Virusuri

oncogene
 Cuprins
O Epstein Barr virus
O HBV
O HCV
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Cuprins
O Epstein Barr virus
O HBV
O HCV
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Cuprins
O Epstein Barr
O HBV
O HVC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Cuprins
O Epstein Barr
O HBV
O HCV
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Cuprins
O Epstein Barr
O HBV
O HVC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Cuprins
O Epstein Barr
O HBV
O HVC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Background
O Cancer and AIDS have been closely linked since the
beginning of the AIDS epidemic
O Kaposi sarcoma and non-Hodgkin lymphoma have been
considered AIDS-defining cancers (ADC) since 1982 and
cervical cancer since 1993
O With the advent of the highly active antiretroviral
therapy (HAART), incidence of ADC has dramatically
decreased
O Conversely, the proportion of non-AIDS-defining cancers
(NADC) has increased, recently surpassing ADC
O Among NADC, virus-related cancers account for an
important share of cases
 Methods
Non-AIDS defining virus-
related cancers

HPV-related EBV-related

Tonsil and Nasophary

Nasophary Hodgkin
Hodgkin
Tongue Anus
oropharynx nx
nx lymphoma
lymphoma

Vulva and
Penis
vagina

HBV/HCV-related

Liver
Liver
 Results
O HPV-related: 127 cases (59.1%)
O Tongue: 15 cases (7.0%)
O Tonsil and oropharynx: 19 cases (8.8%)
O Anus: 73 cases (34.0%)
O Vulva and vagina: 14 cases (6.5%)
O Penis: 6 cases (2.8%)
O EBV-related: 66 cases (30.7%)
O Nasopharynx: 8 cases (3.7%)
O Hodgkin lymphoma: 58 cases (27.0%)
O HBV/HCV-related
O Liver: 22 cases (10.2%)
 Conclusion
O Cancer burden among people with AIDS in São
Paulo was similar to that previously described
in high income countries

O Most non-AIDS-defining virus-related cancers

occurred at increased rates in people with AIDS

O Vaccination against HPV and HBV, treatment of

HCV and immediate initiation of HAART could
reduce cancer burden in this population
 Cuprins
O Epstein Barr
O VHB
O VHC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Background – HPV and OPSCC

Evidence for a Causal Association Between HPV and a Subset of

Head Neck Cancers
 62/253 (25%) of the patients displayed HPV DNA in their tumors
 Of these 60 patients had OPSCC and 55% of these had HPV DNA in their
tumors.
 Patients with HPV+ HNSCC had a better outcome than those with HPV -
tumors
Gillison et al 2000 JNCI May 3;92(9):709-20.

HPV in tonsillar cancer

 26/60 (43%) of the patients displayed HPV DNA in their tumors
 Patients with HPV+ tumors had a better outcome than those with
HPV- tumors
Mellin et al, Int J Cancer, 89, 300-304, 2000
HPV in base of tongue
cancer
 10/25 (40%) of the patients displayed HPV DNA in their tumors
 Patients with HPV+ tumors had a better outcome than those with HPV -
tumors
 Dahlgren et al, Int J Cancer 112, 1015-
This was not the case for mobile tongue 9, 2004
The incidence of HPV+ TSCC and BOTSCC is rising/high

OPSCC accounts for 35-40% of all head and neck cancer

 350 cases/year ≈ 220 TSCC- and 50-70 BOTSCC (70-80% HPV+)

and 50 non-tonsillar non base of tongue cancer (16% HPV+)

 No screening or intervention

 800-900 cases/year of head neck squamous cell carcinoma (HNSCC

Cervical cancer

 450 new cases/year (55% HPV-16 and15% HPV-18)

 Around 1 million PAP smears/year (2-4% cellular changes)
 8000 preventive measures/year
47
 Treatment of HPV+ TSCC and BOTSCC

HNSCC has poor

prognosis in general
Intensification of treatment with:

- chemotherapy (induction or concomitant)

-hyperfractionated radiotherapy

given to an increasing number

of patients with
HPV+ TSCC and BOTSCC

More serious side effects 48

 Treatment of HPV+ TSCC and BOTSCC

• Most HPV+ TSCC and BOTSCC patients may not need intensified
therapy

• Less intensive treatment would be of benefit

• To de-intensify therapy, patients with HPV+ TSCC and BOTSCC

that
respond well to less intensive therapy must be identified

Additional predictive markers are needed

49
Workflow

50
Multi-step process of cervical carcinogenesis

hr-HPV infection precancer

no yes

normal cervix CIN1 CIN2 CIN3 Carcinoma

productive HPV infection

transient HPV infection

persistent HPV infection > transformation > progression to cancer

HPV integration
HPV18 100%
HPV16 ~80%
 HPV integration - Structural and functional
hallmarks
• Disruption/deletion of E2 and E1
 Inactivation of E2 repressor
 Inactivation of E1 replication factor
• URR (promoter-enhancer & ori)
and E6-E7 oncogenes retained
• Viral-cellular fusion transcripts
 HPV-driven Head and Neck Cancer –
Characteristics
• high viral load (≥ 1 copy/cell), viral oncogene
expression (RNA+), p16 overexpression
• better prognosis  tumor-specific and progression-free
survival
• better response to radiation therapy

• mainly oropharynx  tonsils and base of tongue

• increasing incidence

 reported HPV16 integration 0-100%

 Innovative TEN16 multiplex strategy for
identification of 3’ junctions of HPV16
integration sites
p 1: Nextera Tagmentation = DNA fragmentation and adapter tagging of genomic sample DN

Step 2: HPV16 DNA enrichment by multiplex PCR

Step 3: Illumina next-generation DNA sequencing

 100-200 million paired-end sequences

Step 4: Data processing (sorting & mapping) of 1 2 3 4 5

paired-end sequences
 Selection of viral-cellular junction
sequences (~200 000)
 Selection of candidate junction sequences
(~100)
Step 5: Validation of each candidate sequence
by junction-PCR

 Comprehensive mapping of HPV integration sites in HPV-

induced cancer
 Results – HPV16 Integration Frequencies
Integration
Total
OPSCC Yes No
N=48 N (%) N (%)

HPV-driven1 39 20 (51) 19 (49)

HPV non-driven2 9 0 (0) 9 (100)

1
HPV16 DNA+, viral load > 0.5 copies/cell, HPV16 RNA patterns positive
2
HPV16 DNA+, viral load < 0.03 copies/cell, no HPV16 mRNA transcripts

 HPV16 integration in OPSCC only 51%

 Summary and Conclusions
HPV16 integration in OPSCC
O is less frequent (51%) than in cervical cancer
(80%) using the same detection method.
O is not necessary for viral transformation 
deregulation of HPV without integration
remains to be explained.
 No biological or clinical differences in HPV-
driven OPSCC with and without HPV integration.
 VCJs present in the primary tumors are not
necessarily present in the corresponding lymph
node metastases  other VCJs in the lymph
nodes?
 HPV and Oropharyngeal cancer (OPSCC)

 HPV is acknowledged as a risk factor for OPSCC

O Dominated by tonsillar- (TSCC) and base of tongue cancer

(BOTSCC)

 The incidence of HPV-positive (HPV+) TSCC and BOTSCC has

increased in many Western countries

• About 80% of TSCC and BOTSCC cases in Sweden are HPV-
positive

Cecilia Nordfors Sunday, April 2 57

6, 2020

http://msk-anatomy.blogspot.se/2012/06/pharynx-
 Diagnosis of HPV+ TSCC and BOTSCC

 Today; Suspicion of HNSCC  Diagnostic biopsy

 HNSCC (including HPV+ TSCC and BOTSCC) often presents with

an enlarged cervical lymph node on the neck as the first symptom

Our hypothesis

 By detecting HPV in fine needle aspiration cytology (FNAC)  to

determine possible presence of an HPV+ TSCC or BOTSCC

 Spare the patient the diagnostic biopsy or neck dissection

Cecilia Nordfors Sunday, April 2 58

6, 2020
 Material and methods

 FNACS from 66 patients with neck masses

O Karolinska University Hospital 2013-2016

 Analysed for 27 types of HPV using a bead-based multiplex assay

• DNA (and mRNA if quality was good)

 Results were correlated to;

• HPV-status obtained from histopathological specimens
• p16 Immunohistochemistry
• Final diagnosis

Cecilia Nordfors Sunday, April 2 59

6, 2020
 Conclusions

General conclusions
 HPV-DNA was not present in malignancies other than TSCC and

BOTSCC
 HPV-DNA detection in cytology material was as reliable as in

histopathological material

Take home message

 HPV16 DNA-detection in fine-needle aspirates from a neck mass is a

reliable indicator of an HPVDNA+ TSCC and BOTSCC

Cecilia Nordfors Sunday, April 2 60

6, 2020
Incidence of HPV-associated cancers - USA

Jemal et al. JNCI Slide courtesy Aimee

2013 Kreimer
 HPV Types in OPSCC
533 OPSCC
Germany, Italy,
USA

111 (21%) HPV-

driven
HPV antibodies
Case # HPV DNA HPV RNA p16 E6 E7
101 (91%)
18 18 nd nd nd
HPV16
01
HPV type %
10 (9%) Non- 02 26 26 ↑ nd nd

16 16 91 03 33 33 ↑ + -

35 4 04 33 33 nd nd nd
05 33 33 ↑ + -
33 3 06 35 35 ↑ + +
18 1 07 35 35 nd + -

26 1 08 35 35 nd + +
09 35 35 ↑ nd nd
58 1
10 58 58 nd + +
 Tim Waterboer
Multiplex Serology
Peter Sehr
Kristina Michael

Bead- based (Luminex)

Affinity purified full length viral proteins
fused to GST
Up to 100 antigens in parallel
> 1000 sera analyzed within days

multiplex serology
GST capture ELISA
multiple bead sorts
multiple wells
1 well

Waterboer et al., Clin Chem 2005

Waterboer et al., J Immunol Meth 2006
Low HPV prevalence in head and neck cancer: results from two large
case-control studies in high incidence regions
K Braga Ribeiro, JE Levi, M Pawlita, S Koifman, E Matos, J Eluf-Neto, V Wunsch-Filho, M Curado, O Xangina, D
Zaridze, N Szeszenia-Dabrowska, J Lissowska, , A Daudt, A Menezes, V Bencko, D Mates, L Fernandez, E
Fabianova, T Gheit, M Tommasino, P Boffetta, P Brennan, T Waterboer
Int J Epidemiol 2011
Hospital-based case–control studies
Central Europe (CE) and Latin America (LA)
2214 cases, 3319 controls, collected 1998-2003

HPV16 L1, E6, and E7 antibodies and risk of HNSCC and individual
tumor
Study sites
All cases Controls Oral cavity Oropharynx Hypopharynx/Larynx

+/- (Prev %) +/- (Prev %) OR*(95%CI) +/- OR (95%CI) +/- OR (95%CI) +/- OR (95%CI)
L1 LA 139/1531 (8.3) 111/1177 (8.6) 1.06 (0.79-1.42) 45/486 1.08 (0.73-1.62) 31/329 1.19 (0.74-1.93) 63/716 1.11 (0.77-1.60)
CE 33/511 (6.1) 128/1903 (6.3) 0.91 (0.58-1.42) 6/150 0.47 (0.19-1.14) 4/75 0.79 (0.27-2.32) 23/286 1.23 (0.73-2.07)
6.5%
E6 LA 46/1624 (2.7) 8/1280 (0.6) 7.90 (3.38-18.5) 8/523 2.18 (0.69-6.86) 22/338 42.9 (12.8-144) 16/763 5.17 (1.86-14.4)
CE 10/534 (1.8) 21/2010 (1.0) 1.67 (0.70-3.98) 3/153 1.75 (0.46-6.61) 2/77 2.6%1.97 (0.38-10.2) 5/304 1.38 (0.46-4.17)

E7 LA 61/1609 (3.6) 27/1261 (2.1) 2.27 (1.35-3.81) 19/512 1.79 (0.92-23.5) 25/335 5.75 (2.88-11.5) 17/762 1.26 (0.63-2.52)
CE 9/535 (1.6) 31/2000 (1.5) 0.98 (0.42-2.27) 3/153 2.05 (0.53-7.94) 1/78 0.83 (0.09-7.01) 5/304 0.80 (0.28-2.26)

E6 + E7 Pooled 22/2110 (1.0) 3/3235 (0.1) 19.0 (5.24-69.0) 1/655 1.82 (0.09-37.1) 16/405 179 (35.8-899) 5/1050 14.9 (2.92-76.1)
* OR adjusted by age, sex, smoking, alcohol drinking, and country; ** p value for heterogeneity; *** LA = Latin America; CE = Central Europe
† Comparison of double HPV16 E6 and E7 positives against double HPV16 E6 and E7 negatives
HPV Antibodies in Head-and-Neck Cancers
Conclusions
Oropharynx
O Patients with HPV-driven oropharyngeal cancer almost always develop
strong and stable antibody responses to HPV early proteins, especially E6
O Antibodies to 16 E6 and other early proteins can develop more than 10
years before cancer diagnosis, indicating that yet unidentified HPV-
specific lesions exist many years prior to oropharyngeal cancer diagnosis
O The strong immune response to HPV induced years before tumor
diagnosis is not protective
O HPV E protein seropositivity predicts better survival
O HPV E protein antibodies may provide a clinically useful marker for
identification of HPV-driven OPSCC
O HPV E protein antibodies could be used as screening marker for HPV-
induced OPSCC

Oral Cavity, Larynx, Hypopharynx

O Significant, but much weaker serological associations with
incident/prevalent cases, but not with prospective cases
O Seropositivity associated with involvement of lymphoid tissues in
Waldeyer‘s ring
O Misassignment of tumor site cannot always be excluded
 Checklist: Screening for HPV-driven OPSCC

Test that detects cancer before symptoms Yes

Identifiable precancerous state No

Effective treatment Yes

Evidence of reduced incidence/mortality Missing

Benefits outweigh risks and costs Missing

 HPV-Driven Head and Neck Cancer –
Open Questions 2016
O Understanding the temporal and geographic
differences in HPV-driven OPSCC incidence
What are the risk factors? Changes over time and
geography?

O Epidemiology and natural history of oral HPV

infection
Who gets oral HPV infection, why, when, where, for
how long?

O Precursor lesion of HPV-driven OPSCC

What is it, where (tissue context), why is it inducing
more antibodies and earlier than cervical cancer?

O Understanding biological differences of HPV+ vs

HPV- oropharyngeal cancers
Why is there early lymph node involvement, why
better survival?

O HPV+ tumors in other oral sites

 Outline
• Background:
• Epidemiology of keratinocyte cancer (KC)
• Cutaneous viral infections
• Possible interaction with UV exposure
• VIRUSCAN Study Design and Methods
• Year 1 Baseline Results:
• Prevalence of cutaneous infections by UV
• Viral DNA in KC tumors and solar elastosis
• Conclusions
• Future Directions
Model of the interaction between UV
and HPV in skin carcinogenesis

(-)
HPV38 and others (?) DNp73 p53 and p73

UV exposure Normal
Cell cycle arrest and skin
DNA repair
DNA or
damag apoptosis
e

Normal
skin
Cancer

Slide courtesy of Massimo

Beta-HPV DNA in SCC tumors by
anatomic site
n 1+ types 3+ types
80 64% 25%

45 73% 38%
15 33% 13%

9 33% 0%
p=0.01 p=0.03
 Complex etiology of KC
Keratinocyte cancer (KC)

Cutaneous human KC risk factors:

papillomavirus - Ultraviolet radiation
(HPV) and - Smoking
polyomavirus (PyV) (VIRUSCAN - Steroid use
infections Study)
   

 
 HPV38 cooperates with UV in the
development of actinic keratosis
UVB doses
(mJ/cm ) 2

and SCC
Chronic UV irradiation
120
450
End of
the
of WT or HPV38 E6/E7 experime
transgenic mice nt
Weeks 0 1 2 3 15 16 17 18 19 20 29 30

WT Tg-183 WT Tg-183
1.0

1.0
183
187
0.8

0.8
FVB/N

SCC incidence
0.6

0.6
0.4

0.4
Tg-2
187
Tg-1
183
0.2
0.2

WT
0.0
0.0

Normal AK Normal SCC 00 5

5 20
20 25
25 30
30

Weeks of UV
Week 24 Week 29 irradiation

Viarisio et al. 2011 PLoS Pathogens

 Geographical Variation of HPV DNA+ OPSCC

HPV+ tonsillar
~Ca
79%
~ ~
70% 40% < 5%

HPV+
oropharynx < 5%
Ca
Based on data
from
Smith et al., 2008
Gillison et al.,
2008 Dalianis et
al., 2009 Jung et
al., 2010
Ribeiro et al.,
2010
Holzinger et al.,
2012
 Cuprins
O Epstein Barr
O VHB
O VHC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus
Type 1
 Cuprins
O Epstein Barr
O VHB
O VHC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Cuprins
O Epstein Barr
O HBV
O HVC
O Kaposi sarcoma herpesviruses
O HIV – 1
O HPVs
O Human T-cell lymphotropic virus Type
1
 Risk factors for cancer in the GPC
Table 1: Prevalence of oncogenic infections among adults (13+ years) in the General Population
Cohort (GPC) in rural Ugandan.

Class 1 Carcinogens Prevalence (95% CI) among adults (13+ years) in the GPC
(number positive/total)

Epstein Barr Virus (EBV) 94% (93-94%) 5665/6058

Kaposi’s Sarcoma associated herpesvirus 94% (93-95%) 5693/6058

(KSHV)

Hepatitis B virus (chronic infection) 11% (10-11%) 639/6058

Hepatitis C virus < 1%

Human Papillomavirus

- All high risk mucosal types 54% (53-56%) 3035/5586

- HPV 16 21% (20-22%) 1179/5586

- HPV 18 23% (22-24%) 1281/5586

Human Immunodeficiency virus-1 (HIV) 9% (9-10%) 561/5974

Human T-cell lymphotropic virus-1 (HTLV) Not assessed

Helicobacter Pylori (H. pylori) 97% (96-97%) 5398/5586

Schistosomiasis haematobium Not assessed

Class 2.1 Carcinogens

Merkel cell polyomavirus (MCV) 100%

Plasmodium falciparum malaria Not assessed but ubiquitous

 Risk factors for cancer in the GPC
Table 2: Prevalence of selected non-infectious risk factors for cancer among adults (13+ years) in the General Population Cohort (GPC)
in rural Uganda.

Exposure/risk factor Prevalence of exposure Comments

Tobacco (current daily smoker) 6.5% Males - 13.1%, females - 1.3%; >80% of smokers
report consuming < 5 cigarettes per day

Alcohol (at least weekly) 11% Males - 19.1%, females - 5.7%

Overweight or obese 11.8% Males: 5.2%, females - 16.9%

Low consumption of fresh fruit and vegetables 75.8% Males – 74.5%, females – 76.9%

Low levels of exercise 29.8% Males - 20.8%, females - 36.7%

Aflatoxin (detectable in serum) 100% 25% had serum levels of aflatoxin-albumin adduct >
15.1pg/mg albumin

Overweight: body mass index > 25kg/m2; low fresh fruit/vegetable: < 5 servings per day; low exercise: < 5 days per week of any combination of
walking, moderate- or vigorous-intensity activities and <600 minutes of physical activity per week. For further details, see references: 1-3.
Oncogenic infections in the GPC

H. pylori EBV

HBV
KSHV
 Kaposi’s Sarcoma
1 2 4
. . .

3
.

1. UCSF, 2007 photograph, viewed 23 May 2016 ,

http://www.dermatology.ucsf.edu/skincancer/professionals/types.aspx
2. Lee, S., Abrahamian, F. and Stroger, J. H. (2012) Gastrointestinal Kaposi’s sarcom
107, 2012.
 Kaposi’s Sarcoma
Classic

Iatrogenic
Endemi
c

AIDS-related

Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M,

Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and
Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, Fr. Int. Agency
Kaposi’s Sarcoma-associated
Herpes Virus (KSHV)

Boshoff, C. (2012) Ephrin receptor: a door to KSHV infection. Nat. Med.,

Nature Publishing Group 18, 861–863.
Kaposi’s Sarcoma-associated
Herpes Virus (KSHV)
• KSHV was first isolated
from a KS lesion in 1994
(Chang et al.)
• Causative agent of KS,
Primary effusion lymphoma
and Multicentric
Castleman’s Disease.
• Necessary, but not
sufficient, for KS
development- requires
precipitating factors

Boshoff, C. (2012) Ephrin receptor: a door to KSHV infection. Nat.

Med., Nature Publishing Group 18, 861–863.
Kaposi’s Sarcoma-associated
Herpes Virus (KSHV)
• γ2 herpesvirus
• Double stranded DNA
genome (160kb)
• 100 ORFs
• Capsid
• Tegument
• Lipid envelope

Boshoff, C. (2012) Ephrin receptor: a door to KSHV infection. Nat.

Med., Nature Publishing Group 18, 861–863.
 Global KSHV/KS Epidemiology

KS
incidence

KSHV
prevalence

Mesri et al. (2010) Nature Reviews Cancer 10, 707-719

 KSHV epidemiology

Uninfected Environment

KSHV

Latently Infected KSHV

Reactivation
KSHV Lytic Cycle
Increased KSHV viral load virus
Healthy, low shedding
viral load

Increased KSHV
transmission

Low prevalence
High prevalence
 The microbiome in
Chronic carcinogenesis
Intrinsic /
inflammatio oncogene Anti-cancer
n induced immune Response to
(infections, inflammatio response therapy
aseptic) n Co-
morbidities
Tumor Immuno
Genomic promotion evasion
mutations

Primary tumor

Predisposing conditions Cancer Tumor

(obesity, metabolic associated growth
syndrome) inflammatio Angiogenesi
n s
Tissue
remodeling Met
COMMENSAL MICROBIOTA Infiltration
and
s
 Polyomaviruses

VP1 / VP2/3
• non-enveloped, Ø 40-45
nm

• chromatinized dsDNA
genome (5-6kB)

• >200 types

• high prevalence, mostly

asymptomatic

White et al. (2013) PLoS path. 9, e1003206

Merkel Cell Polyomavirus
• detected in Merkel cell carcinoma (2008)

• about 60% prevalence

• continuously shed from the skin

• currently no viral propagation in cell

culture

• diverges from prototypic

polyomaviruses
• PsV infection studies possible, best
system: human lung carcinoma cells

• uses TWO receptors

• heparan sulfate proteoglycans
• gangliosides
 Human Cytomegalovirus (HCMV)
O β-Herpesvirus
O ~235kb dsDNA genome
O Lytic or latent infections
O 40% - 99% of population infected
O Causes disease in the absence of full immune function
O Does not transform cells in vitro
O Present in human cancers (e.g. glioblastoma and
breast cancer)
FrozenGBMs
UL111A
UL123
UL144
UL17
UL18
UL19

US11
US28
UL98
UL27
UL36
UL44
UL55
UL69
UL82
UL96
UL97

US2
UL1

1
2 qPCR from GBMs averages
3
4 1 viral genome / 160 copies of actin
5
6
7
8 Ranganathan, P. et al 2012
9
10
J. Virol. 86(2):854-864
11
12
 Merkel Cell Carcinoma
Merkel cell carcinoma (MCC)
Highly aggressive skin cancer
2,000 new cases/year in USA
40% mortality
Melanoma (invasive) 77,000/yr
13% mortality

Risk factors
Age: median 74 years
UV/sun exposure
Immune compromise

Treatment
Surgery plus adjuvant radiation therapy
Cisplatin, etoposide for metastatic disease
Checkpoint Blockade: Pembrolizumab (PD1) or Avelumab (PDL1)