ΟΙΚΟΝΟΜΟΠΟΥΛΟΥ ΠΑΝΑΓΙΩΤΑ ΠΑΝΕΠΙΣΤΗΜΙΑΚΗ ΥΠΟΤΡΟΦΟΣ Ο ΡΟΛΟΣ ΤΟΥ ΙΟΥ HPV ΣΤΟΝ ΚΑΡΚΙΝΟ ΚΕΦΑΛΗΣ ΚΑΙ ΤΡΑΧΗΛΟΥ (ΣΤΟΜΑΤΟΦΑΡΥΓΓΑ-ΚΣ) ΟΙΚΟΝΟΜΟΠΟΥΛΟΥ ΠΑΝΑΓΙΩΤΑ ΠΑΘΟΛΟΓΟΣ ΟΓΚΟΛΟΓΟΣ ΠΑΝΕΠΙΣΤΗΜΙΑΚΗ ΥΠΟΤΡΟΦΟΣ Π.Γ.Ν. ΑΤΤΙΚΟΝ

Many subsites Heterogeneous group of tumours of varying primary sites 95% are HNSCC Oral cavity Oropharynx/hypopharynx Larynx Nasopharynx Other anatomic sites Paranasal sinuses Salivary glands Lip The predominant histology has been and remains squamous cell carcinoma, and at least in the early stages of disease, 2

ΚΑΡΚΙΝΟΣ ΣΤΟΜΑΤΟΦΑΡΥΓΓΑ: AITIOΛΟΓΙΑ

O ΙΟΣ ΤΩΝ ΠΑΠΙΛΛΩΜΑΤΩΝ: HPV Καρκίνος τραχήλου μήτρας Καρκίνος στοματοφαρυγγα

ΑΠΟΔΕΙΞΗ ΑΙΤΙΟΛΟΓΙΚΗΣ ΣΥΣΧΕΤΙΣΗΣ ΕΠΙΔΗΜΙΟΛΟΓΙΚΗ ΣΥΣΧΕΤΙΣΗ ΜΟΡΙΑΚΗ/ΠΑΘΟΛΟΓΟΑΝΑΤΟΜΙΚΗ ΣΥΣΧΕΤΙΣΗ ΠΕΙΡΑΜΑΤΙΚΑ (IN VITRO) ΔΕΔΟΜΕΝΑ ΜΕΛΕΤΕΣ ΣΕ ΖΩΙΚΑ ΜΟΝΤΕΛΑ Carbone et al: “Modern Criteria to Establish Human Cancer Etiology” Clin Can Res, 2004

ΕΠΙΔΗΜΙΟΛΟΓΙΚΑ ΔΕΔΟΜΕΝΑ Με βάση τα στοιχεία της SEER βάσης δεδομένων, η επίπτωση του καρκίνου στοματοφαρυγγα αυξήθηκε από 3.9% το χρόνο, μεταξύ των ετών 1973-2001, σε λευκούς άντρες και γυναίκες 20-44 ετών, ενώ η επίπτωση των υπολοίπων καρκίνων κεφαλής-τραχήλου μειώθηκε Η οροθετικότητα για τον ιό Herpes simplex-2 (ένα δείκτη υψηλού κινδύνου σεξουαλικής συμπεριφοράς) αυξήθηκε κατά 30% για τις περιόδους 1976-1980 και 1988-1994

ΚΑΡΚΙΝΟΣ ΣΤΟΜΑΤΟΦΑΡΥΓΓΑ ΚΑΙ ΣΕΞΟΥΑΛΙΚΗ ΣΥΜΠΕΡΙΦΟΡΑ Συγκεκριμένες σεξουαλικές συνήθειες αυξάνουν τον κίνδυνο ΚΣ (oral sex, oral-anal contact) Στους άνδρες, νεαρή ηλικία έναρξης της σεξουαλικής δραστηριότητας, ο αριθμός σεξουαλικών συντρόφων και ιστορικό κονδυλωμάτων αυξάνει σημαντικά τον κίνδυνο ΚΣ Στις γυναίκες, ο αυξημένος αριθμός σεξουαλικών συντρόφων αυξάνει τον κίνδυνο ΚΣ

ΚΑΡΚΙΝΟΣ ΣΤΟΜΑΤΟΦΑΡΥΓΓΑ ΚΑΙ ΣΕΞΟΥΑΛΙΚΗ ΣΥΜΠΕΡΙΦΟΡΑ Άτομα αυξημένου κινδύνου να αναπτύξουν ΚΣ είναι τα ακόλουθα: Άτομα με ιστορικό HPV-σχετιζόμενου γυναικολογικού καρκίνου Σύζυγοι γυναικών με situ η διηθητικό καρκίνο τραχήλου μήτρας Άτομα με HIV λοίμωξη

ΔΕΙΚΤΕΣ HPV ΛΟΙΜΩΞΗΣ ΚΑΙ ΑΥΞΗΜΕΝΟΣ ΚΙΝΔΥΝΟΣ ΚΣ Ο ΚΣ συνδέεται με στοματική λοίμωξη από HPV και HPV-16 L1 οροθετικότητα σε ασθενείς με η χωρίς ιστορικό κατάχρησης καπνού η αλκοόλ HPV DNA έχει ανιχνευθεί σε πληθυσμούς ασθενών με ΚΣ και σχετίζεται με καλύτερη πρόγνωση D'Souza G et al: N Engl J Med 356:1944-56, 2007

Epidemic of HPV-associated OPC Incidence rates for overall oropharyngeal cancer, HPV-positive oropharyngeal cancer, and HPV- negative oropharyngeal cancer from 1988 to 2004 in Hawaii, Iowa, and Los Angeles Estimated age-standardised incidence of human papillomavirus (HPV)-positive and HPV-negative tonsillar cancer squamous cell carcinoma cases per 100,000 person-years, Stockholm, Sweden, 1970- 2006 Chaturvedi AK, et al. J Clin Oncol 2011;29:4294–301. Reprinted with permission. © 2011 American Society of Clinical Oncology. All rights reserved Error bars indicate 95% CI. Näsman A , et al. Int J Cancer 2009;125:362–6

ΑΠΟΔΕΙΞΗ ΑΙΤΙΟΛΟΓΙΚΗΣ ΣΥΣΧΕΤΙΣΗΣ Η ανίχνευση HPV DNA στον όγκο δεν αποδεικνύει αιτιολογική συσχέτιση Αυτό αποδεικνύεται όταν μοριακές τεχνικές επιβεβαιώσουν ότι ο ιός είναι απαραίτητος για την κακοήθη εξαλλαγή των κυττάρων

HPV PROTEINS E1: Viral replication; maintains episome E2: Transcriptional regulation, co-factor for viral replication E4: Disrupts cytokeratins E5: Interacts with growth factor receptors E6: Transforming protein; p53 degradation E7: Transforming protein; Rb binding Genes L1 and L2 are coding for major and minor capsid proteins respectively Human papillomaviruses are small non enveloped DNA viruses that replicate their genomes within the nuclei of infected host cells. The double stranded circular DNA of all HPVS are approximately 8 KB in size and The viral genome organization is very conserved To date, over 120 different viral types have been identified. There is a specific tropism for squamous epithelia but seems that sequence Variation determines tropism and disease potential This is an overview of HPV genome The papillomavirus E1 play important roles in viral genome replication. HPV E2 protein is a transcroptional regulator and a co factor for viral replication The functions of E4, E5 proteins are not yet fully understood. E4 disrupts cytokeratin and E5 interacts with growth factor receptors. The E6 and E7 proteins of high risk HPV types act as viral oncoproteins. E6 protein binds and degrades p53 E7 protein binds and degrades pRb The L1 and L2 proteins are assembled late and form icosahedral capsids. Following viron assembly, mature viruses Are released from the uppermost layers of the epithelium

Η καρκινογένεση από ιό HPV Δυο βασικές HPV ογκοπρωτεΐνες: E6, E7 Δρα μέσω αδρανοποίησης των ογκοκατασταλτικών μονοπατιών p53 και pRb X X In this slide, we can see a simplified model of hpv carcinogenesis in cervical cells. High risk E6 binds the p53 tumor suppressor protein as part of a trimeric complex with the cellular ubiquitin ligase E6AP, Leading to the rapid turnover of p53. Another major function of the high risk E6 proteins that is important for immortalization is Their ability to activate the expression of the catalytic subunit of telomerase, hTERT E7 binds to the retinoblastoma (Rb) family of tumor suppressors resulting in release of Rb from E2F transcription factor complexes. That leads to constitutive activation of transcription of genes that control the progression from G1 to S cell cycle phase Loss of Rb results in dysregulation of cell-cycle control and subsequent increase in p16 levels.

Ο HPV ΚΑΙ Ο ΚΑΡΚΙΝΟΣ ΤΟΥ ΤΡΑΧΗΛΟΥ ΤΗΣ ΜΗΤΡΑΣ E6 και E7 είναι τα κύρια HPV ογκογονίδια E6 και E7 ογκογονίδια εκφράζονται από τα καρκινικά κύτταρα και η συνεχής έκφραση τους είναι απαραίτητη για τη συντήρηση του κακοήθους φαινοτύπου

Καταστολή της μεταγραφής E6 και E7 σε HeLΑ καρκινικά κύτταρα Η E2 πρωτεΐνη καταστέλλει τη μεταγραφή E6 και E7 Αυτή η καταστολή έχει ως αποτέλεσμα την ενεργοποίηση p53 και pRb και αναστολή της ανάπτυξης των καρκινικών κυττάρων Η αναστολή οδηγεί στη γήρανση των κυττάρων Η καταστολή της έκφρασης Ε6 και Ε7 σε καρκινικά κύτταρα τραχήλου της μήτρας έχει ως αποτέλεσμα σημαντική μείωση του κυτταρικού πολλαπλασιασμού Goodwin EC, Dimaio D: Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways PNAs, 2000 Psyrri et al: Role of the retinoblastoma pathway in senescence triggered by repression of the human papillomavirus E7 protein in cervical carcinoma cells Cancer Res, 2004 As I have told you previously, E2 viral protein is a transcription regulator. Prof DiMaio has shown in his lab that In hela cells, introduction of heterologous expression vectors for E2 results in a suppression of transcription of the endogenous e6 and e7 genes leading to senescence.. This indicates that the continued expression of E6 and E7 genes is required to maintain the transformed phenotype of Cervical cancer cells.

Καταστολή της μεταγραφής E6 και E7 σε καρκινικά κύτταρα ΚΣ Χρησιμοποιήσαμε σύστημα με retrovirus-βασισμένο E6/E7 σύστημα καταστολής για να μελετήσουμε τις φαινοτυπικές και βιοχημικές συνέπειες της καταστολής E6 και E7 σε HPV16+ καρκινικά κύτταρα ΚΣ

ΣΥΜΠΕΡΑΣΜΑ Η συνεχής έκφραση E6 και E7 είναι απαραίτητη για τη συντήρηση του κακοήθους φαινοτύπου των HPV+ καρκινικών κυττάρων ΚΣ Καταστολή της έκφρασης E6 και E7 ογκογονιδίων έχει ως αποτέλεσμα την ενεργοποίηση των p53 and Rb ογκοκατασταλτικών οδών και την απόπτωση Θεραπευτικές στρατηγικές που στοχεύουν τον HPV μπορούν να οδηγήσουν στην ίαση ασθενών με HPV16+ ΚΣ Rampias et al JNCI, March 2009

ΚΣ: Δυο νοσολογικές οντότητες HPV positive HPV negative Tumor site Tonsil/BOT All sites Histology Basaloid keratinized Age Younger Older SE status High Low Risk Factors Sexual ETOH/tobacco Survival Improved Dismal Incidence increasing decreasing

Molecular classification of oropharynx cancers A cohort of 107 oropharyngeal SCC A cohort of 107 patients with oropharyngeal SCC treated at Yale University from 1980-1999 was assembled HPV 16 DNA viral load was determined using real-time quantitative PCR with primers specific for E6 target gene Expression of p53, p16 and retinoblastoma (Rb) proteins was determined using quantitative fluorescent immunohistochemistry (IHC) on a tissue array composed of the cohort of oropharyngeal cancers. For p16, conventional IHC was also performed

Outcomes by HPV DNA status This is a survival curve showing local recurrence. On the vertical axis is the % patients experiencing a local recurrence. On the horizontal axis, time in months to 5 years. We compared all patients who had HPV + tumors (pink) to the HPV negative patients (green). There was no difference in recurrence between these groups.

Based on HPV positivity and p16 protein expression tumors were classified into 3 categories Class I: HPV negative, p16 low Class II: HPV positive, p16 low Class III: HPV positive, p16 high

Local Recurrence: by p16/HPV This is a survival curve showing on the vertical axis % patients experiencing local recurrence. On the horizontal axis is time in months to 5 years. As opposed to the analysis comparing all HPV positive cases to HPV negative, we can see clear distinctions in local recurrence rates when compared by p16 AND HPV grouping. The p16 HIGH group (HPV active) had very low recurrence rates, around 17%. HPV negative cases recurred at around 42%, while the p16 Low/HPV positive cases had over an 80% local recurrence rate. This was significant at p=0.006.

Survival outcomes by HPV class Weinberger PM, A Psyrri JCO 2006

HPV and Survival The relative survival for HPV positive patient is independent of therapy as long as this therapy is within the current standard of care Risk of death is consistently less than 60% that of HPV negative cancers The absolute survival difference is consistently higher than 30%

Therapy response in relation to HPV status Ten percent of the relative survival benefit can be attributed to favorable prognostic profile Second primary cancers less common Tobacco has a negative effect on response Radiosensitive Accelerated RT beneficial Increased response to induction chemotherapy

Clinical Utility NCCN guidelines: “HPV testing recommended for all oropharynx tumors” National Cancer Institute US, CTEP: “HPV status must be included as stratification factor for trials including oropharynx cancer patients” U.S Cooperative Groups and European Organization for Research and Treatment of Cancer: “HPV-positive oropharynx cancer is a distinct disease entity”

Treatment of HPV-associated HNSCC Guidelines do not currently recommend using HPV status to direct treatment However, strategies to treat HPV+ LA-SCCHN have been proposed that take advantage of its tendency to respond to treatment, and are being investigated

Implications for clinical trial design P16 positive patients should be studied in separate clinical trials The superior outcome of HPV positive low-risk disease implies that treatment deintensification should be explored in order to reduce toxicity

Treatment Deintensification Aims to reduce treatment-related morbidity and improve patient quality of life without compromising treatment effectiveness Patients with HPV+ OPSCC are younger and expected to live long; therefore, morbidity resulting from late toxicity is a concern in these patients

Deintensification Strategies RT alone Reduced RT dose Targeted Therapy

QUARTERBACK: TPF → CT + reduced or standard dose RT in HPV+ SCCHN (OPC, unknown or NP) Lead investigator: M Posner Mount Sinai School of Medicine Carboplatin + R A N D O M I Z T T P F RT 5600 cGy Oropharynx, nasopharynx or unknown primary Stage III–IV, M0 HPV+ (PCR) and p16+ (IHC) CR/PR N=365* Q3 wks x3 cycles Carboplatin + RT 7000 cGy Standard dose CRT Primary endpoint: LRC (3 yr) Secondary endpoints: OS (5 yr), toxicity (5 yr), QoL Biological profiling? Details TBC *Estimated number TPF, docetaxel + cisplatin + 5-FU www.clinicaltrials.gov/ct2/show/NCT01706939

ADEPT A phase 3 trial that seeks to deintensify adjuvant therapy following surgery in patients with p16+ oropharyngeal tumors with extracapsular spread in their lymph nodes Following surgery, patients are randomized to either IMRT alone or weekly cisplatin/IMRT. Primary endpoints are DFS and LRC. Secondary endpoints are rates of distant failure, DSS, toxicity, and QoL

Deintensification by substituting chemo with cetuximab

Late toxicity (cis vs cetuximab) Variable Cisplatin No =58 % Cetuximab No=56 Residual Renal Dysfunction 13 22.4 Grade III-IV toxicity Mucosal 2 3.5 1 1.8 Xerostomia 6 10.3 8 5.9 Subcutaneous Fibrosis 4 7 1 2 Neuropathy 2 3.4 0 0 Laryngoesophageal 5 8.6 5 9 Lefebvre et al JCO March 2013

Do HPV positive patients respond to cetuximab?

RT +/- Cetuximab (“Bonner Study”) Lancet Oncol. 2010 Jan;11(1):21-8

A. Psyrri*, L. Licitra, B. de Blas, I. Celik, J. B. Vermorken Safety and Efficacy of Cisplatin plus 5-FU and Cetuximab in HPV-positive and HPV-negative Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck (R/M SCCHN): Analysis of the Phase III EXTREME Trial A. Psyrri*, L. Licitra, B. de Blas, I. Celik, J. B. Vermorken *University of Athens Medical School, Attikon University Hospital, Athens, Greece

Overall Survival by p16 Status p16+ patients p16− patients HR (95% CI) 0.82 (0.65–1.04) p-value 0.11 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 3 6 9 12 15 18 21 24 27 CT + cetuximab (n=18) CT (n=23) CT + cetuximab (n=178) CT (n=162) 11 10 8 4 1 23 17 7 2 178 150 126 93 61 40 19 162 128 92 56 47 33 HR (95% CI) 0.63 (0.30–1.34) p-value 0.22 Overall survival Overall survival Months Months Number of patients at risk Number of patients at risk HRs are CT + cetuximab vs CT CI, confidence interval; HR, hazard ratio

Progression-free survival Objective response rate Prognostic Effect Overall survival Progression-free survival Objective response rate Treatment p16+ p16− CT + cetuximab (n=196) Median (months) / Rate 12.6 9.7 5.6 5.7 50.0% 36.5% HR*/ Odds ratio† 0.59* 1.17* 1.74† 95% CI 0.32–1.10 0.69–2.01 0.66–4.60 p-value 0.09 0.56 0.26 CT (n=185) 9.6 7.3 3.6 3.1 21.7% 17.3% HR*/ Odds Ratio† 0.83* 0.87* 1.33† 0.50–1.36 0.53–1.43 0.46–3.88 0.45 0.59 0.60 HRs and odds ratios are p16+ vs p16−

Conclusions Globally, 88.3% of patients receiving CT + cetuximab were evaluable for p16 as a surrogate marker for HPV in EXTREME, with 9.2% of R/M SCCHN patients having p16+ tumors All subgroups were comparable regarding demographics and baseline characteristics Patients, independent of tumor p16/HPV status, benefited from the addition of cetuximab to platinum-based chemotherapy The data suggest that p16 expression may be a positive prognostic factor in R/M SCCHN No new safety findings in any of the subgroups These results do not confirm the findings of the SPECTRUM trial presented at ESMO 20111 1Vermorken J et al. Eur J Cancer 2011;47(Suppl 2):25 . .

Trans-Tasman Radiation Oncology Group Ongoing Phase 3 studies to assess cetuximab + RT vs CRT in unresectable HPV-associated OPC Investigator-sponsored studies ongoing in p16+ OPC in Europe and the US to evaluate the combination of cetuximab + RT vs CRT De-ESCALaTE1 H Mehanna Cancer Research UK/ University of Warwick (toxicity) CRT ERT TROG 12.012 D Rischin Trans-Tasman Radiation Oncology Group (symptom severity) CRT ERT RTOG-10163 AM Trotti Radiation Therapy Oncology Group (OS) CRT ERT 2016 2017 2018 2019 2020 1. www.clinicaltrials.gov/ct2/show/NCT01874171 2. www.clinicaltrials.gov/ct2/show/NCT01855451 3. www.clinicaltrials.gov/ct2/show/NCT01302834

Are all HPV+ oropharyngeal cancers patients the same in terms of prognosis?

Oropharynx: Classification of patients into risk-of-death categories Oropharyngeal cancer (n=266) HPV-positive (n=178) HPV-negative (n=88) ≤10 pack-years (n=88) >10 pack-years (n=90) ≤10 pack-years (n=23) >10 pack-years (n=65) N0-N2a (n=26) N2b-N3 (n=64) T2-T3 (n=15) T4 (n=8) Ang KK et al. N Engl J Med 2010;363:24-35. 42.9% at low risk 3 year OS = 93.0% 29.7% at intermediate risk 3 year OS = 70.8% 27.4% at high risk 3 year OS = 46.2% Recursive-partitioning analysis identified prognostic factors with the most predictive significance Ang KK, et al. NEJM 2010;363:24–35

Conclusions Response rates and survival outcomes are clearly better for the HPV positive patients Relative survival does not depend on therapy but the relationship between choice of therapy and absolute survival is unknown At this point these patients should be treated similarly to stage-matched HPV negative patients We need to enroll patients on clinical trials to address these issues