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
9.
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
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
15.
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.
19. 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 :
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• 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.
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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
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.
Notes de l'éditeur
The vaccine recommended for those who are at risk of acquiring HBV infection is recombinant DNA vaccine either Engerix-B or HB-VAX II. Both recombinant vaccines2 contain nonglycosylated hepatitis-B surface antigen (HBsAg) particles that have been purified, adsorbed on aluminium hydroxide and preserved with thimosal. Manufactures`s data claim more than 90% response to hepatitis-B vaccination. However, the response rate is much lower in clinical practice. Males aged 15 to 20 shows an 80 % response and that in ages 50 or above show 60% response. The response rate is better in females with 97% and 82% in the similar age groups.(ref) What is non-response? Vaccine response is measured by the titre of hepatitis-B surface antibody (HBsAb). The sensitivity of assay is 2 to 5mIU/ml. A level of 10 mIU/ml is taken as a measurable response. Less than 10 are considered as non-response, 10 to 100 is considered as hypo-response and above 100 mIU/ml as adequate response. In peoples with a titre level less than 10, current recommendation is to revaccinate. An additional booster is recommended for hyporesponders. At levels over 100, a booster is still advised after 3 to 5 years. However, the need for booster vaccination after decay in antibody is a subject of debate world-wide. Causes of non-response Several factors can be responsible for non- or hypo-response3,4 (table ). A recent study showed lower response in healthy homosexual men compared to healthy healthcare workers4. An association with different HLA-DR alleles has been found in different studies. Those with HLA phenotype of B44, DRB1*0701, DQB1*0201 were nearly four times likely to be non-responders compared to responders. It is possible that 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 the major histocompatability complex (MHC). Much effort has been devoted to overcome the class II linked non-responsiveness to hepatitis-B vaccine. Several immunomodulators in the form of interferons have been tried with vaccination but the results are conflicting and without significance(1). Kinetics of Vaccine response The HBsAb titre declines after the vaccination(3). A booster injection results in a rapid increase in antibody titre (HBsAb) within 4 days reaching the highest titre during a month followed by rapid decline in the next 12 months and more slowly thereafter. Revaccination with different vaccine A significant shift in antibody levels has been observed with HB-VAX II in previous non-responders to Engerix-B5. Nearly 60% of non-responders or hyporesponders developed adequate antibody response. However, there is no randomised controlled trials looking into the difference in efficacy of these two products and from a practical point of view, both of them are equally immunogenic and are interchangeable2. A new triple S antigen recombinant hepatitis-B vaccine which incorporated the pre-S1 and pre-S2 components of the surface antigen overcame the non-response in 69% of healthcare workers with a history of persistent non-response to conventional hepatitis-B vaccines6. However, this vaccine is not licensed for use in the UK.
In mathematics, the geometric mean is a type of mean or average, which indicates the central tendency or typical value of a set of numbers by using the product of their values (as opposed to the Arithmetic mean which uses their sum). A geometric mean is often used when comparing different items – finding a single "figure of merit" for these items – when each item has multiple properties that have different numeric ranges.[1] For example, the geometric mean can give a meaningful "average" to compare two companies which are each rated at 0 to 5 for their environmental sustainability, and are rated at 0 to 100 for their financial viability. If an arithmetic mean was used instead of a geometric mean, the financial viability is given more weight because its numeric range is larger- so a small percentage change in the financial rating (e.g. going from 80 to 90) makes a much larger difference in the arithmetic mean than a large percentage change in environmental sustainability (e.g. going from 2 to 5). The use of a geometric mean "normalizes" the ranges being averaged, so that no range dominates the weighting, and a given percentage change in any of the properties has the same effect on the geometric mean. So, a 20% change in environmental sustainability from 4 to 4.8 has the same effect on the geometric mean as a 20% change in financial viability from 60 to 72. The geometric mean is similar to the arithmetic mean, except that the numbers are multiplied and then the n th root (where n is the count of numbers in the set) of the resulting product is taken. For instance, the geometric mean of two numbers, say 2 and 8, is just the square root of their product; that is 2√2 × 8 = 4. As another example, the geometric mean of the three numbers 4, 1, and 1/32 is the cube root of their product (1/8), which is 1/2; that is 3√4 × 1 × 1/32 = ½ . More generally, if the numbers are , the geometric mean satisfies and hence The latter expression states that the log of the geometric mean is the arithmetic mean of the logs of the numbers. The geometric mean can also be understood in terms of geometry. The geometric mean of two numbers, a and b , is the length of one side of a square whose area is equal to the area of a rectangle with sides of lengths a and b . Similarly, the geometric mean of three numbers, a , b , and c , is the length of one side of a cube whose volume is the same as that of a cuboid with sides whose lengths are equal to the three given numbers. The geometric mean applies only to positive numbers.[2] It is also often used for a set of numbers whose values are meant to be multiplied together or are exponential in nature, such as data on the growth of the human population or interest rates of a financial investment. The geometric mean is also one of the three classical Pythagorean means, together with the aforementioned arithmetic mean and the harmonic mean. For all positive data sets containing at least one pair of unequal values, the harmonic mean is always the least of the three means, while the arithmetic mean is always the greatest of the three and the geometric mean is always in between (see Inequality of arithmetic and geometric means.)
22% of non-responders experienced clnical AIDS or death compared with 5% responders. Non-response to HBV vaccine was associated with a greater than 2x increased risk of clincial AIDS or death. Thus, the ab response to the HBV vaccine may provide a measure of immune status that cannot be captured by assessing the CD4 count, VL, so may serve as a tool for risk stratification of HIV infected patients. The proportion of adults who achieve seroprotection (≥10 mIU/mL antibody to hepatitis B surface antigen [anti-HBs]) after receipt of the 3-dose vaccine series decreases with age, obesity, smoking, immunosuppression, and comorbid conditions including diabetes. When the antibody responses among older adults with and without diabetes are compared, the response might be reduced among those with diabetes. A synthesis of available literature suggests a protective response is achieved after completion of the hepatitis B vaccine series in ≥90%, 80%, 65%, and <40% of adults with diabetes lacking comorbid conditions aged ≤40 years, 41 through 59 years, 60 through 69 years, and ≥70 years, respectively (CDC, unpublished data, 2011). Revaccination with 1–3 additional doses of hepatitis B vaccine safely increases the proportion of adults who achieve a protective level of anti-HBs (≥10 mIU/mL) ( 5 ). The duration of protection against symptomatic and chronic HBV infection lasts >22 years among healthy vaccine responders ( 9 ); duration of immunity among persons with diabetes is unknown. HBV may have utility in assessing functional immune status and risk stratificating HIV infected individuals, including thoes with a CD4 count>500cells/mm3
