzini archival repository should commensurately rise in value “ (B. Alex Merrick, Ph.D., DHMRI NextGen Sequencing Contract, Project Officer, NIEHS, 2013)Project Officer, NIEHS, 2013)

Molecular biology: CMCRC projects proposed to NIEHS

NextGen sequencing when improved will allow to perform:

� Evaluation of gene expression in mammary cancers

observed as outcomes for synergistic effects of ELFEMF

and gamma radiation (AFPE* and frozen tissues)

� Comparative gene expression of different compounds � Comparative gene expression of different compounds

studied at the CMCRC, which are related to formaldehyde

directly or as a metabolite (formaldehyde, MTBE, methanol,

aspartame) and where an increase of lymphomas was

observed (AFPE and frozen tissues for aspartame and

formaldehyde)

*AFPE: alcohol fixed/paraffin embedded14

Molecular biology: CMCRC projects proposed to NIEHS

� The study of non monotonic curves (AFPE and frozen tissues)

doing some additional short term dosing studies to look for possible reasons

VDC

� The “exposome” concept encompasses the totality of exposures from conception onwards, complementing the genome. We are convinced that our human-equivalent model could represent a very appropriate experimental model to assess and study “exposome”

*AFPE: alcohol fixed/paraffin embedded15

Conclusions

“The reward of great men is that, long after they have died,

one is not quite sure that they are dead”

Jules Renard , 1864 -1910

Conclusions

“The reward of great men is that, long after they have died,

one is not quite sure that they are dead”

Jules Renard , 1864 -1910

• Robert R. Maronpot, DVM, MPH (EPL, Durham, NC)

• QA Pathologists

• Jerry F. Hardisty, DVM (EPL, Durham, NC)

• Peter C. Mann, DVM (EPL, Durham, NC)

• PWG Members

2011

• Charles Clifford, DVM, PhD (Charles River Labs)

• Sabine Francke-Carroll, DVM, PhD (U.S. Food and Drug Administration)

• Peter Greaves, MBChB (Leicester University, United Kingdom)

• James B. Nold, DVM, PhD (Biotechnics, Hillsborough, NC)

• Observers Steven Mog, DVM (U.S. Food and Drug Administration)

• RI Observers Fiorella Belpoggi, PhD, Morando Soffritti, MD Eva Tibaldi, PhD, Laura Falcioni, DVM