Opportunities for Infectomic Investigation in Childhood Cancer

Dr Tarik GHEITInfections and Cancer Biology Group

International Childhood Cancer Cohort Consortium (I4C)

This Talk

1) Development of efficient and sensitive diagnostic tools for the

detection of infectious agents

2) Examples of studies

Infectious agents, especially viruses, account for up to 20% of all cancers

H.pylori : 5.5%

HPV: 5.2%

HBV & HCV: 4.9%

EBV: 1%

HIV/HHV-8: 0.9%

Other: 0.15%

• Several independent studies support the hypothesis of the involvement of infectious agents in the childhood carcinogenesis.

• Acute leukemia (AK) is the most frequent malignancy in children, however the etiological factor(s) remain(s) almost unknown.

• Approximately 80% of AK have a B-cell origin, a type of cancer with an increasing incidence in the Western world.

• The fact that the increased incidence of this pathological condition correlates with the increaseof other childhood-diagnosed immune-related illnesses (e.g. allergy, asthma, and type 1 diabetes)

supports the possible role of early childhood infections in carcinogenesis.

• Many viruses have a tropism for lymphocytes and monocytes, such as HTLV-1 and 2, HIV, EBV, CMV, HHV6, HHV7, several members of the polyomavirus family (e.g. JCV) and HBV.

• Some of these viruses have been clearly associated with carcinogenesis. For instance, HTLV-1 is the etiological factor of adult T leukemia (ATL) and EBV infection is associated with several human B cell malignancies, including Burkitt's lymphoma (BL), a very common cancer of children in equatorial Africa.

How ICB group can contribute to determine whether there is an association between a broad spectrum of known infectious agents

and childhood cancer?

A – Characterization of the transforming activities of viral proteins in vitro and in vivo models

Efficient and sensitive diagnostic tools

To study the association of infectious agents with human cancers

B – Prevalence determination of infectious agents in human specimens

Strategies:

Virus / Cancer association

The existing diagnostic tools use degenerate / consensus primers that have low / high affinity for specific infectious agents (i.e. HPV genotyping assays: GP5+/6+, PGMY, MY09/11)

Diagnostic tools for the detection of infectious agents in a context of multiple infections

The use of specific primers increases specificity and sensitivity

Advantage : Single PCR

Disadvantage : Their performance in detecting multiple infections ?

Disadvantages : Problems due to performing several PCR reactions (time consuming, cross-contaminations, biological samples limitation)

SENSITIVITY (specific primers)

SINGLE PCR REACTION (in one tube)

Development of multiplex PCR

• In case of multiple infections, different infectious agents will not compete for the same primer set, which is in contrast to usual detection methods that use only one primer set

Bead-based hybridisation assay (LUMINEX)Bead-based hybridisation assay (LUMINEX)

Use of high throughput technology for the identification of infectious agents

1. Array reader

2. Microplate platform4. PC

3. High-throughput fluidics (HTF)

The Bio-Plex® 200 system is a suspension array system that permits analysis of up to 100 biomolecules in a single sample:

4. PC and monitor / Bio-Plex Manager™ software — controls the instrument, data acquisition, and data analysis

1. Array reader — combines 2 lasers, high-throughput fluidics, and real-time digital signal processing to distinguish up to 100 different color-coded bead sets, each representing a different assay

2. Microplate platform — automates the reading of 96-well plates

3. High-throughput fluidics (HTF) — delivers up to 20 L of sheath fluid (40 plates) without user intervention

5.6 µm

polystyrene bead

Luminex technology

100 bead sets

• The beads are filled with two fluorescent dyes in various ratio : 100 different bead sets

100 color-codes = 100 simultaneous tests

Multiplex PCR in one tube (+ β-Globin primers) / biotinylated primers

A

B

C

D

E

F

X

…

probe

bead

Design of specific probes for the identification of the different amplicons generated by the multiplex PCR

Specific probes coupled to distinct coloured Luminex bead sets (one set per infectious agent)

Mix beads-probes with PCR products

Complex biotin / streptavidin-R-phycoerythrin

Beads are analyzed in the Luminex reader, which contains two lasers to identify the bead set and to quantify the reporter fluorescence on the bead

biotinylated PCRproduct/probe complex

bead

• Microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by separate laser beams

A red laser excites the dyes in each bead to identify them

A green reporter laser excites the fluorescent reporter molecule to detect and quantify the captured target

The Luminex system takes advantage of flow cytometry technology, allowing rapid measurements

The Luminex analyzer allows for the simultaneous measurement of up to 100 analytes using small sample volumes

Each line represents a single well where the PCR product is mixed with 1-100 distinct bead sets

The results are expressed as median fluorescence intensity (MFI) which represents the median signal intensity measured per microsphere set

Infectious agents detected with Luminex assays at IARC (n = 129)

LR, pHR, HR-HPV mucosal types (n=21) 6, 11,16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68a, 68b, 70, 73 and 82.

Gamma HPV (n=30) 4, 65, 95, 60, 48, 50, 88, 95, 101, 103, 108, 109, 112, 116, 119, 121, 123, 126, 127, 128, 129, 130, 131, 132, 133, 134, 148, 149, 156, SD2

Cutaneous beta HPV types (n=43) 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, 96, 98, 99, 100, 104, 105, 107, 110, 111, 113, 115, 118, 120, 122, 124, 143, 145, 150, 151

HPV alpha, mu and gamma / warts (n = 7) 1, 2, 3, 4, 10, 27, 57

Polyomaviruses (n=12) BKV, WUV, KIV, MCV, JCV, HPyV6, HPyV7, HPyV9, TSV, HPyV10,

HPyV12 and SV40

Herpesviruses (n = 8) HSV1, HSV2, VZ, EBV (1 and 2), CMV, HH6, HH7, HH8,

Other infectious agents (n = 8) Chlamydia T., HBV, Fusobacterium, MMTV, Helicobacter Pylori, Schistosoma (haematobium, mansoni, japonicum)

Multiplex PCR with HPV type specific primers (+ β-Globin primers)

HPV16

HPV18

HPV31

HPV45

HPV51

HPV52

HPV82

…

• 31 HPV type E7 specific primers were used in one multiplex PCR reaction

Gheit et al., 2006. JCM.

Design of specific probes for the identification of 19 pHr / Hr HPV types

Specificity of 20 HPV type-specific probes in multiplex HPV genotyping (each line represents a single well with the PCR product hybridized to a mixture of 20 distinct bead sets)

DNA detection of 19 mucosal HPV types examined is highly specific Addition of HPV 6, 11

Schmitt et al., 2010. JCM.

Results are expressed as MFI values (Median Fluorescence Intensity)

The analytical sensitivity of the multiplex TS-PCR E7 primers was determined with a 10-fold dilutions series of plasmids containing genomic DNA from HPV types in the presence of 100 ng of human placenta DNA or HPV-negative cervical cell line C33A DNA.

Analytical sensitivity of TS-MPG.

Schmitt et al., 2010. JCM.

The E7 multiplex TS-PCR primer set detected all HPV genotypes analyzed, with the sensitivities ranging from 10 to 1,000 copies of the viral genome.

• Comparison of TS-MPG with BS (GP5+/6+)-MPG for the detection of 19 HPV types in samples of cervical exfoliated cells from 649 women in the general population in Mongolia and samples from 86 Mongolian cervical cancer patients (Schmitt et al., 2010. JCM).

VALIDATION

• Cytological specimens from 94 women with atypical squamous cells of undetermined significance (ASC-US) and 86 women with negative Pap tests (TS-MPG vs Roche Linear Array) (Comar et al., 2013. JCM).

The increased sensitivity of the TS-MPG assay for the detection of multiple HPV infections is explained by the use of HPV type-specific primers rather than only one pair of consensus primers as for the LA and GP5+/6+ assays.

The TS-MPG assay detected a statistically significant greater proportion of multiple infections compared to the LA or GP5+/6+ assay (Up to nine HPV types were detected in a single sample by TS-MPG).

Higher sensitivity of the TS-MPG assay compared to conventional HPV amplification methods.

OTHER ASSAYS . . .

25 Beta HPV types / LUMINEX

DNA detection of 25 Beta HPV types examined is highly specific Addition of primers and probes for the detection of 18 new Beta HPV types

POLYOMAVIRUSES

Type BKV KIV JCV WUV MCV HPyV7 HPyV6 SV40 TSV Beta Globin

KIV 2 481 1 1 1 2 0 2 1 3

JCV 4 9 59 1 2 2 3 3 2 4

WUV 3 8 1 404 1 2 1 3 2 3

MCV 3 10 1 2 1894 1 1 3 1 2

PyV7 3 8 1 2 2 119 1 2 2 2

PyV6 4 9 1 1 2 1 41 3 2 4

SV40 3 8 1 2 2 2 1 77 2 4

BKV 33 9 1 1 2 1 2 3 1 3

BKV 50 9 1 1 1 1 1 2 1 3

• Addition of HPyV9, HPyV10 and HPyV12 probes

• Polyomavirus / LT Expression

Approximately 100ng of total human DNA is sufficient to perform each Luminex assay.

Biological materials required for detection DS DNA viruses

Skin cells collected using multiple swabbing techniques

So far, we have been able to extract DNA and perform the Luminex assays from the following specimens:

Formalin fixed tissues in paraffin blocks

Frozen tissues

Exfoliated cervical cells in PBS or fixative (e.g. ThinPrep)

Urine

Oral brushes

Breast ductal lavages

Eyebrow hairs

Saliva

Blood

EZ1 Advanced XL robot for DNA extraction / Qiagen

High-throughput DNA purification

1) Development of efficient and sensitive diagnostic tools for the

detections of infectious agents

2) Examples of studies

This Talk

Infectious agents Anatomic regions Collaborators Countries

Mucosal and Cutaneous HPV Ductal lavage of the breast Pr Umberto Veronesi, Dr Cazzaniga Massimiliano

Italy

Mucosal and Cutaneous HPV Cells from oral cavity Dr Gary Clifford, Dr Silvia Franceschi France

Mucosal and Cutaneous HPV

Polyomaviruses

Non-melanoma skin cancer Dr Dana Rollison USA

Mucosal and Cutaneous HPV Milk / colostrum / Breast cancer Pr Suminori Akiba, Mrs Noureen Khan Japan and Pakistan

Mucosal HPV Cervical cancer cases Pr Nitin Gangane, Dr Mario Sideri India and Italy

Mucosal HPV Oesophageal cancer cases Dr Silvia Franceschi, Dr Min Dai, Dr Pierre Hainaut

China and Kenya

Mucosal and Cutaneous HPV Cutaneous melanoma Pr François Aubin France

Mucosal HPV Cervical scrapings Dr Bolormaa Dondog, Dr Michael Pawlita Mongolia

Cutaneous HPV Plucked eyebrow hairs from immunosuppressed organ transplant recipients

Pr François Aubin France

Mucosal HPV CIN I, CIN II, CIN III and cervical cancer cases

Dr Sankar India

Detection of infectious agents in different anatomic regions

Anatomic regions : Lung, colon, breast, cervix, skin, parotide gland, head and neck, penil, vulva, bladder, œsophagus, prostate…

Transfer of the Luminex technology in other country such as India

Monitor clinical trials : 2 doses vs 3 doses of HPV vaccine in India (Pr. Radhakrishna Pillai (RGCB), Drs Sankaranarayanan (IARC) / Tommasino (IARC) / Pawlita (DKFZ))

• 20,000 healthy, unmarried girls aged 10-18 years

• HPV DNA (IARC) and HPV serology (DKFZ) methods / LUMINEX

Transfer of the Luminex technology to Brazil, Colombia, Italy, Czech republic, …

CONCLUSION

One PCR amplification is performed in a single tube : we avoid the risk of contamination

Detection of infectious agents with high sensitivity in single and multiple infections

Therefore our assays could be used to study the association between infections by known infectious agents and cancer

in child

The Luminex technology fullfill requirements to perform epidemiological studies : high sample throughput (several hundred analysis per day), time-saving & cost-effective

Luminex assays are powerful for detection of infectious agents and a valid and versatile tool to study the association between infections and cancer.

We have developed assays (degenerated primers) for the identification of unknown viruses belonging to Polyomavirus and Herpesvirus families.

International Agency for Research on Cancer (IARC) Lyon, France

Massimo Tommasino

Sandrine McKay-Chopin

ICB group

German Cancer Research Center (DKFZ) Heidelberg, Germany

Michael Pawlita

Markus Schmitt

THANK YOU FOR YOUR ATTENTION

Infectious agents detected with Luminex assays at IARC (n = 129)

LR, pHR, HR-HPV mucosal types (n=21) 6, 11,16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68a, 68b, 70, 73 and 82.

Gamma HPV (n=30) 4, 65, 95, 60, 48, 50, 88, 95, 101, 103, 108, 109, 112, 116, 119, 121, 123, 126, 127, 128, 129, 130, 131, 132, 133, 134, 148, 149, 156, SD2

Cutaneous beta HPV types (n=43) 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, 96, 98, 99, 100, 104, 105, 107, 110, 111, 113, 115, 118, 120, 122, 124, 143, 145, 150, 151

HPV alpha, mu and gamma / warts (n = 7) 1, 2, 3, 4, 10, 27, 57

Polyomaviruses (n=12) BKV, WUV, KIV, MCV, JCV, HPyV6, HPyV7, HPyV9, TSV, HPyV10,

HPyV12 and SV40

Herpes Viruses (n = 8) HSV1, HSV2, VZ, EBV (1 and 2), CMV, HH6, HH7, HH8,

Other infectious agents (n = 8) Chlamydia T., HBV, Fusobacterium, MMTV, Helicobacter Pylori, Schistosoma (haematobium, mansoni, japonicum)