Must we be fighting fit to fight cancer?

Miss Shree Datta

Contents

BackgroundAimsRationaleMethods

– Recruitment– Vaccination schedule

Results and interpretationFuture work

Background• Women with ovarian cancer usually present with advanced stage

disease • Various chemotherapy drugs have different effects on the immune

system.• Although sensitive to chemotherapy initially, the majority relapse• Resistance to chemotherapy contributes to poor prognosis• Immune based therapies may prolong the response to treatment

and reduce chemotherapy resistance. • These are likely to be most effective when disease is minimal or

subclinical, such as at completion of first line chemotherapy, but depend on a sufficient immune response.

• Currently, little is known about the immune competence of ovarian cancer patients treated with chemotherapy– Surgery/chemotherapy: immunosuppressive– Taxol: induces tumour apoptosis– Tsuda: CD8+ T cell function recovers after chemotherapy

Objectives• To assess the immune competence of patients who have had

chemotherapy for ovarian, fallopian tube or primary peritoneal cancer.– Clinical relevance: to identify an optimal target time for immunotherapy, to reduce the

risk of cancer relapse.

• To investigate the role of PD-L1 as a prognostic marker in women with ovarian cancer undergoing chemotherapy.

– Also look at the expression of PD-L1 in our post-chemo patients to see if there is a relationship between the levels of PD-L1 and the levels of immune response.

HBV Vaccine• Replaces an initial infection but is innocuous and leads to the generation of

memory B cells.• Measuring the cellular and humoral immune response • HLA phenotype of B44, DRB1*0701, DQB1*0201 – 4x likely to be non-responders • Yeast-derived recombinant surface antigen • DNA vaccine: Engerix-B or HB-VAX II • Protective levels of antibodies in up to 95%.• Response rate better in women (ie ≥10mIU/L )• BMI, injection site, age need to be considered

Endpoints

• To measure the serum HBsAb titres in Hepatitis B vaccinated cancer women

• To quantify the immunomodulatory response by analyzing Hepatitis-B specific CD8+ T cells in PBMCs in HLA-A2 positive patients.

• To correlate PD-L1/PD-1 levels with response to chemotherapy, defined clinically

• To measure end of treatment PD-1/PD-L1 levels and correlate to disease free survival

RECRUITMENT

• Medical Oncology clinics at the Hammersmith Hospital• Inclusion and exclusion criteria.• 44/group - based on power calculation using accepted

statistical methods (15% logarithmic difference in outcome with a 5% statistically significance level + 80% power)

• The calculation was based on historical data obtained regarding titre range and geometric mean titre (GMT) of healthy volunteers above 50years

VACCINATION REGIMEVISITS GROUPA

3 MONTHS AFTER CHEMOTHERAPY

GROUPB12 MONTHS AFTER CHEMOTHERAPY

FIRST BLOOD TEST BLOOD TEST

SECOND day0

VACCINATION VACCINATION

THIRD(1 MONTH after 1st vaccine)

VACCINATION AND BLOOD TEST VACCINATION AND BLOOD TEST

FOURTH (6 MONTHS after first vaccine)

VACCINATION AND BLOOD TEST VACCINATION AND BLOOD TEST

FIFTH (7 months after first vaccine)

BLOOD TEST BLOODTEST

Initial results

• Since May 2012, 36 women have been recruited.3 excluded due to relapse

• Women who have completed chemotherapy 3-6 months ago (Group A): 10

• Women who have completed chemotherapy 1 year+ (Group B): 23

• 5 have completed the vaccination course, two from Group A and three from the “control” Group

• Currently, 27 women are undergoing the HBV vaccination. Four have just had their first vaccination.

Month 2

• Group A (n=6): 2 patients have anti-HBs ≥0IU/l: 0.03 and 1

• Group B (n=10): 0 patients have anti-HBs ≥0IU/l

Results: Month 6 and 7 (raw data, anti-HBs titre,IU/l)Group Month 2 Month 6 Month 7A 1 2 50

0 11 6620 14>10000 0 0

0.03 35>10000 1 719

B 0 0 70 0 00 11 1140 0 30 10 4260 260 0 10 1 50 250 0 1

Month 6Group A Month 6 anti-HBs titre level: Group B Month 6 anti-HBs titre level:

Mean=7.3

Patient number

Month 7Group A Month 7 anti-HBs titre level: Group B Month 7 anti-HBs titre level:

(NB n=8)

Mean= 572

Patient number

Mean=70

CONCLUSION: Points to ponder

• n=5 • Group A – 100% immune (n=2); only 33% of

group B patients have HBsAb levels >10 (n=3).• ?raised BMI, although less than 45

• ?Suboptimal immune response eg. Group B Patient 3, age 66, HBsAb= 7mIU/L.

• 100% vaccination success rate in Group A patients: ?normal immune response

• PD-L1 levels ?less after chemotherapy

Future work• Continue recruitment• ?Ex vivo Cell stimulation required• ELISA (plasma)• HBsAb titre levels provide a binary result for vaccination status.

– Measure the geometric mean titre for Group A and Group B to identify whether a statistical difference in the response level to HBV vaccination exists between the two groups.

Acknowledgements

Sadaf Ghaem-MaghamiJay ChatterjeeLynsey WhildingS Saso

Funding

Vandervell Foundation

References• Reference- 1.De Rave et al. Immunogenicity of standard and low dose vaccination using yeast-derived recombinant hepatitis B surface antigen in elderly volunteers. Vaccine 1994; 12(6):

532-534 Office for National Statistics. Cancer Statistics registrations: Registrations of cancer diagnosed in 2006, England. Series MB1 no.37 2008• Bristow RE, Puri I, Chi DS. Cytoreductive surgery for recurrent ovarian cancer: a meta-analysis. Gynaecol Oncol 2009; 112 (1): 265-74• Bristow RE, Tomacruz R, Armstrong DK et al. Survival effect of maximal cytoreductive surgery for advanced ovarian carcinoma during the platinum era: a meta-analysis. J Clin Oncol 2002;

20: 1248-1259• Cannon MJ, O’Brien TJ. Cellular immunotherapy for ovarian cancer. Expert Opin Biol Ther 2009 Jun; 9(6): 677-88• Coleman S, Clayton A, Mason M et al. Recovery of CD8+ T cell function during systemic chemotherapy in advanced ovarian cancer. Cancer Res 2005: 65: 7000-6• Coneja-Garcia J et al. Ovarian carcinoma expresses the NKG2D ligand letal and promotes the survival and expansion o f CD28 anti-tumour T cells. Cancer Res 2004; 64: 2175-82• Tomsova M et al. Prognostic significance of CD3+ tumour infiltrating lymphocytes in ovarian carcinoma. Oncology 2008 415-420• Curiel, T.J., et al., Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med, 2004. 10(9):P.942-9.• De Rave et al. Immunogenicity of standard and low dose vaccination using yeast-derived recombinant hepatitis B surface antigen in elderly volunteers. Vaccine 1994; 12(6): 532-534 • Franceschi S, Levi F, La Vecchia C. Survival after ovarian cancer treatment. JAMA 1993, 270 (10): 1196-7• Hwu P, Freedman RS. The immunotherapy of patients with ovarian cancer. J Immunother 2002 May-Jun; 25(3):189-201• Ilavska S, Horvathova M, Szabova M, Nemessanyi T, Jahnova E, Tulinska J, Liskova A, Wsolova L. Association between the human immune response and body mass index. Human

Immunology 2012; 73 (5) 480-5• Knutson K et al. Immunologic principles and immunotherapeutic approaches in ovarian cancer. Haematol Oncol Clin N Am. 2003. 17: 1051-73 • Leffers N, Daemen T, Helfrich W et al Antigen-specific active immunotherapy for ovarian cancer. Cochrane Database Syst Rev. 2010 Jan 20;(1):CD007287• Leroux-Roels, G., et. al., Correlation between in vivo humoral and in vitro cellular immune responses following immunization with hepatitis B surface antigen (HbsAg) vaccines. Vaccine,

Volume 12, Issue 9, 1994, Pages 812-818.• Looney R, Hassan M, Coffin D, Campbell D, Falsey A, Kolassa J, Agosti J, Abraham G, Evans T. Hepatitis B Immunization of Healthy Elderly Adults: Relationship between naïve CD4+ T cells

and primary immune response and evaluation of GM-CSF as an adjuvant. J Clinical Immunology 2001; 21 (1): 30-36• Machiels JP et al. Cyclophosphamide, doxorubicin and paclitaxel enhance the antitumour response in granulocyte/macrophage-colony stimulating factor-secreting who cell vaccines in

HER-2/neu tolerized mice. Cancer Res 2007; 67:7941-4.• McGuire W, Hoskins W, Brady MF et al. Cyclophosphamide and cisplatin versus paclitaxel and cisplatin: a phase III randomized trial in patients with suboptimal stage III/IV ovarian cancer.

Semin Oncol 1996; 23: 40-47• Mitchell, M.S., et. al., Cell-mediated immunity and blocking factor in ovarian carcinoma. Obstetrics and Gynaecology. 1976 Nov; 48(5): 590-7.• Oei AL, Sweep FC, Thomas CM et al The use of monoclonal antibodies for the treatment of epithelial ovarian cancer (review). Int J Oncol 2008 Jun; 32(6):1145-57• Polak L, Turk JL (1974). Reversal of immunological tolerance by cyclophosphamide through inhibition of suppressor cell activity. Nature 249:654–656• Pollack S, Loggers E, Rodler E et al. Immune-based therapies for sarcoma. Sarcoma (2011) ID 438940• Preston C, Goode E, Hartmann L et al. Immunity and immune suppression in human ovarian cancer. Immunotherapy (2011); 3 (4): 539-56• Taylor DD et al. Modulation of TcR/CD3-zeta chain expression by a circulating factor derived from ovarian cancer patients. Br J Cancer 2001: 84 (12): 1824-9• Ten Berge, R. J., et.al. , Combination Chemotherapy and immune capacity in advanced ovarian carcinoma. Eur J Cancer Clin Oncol. 1984 Jan; 20 (1): 91-8.• Tsuda, Naotake, et al., Taxol increases the Amount and T-cell Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells. Cancer Res, 2007.67 :

( 17).p8378-87• Yamaeu H, et al. Cisplatin treatment renders tumour cells more susceptible to attack by lymphokine-activated killer cells. J Clin Lab Immunol 1991; 35:165-70.• Zhang L, et al. Intra-tumoural T cells, recurrence and survival in epithelial ovarian cancer. N Eng J Med 2003; 348:203-13 •

The way in which results of serological titrations are reported has often been a source of confusion. The term G.M.T.(Geometric Mean Titre) is often used. In fact this is nothing more than the simple arithmetic mean of the logarithms of the last positive dilution of each serum. By using a doubling dilution sequence beginning at 2, the number of the last positive tube or well is equal to the logarithm to the base 2 of the dilution. This makes reading and calculation of the mean titre very simple, and is the reason for the increasing popularity of this type of dilution sequence. The table provided (Table 2) may be useful to those unfamiliar with the calculation of mean titres using this system. In view of the fact that in many cases it is not enough to have good titres - it is also necessary that they should be reasonably uniform within the flock - some measure of variability shoud be included when reporting the results. We have used the standard deviations of the logs of the titres for this purpose.

Immune response in ovarian cancer treatment

• Surgery/chemotherapy: immunosuppressive• Taxol: induces tumour apoptosis• Tsuda: CD8+ T cell function recovers after

chemotherapy

PD-1 and PD-L1• PD-1: 288 amino acid transmembrane protein (Keir ME 2008); member of

the CD28 family (Dulos 1997). • Released by cell activation (Ishida 1992) • Expressed on immune cells including T, B and natural killer cells,

monocytes and dendritic cells (Dong H 2002). • FUNCTION: immune checkpoint inhibitory receptor - reduces T cell activity

in the inflammatory response to infection (Ishida 1992). • PD-1 has two ligands: PD1 ligand (PD-L1 or CD274) and PD1 ligand 2 (PD-

L2) (Latchman Y 2001). PD-L1 is the main ligand of PD-1, with a similar pattern of expression as PD-1 as well as on macrophages and bone marrow derived mast cells (Weber J 2010).

• PD-L1 is a trans-membrane protein with 290 amino acids (Keir ME 2008). Its expression is triggered by histological inflammatory signals (Pardoll DM 2012) and it down-regulates T cell activity upon binding to PD1.

PD-L1 and ovarian cancer• PD-L1 is upregulated in the tumour environment (on TILs)• Association between PD-L1 expression on tumour cells and poor clinical

prognosis (Zou W 2008). • However, other external factors may influence prognosis and up-

regulation of PD-L1.

Work so far: PD-L1 is upregulated in ovarian cancer.• Expressed on CD4 + CD8 T cells in blood and ascites. • Lower expression of PD-L1 was noted on CD4+ lymphocytes in the blood

of patients with borderline tumours vs malignant tumours.

Immunotherapy in ovarian cancer

• Local, regional or systemic cytokine/antibody therapy– Bevacizumab

• Vaccine– Different tumour cells may need to be targeted

Current study areas• Is the high relapse rate secondary to chemo-resistance due to

antagonistic effects on immune mechanisms? • When in their post-treatment phase does the immune system

recover to provide an opportunity for targeting other novel immune therapies?

INCLUSION CRITERIA• BMI ≤ 45 and over 50years • Disease free post chemotherapy with normal CA125• No pre-disposing immunosuppressive medical disorder. • History of ovarian cancer treated with surgery/chemotherapy

or chemotherapy alone 3 or 12 months+ out of treatment • Platelet count ≥ to 100• Adequate renal function (creatinine ≤ 200 micro mol/l)• Adequate hepatic function (bilirubin and ALT < 1.5 times

upper limit of normal)• Haemoglobin ≥ 10.0 g/dl• Haematocrit ≥30%• Lymphocyte count ≥ 1x109/dl

EXCLUSION CRITERIA

• Pre-existing immunological disorder excluding vitiligo• Age less than 50• Previous immunity to Hepatitis B• Previous Hepatitis B Infection• Evidence of disease recurrence or persistence• Inability to give informed consent• Active intercurrent disease• Previous treatment with therapeutic cancer vaccines • HIV positive

CD14/PD-L1 levels

HBV vaccine

• Yeast-derived recombinant surface antigen • DNA vaccine: Engerix-B or HB-VAX II • Protective levels of antibodies in up to 95%.• Response rate better in women (ie ≥10mIU/L)

Non-responseIf titre level < 10, current recommendation is to revaccinate. Causes of non-responseHLA phenotype of B44, DRB1*0701, DQB1*0201 – 4x likely to be non-

responders . ?The response may be specific to HBV surface antigen recognition. The ability to produce antibody in response to a specific protein is controlled

by dominant autosomal class II genes of MHCBMI, injection site, age need to be considered

Revaccination with different vaccine Nearly 60% of non-responders or hyporesponders developed adequate antibody response.