This study aims to assess the immune competence of ovarian cancer patients after chemotherapy treatment by vaccinating them against hepatitis B virus (HBV) and measuring the antibody response. Patients who completed chemotherapy 3-12 months prior will be divided into two groups based on time since chemotherapy. All patients will receive the HBV vaccine and have antibody levels measured over time. The study will investigate if antibody responses differ between the two groups and correlate levels of the immune checkpoint proteins PD-L1 and PD-1 with clinical outcomes. Results so far show higher antibody responses in patients vaccinated sooner after chemotherapy. Continued recruitment is planned along with additional immune analyses.

1. Must we be fighting fit to fight cancer?
Miss Shree Datta

2. Contents
Background
Aims
Rationale
Methods
– Recruitment
– Vaccination schedule
Results and interpretation
Future work

3. 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

4. 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.

5. 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

6. 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

7. 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

8. VACCINATION REGIME
VISITS GROUPA
3 MONTHS AFTER
CHEMOTHERAPY
GROUPB
12 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

10. 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.

11. 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

12. Results: Month 6 and 7 (raw data,
anti-HBs titre,IU/l)
Group Month 2 Month 6 Month 7
A 1 2 50
0 11 662
0 14>1000
0 0 0
0.03 35>1000
0 1 719
B 0 0 7
0 0 0
0 11 114
0 0 3
0 10 426
0 26
0 0 1
0 1 5
0 25
0 0 1

13. Month 6
Group A Month 6 anti-HBs titre level: Group B Month 6 anti-HBs titre level:
Mean=7.3
Patient number

14. Month 7
Group A Month 7 anti-HBs titre level: Group B Month 7 anti-HBs titre level:
(NB n=8)
Mean= 572
Patient number
Mean=70

16. 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

17. 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.

18. Acknowledgements
Sadaf Ghaem-Maghami
Jay Chatterjee
Lynsey Whilding
S Saso
Funding
Vandervell Foundation

19. References
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•

20. 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.

21. Immune response in ovarian cancer treatment
• Surgery/chemotherapy: immunosuppressive
• Taxol: induces tumour apoptosis
• Tsuda: CD8+ T cell function recovers after
chemotherapy

22. 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.

23. 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.

24. Immunotherapy in ovarian cancer
• Local, regional or systemic cytokine/antibody
therapy
– Bevacizumab
• Vaccine
– Different tumour cells may need to be targeted

25. 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?

26. 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

27. 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

28. CD14/PD-L1 levels

29. 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)

30. Non-response
If titre level < 10, current recommendation is to revaccinate.
Causes of non-response
HLA 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 MHC
BMI, injection site, age need to be considered
Revaccination with different vaccine Nearly 60% of non-responders or
hyporesponders developed adequate antibody response.