Johns Hopkins University

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1 HPV in HNSCC: clinical and epidemiological considerations Wayne M. Koch, MD Johns Hopkins University

2 Financial Disclosure Research Support from NIDCR/NIH RO1, SPORE Acknowledgements Maura Gillison William Westra David Sidransky Joe Califano PtikH Patrick Ha Gypsamber D Souza Shahnez Begum many in lab Educational objectives Participants will know the distinctive clinical factors for HPV related head and neck cancer Participants i will be aware of the increasing i incidence of HPV related tonsil cancer in recent decades

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4 The FACTS about Tonsil Cancer 5-10,000 cases in USA annually? Global incidenceaa 50% of the cases I see!!! Tonsil and tongue base Mostly men Increasing 5-10% each year No warning signs Treat with chemotherapy and radiation (?? ) Role for TORS (Trans-oral Robotic Surgery) HPV-HNSCC is INCREASING 2% increase per year since 1973 in USA Increasing cases at JHU Dominating clinical trials around the world: Chemorad athletes Tonsil/BOT 50 Larynx 40 Oral Cavity 30 NP

5 World-wide patterns of HPV-related OP cancer 47% of all HNSCC in USA, Asia 36% in South America, Africa, Australia 28% in Europe, but increasing i Human Papillomavirus an etiologic factor in HNSCC HPV DNA in 62/253 tumors (25%) In Situ hybridization: tumor cell nuclei involved viral integration - Southern Blot Gillison, et al. JNCI 92:709, 2000

6 HPV-HNSCC is site specific Concentrated in Oropharynx (Tonsil) 34/60 (55%) oropharynx cases - HPV + Not all OP is tonsil! 32/52 (62%) tonsillar tissue - HPV+ Lymphoepithelium Not all tonsil is called OP (oral tongue, supraglottis, HP) Oral cavity HPV controversy 5-10% at JHU and IARC Higher in other series (technique vs. demographics?) HPV Detection: Technical notes In situ hybridization is gold-standard for specific cancer-related HPV Only areas with dysplasia or cancer display signal Begum, et al. Clin Ca Res 2005; 11(16): 5694 Standard PCR may pick up irrelevant HPV infection Real-time quantitative PCR is accurate Ha, et al. Clin Ca Res 2002; 8: 1205

7 HPV-HNSCC is clinically distinct More likely to be nonsmoker/nondrinker (but many have smoked) More likely to be of younger age Basisquamous histology Cystic lymph nodes Unknown primary Why tonsil lymphoepithelium? Reticulated (net-like) epithelium transport Basement membrane is porous and disrupted Direct passage of lymphocytes Superficial layer this and fragile complete desquamation occurs with infection Microtrauma? Perry, ME J. Anat. 185: 111, 1994.

8 HPV-HNSCC has better prognosis: Why? Gillison, JNCI 2000 Why do HPV + tumors do better? overall health, less smoking, less second primary risk (absence of field effect) 0-2% in HPV+ vs % in HPV - Immune surveillance to viral-specific ags Intact p53 mediated apoptotic response to radiation and chemo? Less calamitous genetic profile

9 HPV and chemoresponse ECOG 2399 Retrospective analysis of 96 patients OP 60 /La - 36 Stage III-IV Induction paclitaxel + Carboplatin then chemort 38/60 Oropharynx HPV + 0/36 Laryngeal cases HPV + group: more whites, better performance status, less smoking Fahkry, et al: JNCI 2008 Feb 20;100(4): HPV and chemort response HPV + tumors show 84% response HPV (-) tumors 57% Same for HPV (-) La and OP Hazard ratio for progression for HPV= 0.27 Multivariate i analysis HR=0.36 (64% lower risk of death) Fahkry, et al: JNCI 2008 Feb 20;100(4):261-9.

10 Kaplan-Meier curves for overall and progression-free survival stratified by tumor human papillomavirus (HPV) status Risk of progression 73% lower for HPV+ Risk of death 64% lower Fakhry, C. et al. J. Natl. Cancer Inst : HPV -> chemosensitivity? But HPV + pts do better than HPV for all treatment protocols that include at least radiation therapy Licitra Karolinska

11 HPV and Smoking Increased risk for recurrence (distant and local) second primary among EVER smokers HR= 2/3rds of HPV + cases have smoked Current smokers > past smokers risk Mixed etiology (virus + carcinogen) Univ. of Michigan, Clin Ca Research March, 2010 HPV-HNSCC is molecularly distinct Less p53 mutations 30% of HPV+ tumors have p53 mutation ti vs 53% overall Less promoter hypermethylation Less LOH at 3p, 9p, 17p Braakhuis, et al. JNCI 2004; 96: 998 Differential expression of multiple genes

12 Prognosis: os s How we think it will behave Impacts how we approach it Prognosis: A complex concept Endpoints: overall, disease-free, disease specific survival Tumor factors Host factors immune response, overall health Treatment factors

13 Prognostic factors in Head and Neck Squamous Cell Carcinoma Site: Nasopharynx, Oropharynx, Oral cavity, Larynx, Hypopharynx Etiology Anatomic factors: Stage: rate of growth, duration, invasive capability Primary Nodal Phenotype: perineural, lymphovascular invasion, differentiation, angiogenesis, g metastatic ability Genotype: p53, EGFR, Aneuploidy?? Genotype Phenotype Enormous complexity Critical functions: apoptosis, angiogenesis, growth, repair, invasion, metastasis, drug resistance Key pathways: interlacing p53, Retinoblastoma, signal transduction (EGFr), invasion/metastasis Genetic, epigenetic, post-transcriptional events Oncogenome: Hills and mounds

14 Genomic Landscape Sjoblom Science 2006 Cancer pathways Numerous Complex Interwoven en Focus on single marker Unlikely prognostic impact Unlikely therapeutic effect

15 Ample sample size Ample events Statistical Rigor Meaningful follow-up period Accurate and uniform site, stage, treatment, comorbity categories and data Multivariate analysis - independence Clinical vs. Statistical significance ECOG p53 Study Eligibility ibilit Squamous cell cancer of the head and neck Planned resection with curative intent Tumor, margins, peripheral blood sent to lab Tumor tissue microdissected and sequenced p53 GeneChip (Affymetrix) v.2.0 Manual sequence confirmation Poeta, et al: NEJM, 2007; 357:

16 p53 Functions Guardian of the genome - DNA damage cell cycle block for repair OR apoptosis (programmed cell death) Transcription factor for MANY pathways Most Frequent Mutations Are In The Loops That Make Contact With DNA Codon: 175 > 248 > 273 > 282 > 249 > 245 > 220 > 176 From the International Agency for Research on Cancer, Lyon, France

17 Types of p53 mutation Different TP53 mutations have different effects - disruptive/ non-disruptive Loss of DNA binding Transactivation of cell proliferation genes (gain of function) Denaturation of WT allele (dominant negative) Classification of amino acids based on polarity and charges NonPolar, amino acids Polar, Negatively charged, Acidic amino acids Polar, No charge, g, amino acids Phe Asp Cys His Met Glu Asn Lys Trp Gln Arg Polar, Positively Charged Amino acids Ile Val Leu Ala Thr Tyr Ser Gly Pro 242 Cys > Arg = disruptive mutation L2 loop: L3 loop: N-terminus C-terminus Transactivation domain (1-62) Proline Rich (65-100) DNA binding domain ( ) Oligomerization ( ) Regulation ( )

18 1.0 gressio n-free Pro oportion Alive and Pro Log Rank Test p< Years TOTAL FAILED CENSORED MEDIAN Wild type Non-disruptive mutation Disruptive mutation Multivariate analysis Model includes: Site, Stage, TP53 status, treatment Disruptive mutation: HR= p= Nondisruptive Mut HR= 1.14 HP site HR= 1.83 p=0.02 N+ HR= 2.44 p<0.001 Salvage surgery HR= 2.02 p<0.01 Poeta, et al: NEJM 2007; 357:

19 HPV p53 Two molecular Px factors How do they interact? H&E staining HPV insitu hybridization HPV+ HPV(-) Westra, et al: Clin Cancer Res 2008;14(2):

20 HPV and p53 together? Tissue microarray of 96 of the margin study patients Includes 21 with tonsillar CA 12/21 HPV+ (57%) 0/75 non-tonsil HPV+ Tonsil cancers with disruptive p53 mutation All HPV (-) 3 cases with HPV and p53 mutation All with nondisruptive mutations Westra, et al: Clin Cancer Res 2008;14(2): Site p53 mutation (%) HPV16-positive (%) Palatine/lingual tonsils 8/21 (38) 12/21 (57) Disruptive: 1 (13) Nondisruptive: 7 (88) Nontonsillar 37/68 (54) 0/68 (0) Disruptive: 14 (38) Nondisruptive 23 (62) Oral cavity 22/38 (58) 0/38 (0) Disruptive: 10 (45) Nondisruptive: 12 (55) Larynx 11/20 (55) 0/20 (0) Disruptive: 3 (27) Nondisruptive: 8 (73) Hypopharynx 4/6 (67) 0/6 (0) Disruptive: 1 (25) Nondisruptive: 3 (75) Palate 0/4 (0) 0/4 (0) Disruptive: 0 Nondisruptive: 0

21 Tumor Exon Codon Nucldeotide Amino acid Disruptive vs nondisruptive HPV16 status CGT > CAT Arginine > histidine ND TAT > TGT Tyrosine > cysteine ND CGT > CAT Arginine > histidine ND GCC > CCC Alanine > proline ND CGT > CTT Arginine > leucine ND GGC > AGC Glycine > serine ND CCT > ACT Proline > threonine ND CGG > CAG Arginine > glutamine D CAT > CTT Histidine > leucine D CGA > TGA Arginine > stop D CAG > GAG Glutamine > glutamic acid D CCC > -CC Frameshift D ATG > -TG Frameshift D CGA > TGA Arginine > stop D Westra, et al: Clin Cancer Res 2008;14(2): Mechanism of HPV-p53 effect Is HPV superfluous when coexistent? Results suggest possible additive functionnondisruptive mutation and HPV Difference in activity? Px?

22 Mechanism of HPV-p53 effect TP53 mutation in >50%, LOH >50% Cell cycle control and apoptosis response HPV E6 and p53 protein Ubiquitination and proteasomal degradation But some p53 target genes are still activated Apoptotic response to radiation remains Clinical Impact of Molecular Px Factors Treatment selection: High risk /low risk paradigms Time to treat HPV- tumors more aggressively? Cut back on Rx for HPV+ tumors? Study stratification Target selection

23 Cigarettes and HPV + SCC Current smokers HR=5.2 for recurrence vs. non-smokers ever smokers trend to decreased disease- free survival Lung metastasis Risk directly related to pack-years Mechanism of interaction? Univ. of Michigan Clin Ca Res Feb, 2010 BCl-2 and HPV N=68 platinum based chemort BCL2 apoptosis inhibitor BCL2 and HPV both px factors BCL2 (+) HR = 6.1 HPV (+) HR= 0.11 HPV (-) and BCl-2 positive - worst Px HPV (+) BCL2 (-) only 1/32 recurred Rocco Cl Cancer Research April 2010

24 What do we know about HPV? Risk profile for HPV(+) HNSCC: Analogy to uterine cervix SCC Same virus - Sexual history Latency period Infectivity Long period to clear the infection (3 yrs) Prevention

25 Risk Factors Sexual debut before age 18 2x risk More than 6 genital - contact partners 3x 1-5 Oral genital partners 4x More than 6 9x Deep kissing Marijuana? Tobacco? D Souza, Et al New England Journal Medicine 2007 Demographics of HPV Infection (population p statistics) Women after 1 year - ONE partner 28% are infected! 50% after 3 years Causes warts detected by Pap smear Higher in men 62% genital infection after 24 months of activity (just as fast) 4.5 million year olds are infected No signs or symptoms Anal infection rate high (even in heterosexuals) Takes >6 months to clear Overall risk in population 1:100,000 for HPV cancer 16x if infected

26 Infection -> Cancer 4.5 million infected -> 10,000 cancers WHY? Only a few strains cause cancer DNA repair Immune surveillance Cofactors (Marijuana?) Don t know Lengthy incubation years Temporal relationship between HPV exposure and HNSCC Sero-reactivity it for HPV-16 viral capsid in sera stored 10 years prior to HNSCC dx 14x increased risk for HNSCC Mork, et al. NEJM 2001; 344: 1125 Perhaps with first contact

27 HPV and HIV HPV more γ in HIV+ people More likely to persist in HIV+ people HR for HPV OP cancer is 2.6, (much lower than other sites) Aggressive HNSCC in AIDS Lack of HNSCC with ART Kreimer AR, et al. Oral HPV infection is associated with sexual behavior and HIV serostatus. J Inf Dis 2004; 189: 686 Marijuana and HPV HNSCC Independent risk factor Current use => HR 5.2 for HPV+ 2.3 for HPV(-) Increases with intensity, duration of use Perhaps due to immunomodulatory effects Cannaboids bind CB2 receptor on B, T, NK cells Suppresses immune responses Gillison, et al. Distinct risk factor profiles for HPV-16 + and HNSCC. JNCI in press

28 Preventive HPV Vaccine Induces high titers of neutralizing antibody Blocks cellular entry by virus Must be long-lasting Must work at point of contact tonsil Secreted up from basal layer Targets capsid proteins (virus like particles L1) Therapeutic Clear infected cells, eradicate cancer cells Target E6, E7 VACCINE Gardasil: 100% effective against genital warts in women Duration?? (5 years so far) MUST be before exposure By age 16-21, 21% already infected Approved for men in USA (genital warts) Merck trial underway year old hetero + homosexuals Early data looks like boys have better ab response Not tested for protection against oral infection or cancer

29 Gardasil Covers HPV 6,11,16,18 FDA approved for girls 9-26 years old Three doses Must be given before exposure infection viral integration Australia has vaccinated 80% of girls, approved for boys Covers HPV 16,18 Cervarix Conclusions HPV and TP53 status do correspond with clinical outcome HPV and p53 interface is nuanced HNSCC is actually multiple disease states Careful attention to HPV status is needed in planning and interpreting clinical trials

30 Basisquamous epithelium Mixed HPV +/- The HPV ( ) aggressive, rapidly growing with poor outcome HPV + good prognosis