This document discusses tumor cell proliferation and immunotherapy for cancers. It provides details on the cell cycle phases (M, G1, S, G2, G0) and how they relate to tumor growth and response to treatment. It also discusses cell kinetics, the growth fraction, and cancer stem cells. Targeted therapies discussed include those that inhibit angiogenesis by targeting VEGF, as well as EGFR inhibitors. Bevacizumab is highlighted as an anti-angiogenic therapy shown to improve outcomes for ovarian cancer both as a single agent and in combination with other drugs.

1. Tumor cell proliferation and immunotheray

of cancers
Prof. M.C.Bansal
MBBS,MS,MICOG,FICOG
Professor OBGY
Ex-Principal & Controller
Jhalawar Medical College & Hospital
Mahatma Gandhi Medical College, Jaipur.

2. CELL CYCLE
M: MITOTIC PHASE i.e. cell
division
G1: post mitotic phase
( protein & RNA synthesis)
S: sythetic phase ( DNA
synthesis)
G2: post synthetic phase
(Nucleus has diploid no.
chromosomes)
G0: resting phase ( cell may
go in or go out of mitosis)

3. Cell Cycle
 Information on growth patterns and doubling times
relates to the growth of the tumor mass as a whole.
The kinetic behavior of individual tumor cells has
been well described, and a classic cell cycle model has
been produced .
 M phase (mitotic phase) of the cell cycle is the phase
of cell division.
 G1 phase (postmitotic phase) is a period of variable
duration when cellular activities and protein and RNA
synthesis continue. These G1 cells can differentiate or
continue in the proliferative cycle.

4.  S phase (DNA synthetic phase) is the period in which
new DNA replication occurs.
 G2 phase (postsynthetic phase) is the period in which
the cell has a diploid number of chromosomes and
twice the DNA content of the normal cell. The cell
remains in this phase for a relatively short time and
then enters the mitotic phase again.
 G0 phase (the resting phase) is the time during which
cells do not divide. Cells may move in and out of the
G0 phase.

5.  The generation time is the duration of the cycle from
M phase to M phase. Variation occurs in all phases of
the cell cycle, but the variation is greatest during the
G1 period. The reasons for this variation are complex
and not completely understood.

6.  These cell cycle events have important implications for
the cancer therapist.
 Differential sensitivities to chemotherapy and
radiation therapy are associated with different
proliferative states.
 Dividing cancer cells that are actively traversing the
cell cycle are very sensitive to chemotherapeutic
agents.

7.  Cells in a resting state (G0) are relatively insensitive to
chemotherapeutic agents, although they occupy space
and contribute to the bulk of the tumor.

8. Cell Kinetics
 In cell kinetic studies performed on human tumors,
the duration of the S phase (DNA synthesis phase) is
relatively similar for most human tumors, ranging
from a low of 10 hours to a high of approximately 31
hours.
 The length of the cell cycle in human tumors varies
from slightly more than half a day to perhaps 5 days.
 With cell cycle times in the range of 24 hours and
doubling times in the range of 10 to 1,000 days, it is
clear that only a small proportion of tumor cells are in
active cell division at any one time.

9.  Two major factors that affect the rate at which tumors
grow are the growth fraction and cell death.
 The growth fraction is the number of cells in the
tumor mass that are actively undergoing cell division.
 There is a marked variation in the growth fraction of
tumors in human beings, ranging from 25% to almost
95%.

10.  In the past, it was thought that human tumors
contained billions of cells, all growing slowly.
 In actuality, only a small fraction of cells in a tumor
mass are rapidly proliferating; the remainder are out of
the cell cycle and quiescent.
 Cancer “stem cells” are a very small population of cells
that appear to be relatively chemoresistant; these play
a major role in the development and progression of
cancers.

11. Tumor growth may be altered by the following:
 immunologic therapies, which seem to alter both
generation time and growth fraction .
 cytotoxic chemotherapy, which alters both the generation
time and the growth fraction of a tumor
 hormones, which appear to alter the growth fraction
without changing the generation time
 radiation therapy, which alters both the generation time
and the growth fraction
 alterations in oxygen tension and vascular supply, which
alter the growth fraction without altering generation time

13. INTRODUCTION
 Cancer is caused by a series of events that include the
accumulation of successive molecular lesions and
alterations in the tumor microenvironment .
 Molecular lesions include
overexpression,
amplification,
mutations of oncogenes;
deletion of tumor suppressor genes;
inappropriate expression of growth factors
and their cellular receptors.

14.  In addition to these molecular changes, angiogenesis
and the lack of effective host antitumor immune
responses create a microenvironment that supports
the growth of cancer .

15.  Our improved understanding of these mechanisms
presents an opportunity for the development of novel
therapeutic approaches .
 This presentation provides an overview of
biologic, targeted, and immunotherapeutic strategies
for gynecologic cancers.

16. Biologic and Targeted Therapies
 The growth of cancer cells is crucially dependent on
oncogenic signal transduction pathways.
 Extracellular signals are transmitted to the cancer cell via
transmembrane receptors.
 Activation of the epidermal growth factor receptors (EGFR,
HER2, HER3, and HER4), for example, stimulates a cascade
of intracellular proteins that ultimately lead to changes in
gene expression.
 Novel therapeutics are targeted to modulate these signal
transduction pathways by blocking the extracellular
transmembrane receptors or interfering with intracellular
proteins such as tyrosine kinases further downstream.

17.  This novel therapeutic approach is also termed
molecular targeting .
 It is accomplished by either monoclonal antibodies
that bind to transmembrane receptors and serum
proteins such as vascular endothelial growth factor
(VEGF) or chemical, small-molecule inhibitors that
prevent activation of signal transduction proteins.
 Targeting the signaling cascade inhibits the
proliferation of cancer cells, induces apoptosis, and
blocks metastasis.

18.  The specificity of these molecules is based on the
assumption that cancer cells are over expressing
various proteins in the signal transduction
pathways, therefore presenting a preferred target
compared to normal cells.
 Conceptually, this should result in more cancer cell-
specific therapy and less clinical side effects because of
sparing of normal tissue

21. Angiogenesis
 The formation of new blood vessels (neoangiogenesis) is a
normal process during embryonic development, tissue
remodeling, and wound healing .
 Malignant tumors are able to induce angiogenesis by
secreting paracrine factors that promote the formation of
new blood vessels.
 Angiogenesis is a complex process that is influenced by
various pro-and antiangiogenic factors, including
VEGF, interleukin 8, platelet-derived endothelial cell
growth factor, and angiopoietins.
 Overexpression of these angiogenic factors leads to
neovascularization and increased supply of nutrients and
oxygen to the tumor.

22.  Three main therapeutic strategies that target
angiogenesis are currently being explored for the
treatment of cancer patients .
 One group of agents targets VEGF
(e.g., bevacizumab, VEGF-Trap), the second group
prevents VEGF from binding to its receptor
(pertuzumab), and a third group of agents inhibits
tyrosine kinase activation and downstream signaling
in the angiogenesis signaling cascade
(valatanib, sunitenib).

23. Vascular Endothelial Growth Factor
 VEGF is overexpressed in gynecologic
malignancies, therefore presenting an excellent target
for therapy .
 Inhibition of VEGF-induced angiogenic signaling
decreases tumor microvascular density and causes
death of solid tumors in various preclinical models.
 Several agents are now available for clinical use; all
target the VEGF signaling pathway.
 The most widely used agent at this time is
bevacizumab, a humanized, recombinant monoclonal
antibody that binds to all isoforms of VEGF-A.

24.  In ovarian carcinoma, various clinical trials have
demonstrated the efficacy of bevacizumab treatment.
 In a study by the Gynecologic Oncology Group, 62
patients received single agent bevacizumab 15 mg/kg
intravenously every 21 days .
 Thirteen patients (21%) showed clinical responses
with two complete and 11 partial responses.
 The median response duration was 10 months, and 25
patients (41.3%) survived progression free for at least 6
months.

25.  Bevacizumab has also been used in combination with other
agents.
 In a phase II study of 13 patients with recurrent ovarian or
primary peritoneal carcinoma, combination treatment with
bevacizumab (15 mg/kg i.v. every 21 days) and erlotinib (150
mg/day orally) resulted in one complete response and one
partial response for a total response rate of 15% .
 Seven patients had stable disease.
 Another trial investigated the combination of bevacizumab
(10 mg/kg every 14 days) and oral cyclophosphamide (50
mg/day orally) in 70 patients with recurrent ovarian cancer.

26.  The Gynecologic Oncology Group has initiated a
clinical trial that will evaluate the addition of
bevacizumab to first-line chemotherapy after primary
tumor debulking.
 A similar trial by the Gynecologic Cancer InterGroup is
designed to evaluate the safety and efficacy of adding
bevacizumab to standard chemotherapy (carboplatin
and paclitaxel) in patients with advanced epithelial
ovarian or primary peritoneal cancer .

27. Epidermal Growth Factor Receptor
Inhibitors
 The epidermal growth factor receptor family consists
of four members including EGFR
(HER1), HER2, HER3, and HER4 .
 EGFR overexpression has been reported in 35% to
70% of patients with epithelial ovarian cancer .
 In endometrial cancer, EGFR is overexpressed in 43%
to 67% of tumors and is associated with shortened
disease-free and overall survival .
 In addition, amplification of the HER2 gene is
commonly found in endometrial carcinoma.

28.  Various agents directed against epidermal growth
factor receptors are available .
 Trastuzumab is a humanized monoclonal antibody
that binds to the extracellular domain of HER2 .
 Blockade of HER2 affects various molecules that
ultimately decreases cell proliferation.
 Pertuzumab is another humanized monoclonal
antibody that binds to a different epitope of HER2
compared to trastuzumab.
 Binding to HER2 prevents dimerization of the
receptor, which is required for its function .

30. Epidermal Growth Factor Receptor
 Inhibition of EGFR signaling is accomplished by using
either monoclonal antibodies against the extracellular
receptor or small-molecule inhibitors against the
intracellular kinase domain.
 Both strategies results in inhibition of
phosphorylation or receptor activation.
 Erlotinib is a potent reversible inhibitor of EGFR
tyrosine kinase that blocks receptor
autophosphorylation and has been used for the
treatment of ovarian carcinoma.

31.  Erlotinib has been used in combination with docetaxel and
carboplatin as first-line treatment after surgical
cytoreduction in patients with ovarian, fallopian tube, and
primary peritoneal cancers .
 Cetuximab (C225, Erbitux) is a chimerized monoclonal
antibody against EGFR.
 Cetuximab in combination with carboplatin resulted in
three complete (10.7%) and six partial (21.4%) responses in
28 patients with recurrent ovarian cancer.
 Twenty-six of these 28 patients (92.8%) had EGFR-positive
tumors.


32.  The combination of paclitaxel, carboplatin, and
cetuximab for first-line chemotherapy of stage III
ovarian cancer patients resulted in progression-free
survival of 14.4 months and was therefore not
significantly prolonged compared to historical data.

33.  Gefitinib (ZD1839 Iressa) is a low molecular weight
quinazoline derivative that inhibits the activation of EGFR
tyrosine kinase via competitive binding of the ATP-binding
domain of the receptor.
 Treatment of patients with recurrent ovarian cancer using
the combination of gefitinib, carboplatin, and paxitaxel
resulted in a high overall response rate of 63% .
 Interestingly, antitumor responses were observed in 35% of
patients with platinum-resistant disease compared to a
73% response rate in patients with platinum-sensitive
disease.

34.  Gefitinib has also been used in combination with
tamoxifen.
 In squamous and adenocarcinoma of the cervix, gefitinib
(500 mg/day) treatment resulted in disease stabilization in
six of 28 patients (20%) but no clinical responses .
 Lapatinib is a small-molecule inhibitor of both the HER2
and EGFR tyrosine kinase receptor.
 The rationale for using lapatinib in endometrial carcinoma
is supported mainly by studies in human cancer cell lines.
Its efficacy in endometrial cancer is being investigated
currently in clinical trials .

35. HER-2/neu
 The HER-2/neu receptor is activated by homo- or
heterodimerization, resulting in tyrosine phosphorylation
and subsequent activation of various downstream signals
that among other functions control cellular
proliferation, migration, and invasion.
 Trastuzumab is a recombinant, humanized IgG1
monoclonal antibody that is specific for the extracellular
domain of HER-2/neu.
 Binding of the antibody to HER-2/neu prevents activation
of the receptor with a subsequent increase of apoptosis in
vitro and in vivo, impaired DNA damage repair, and
inhibition of tumor neovascularization.

36.  The HER-2/neu oncogene is overexpressed in several
gynecologic malignancies, including 20% to 30% of
ovarian cancers.
 HER2/neu overexpression is infrequent in cervical
cancer.
 In uterine papillary serous carcinoma, 12 of 68 (18%)
tumors showed HER2/neu overexpression; this was
associated with a worse overall prognosis .

37.  Mitogen-Activated Protein Kinase Pathways
 The mitogen-activated protein (MAP) kinase cascades
are activated by various cofactors, inflammatory
cytokines, and stress.
 Sorafenib is among the first of the agents with
clinically proven efficacy. Sorafenib is a competitive
inhibitor of raf that has been approved for treatment
of renal cell carcinoma and hepatocellular carcinoma.
 Besides targeting raf, sorafenib also inhibits VEGFR2
and VEGFR3, FT3, c-kit, and PDGFR-β.

38. The PI3-kinase/Akt/mTOR Pathway
 The phosphoinositide3-kinase (PI3-kinase)/Akt/mTOR
pathway is a major oncogenic signaling pathway in various
cancers.
 Activation of this pathway can be demonstrated in more
than 80% of endometrial cancers, 50% to 70% of epithelial
ovarian cancers, and approximately 50% of cervical
cancers.
 Several inhibitors of PI3-kinase/Akt/mTOR signaling are
currently in clinical trials.
 Rapamycin or rapamycin analogues, for example, block the
activity of mTOR, a protein complex responsible for
increasing protein synthesis and cellular proliferation.

39.  Several mTOR inhibitors, including RAD001 and
CCI779, and specific PI3-kinase inhibitors are
currently under development in preclinical models
and clinical trials. PI3-kinase/Akt/mTOR inhibitors
have been used in endometrial cancer with limited
benefit.

40. Immunotherapy
 Failure of functional immunity contributes to the
genesis of virus-associated cancers, such as those
caused by human papilloma virus (HPV) or Epstein-
Barr virus.
 The greatest success story involving the enhancement
of immunity to combat gynecologic cancer is the
development of vaccines against HPV, which are highly
effective for the prevention of cervical dysplasia and
cancer .

41.  Some researchers suggest that immune responses are
mainly involved in protection from virus-associated
cancers but not other forms of cancer .
 Cancer is a common disease, and overt immune
deficiency certainly is not necessary for its
development.
 However, recent studies have shown that many
cancers, including those that are not known to have a
viral etiology, are seen with increased frequency in
patients who have dysfunctional immunity like as in
patients of HIV.

42.  In a recent metaanalysis of cancer incidence in
populations known to be immune deficient
(e.g., organ-transplant recipients, patients with HIV
infection), Grulich and co-workers found an increased
incidence of several common cancers, suggesting that
impaired immunity can contribute to the development
of cancer.

43. Components of the Immune System
Involved in Antitumor Responses
 Various types of human immune responses can target
tumor cells.
 Immune responses can be categorized as humoral or
cellular, a distinction based on the observation in
experimental systems that some immune responses
could be transferred by serum (humoral) and others
by cells (cellular).
 In general, humoral responses refer to antibody
responses; antibodies are antigen-
reactive, soluble, bifunctional molecules composed of
specific antigen-binding sites.

44.  Cellular immune responses generally refer to cytotoxic
responses mediated directly by activated immune cells
rather than by the production of antibodies .
 Nearly all immune responses involve both humoral
and cellular components and require the coordinated
activities of populations of lymphocytes operating in
concert with each other and with antigen-presenting
cells.

47. Therapeutic Strategies
 There is great interest in developing effective biologic
and immune therapies for gynecologic malignancies.
 For example, patients with small-volume or
microscopic residual peritoneal ovarian cancer are
attractive candidates for immunotherapy or biologic
therapy, especially approaches based on regional
peritoneal immunotherapy or biotherapy.
 Also, many patients with advanced disease are
immunocompromised, suggesting a role for
immuneenhancing therapeutic approaches.

48.  Dysplastic and cancerous cervical epithelial cells
infected with HPV, an oncogenic virus, also present an
attractive target for immune enhancement-based
therapeutic strategies, including the development of
therapeutic vaccines for HPV.
 Advances in molecular biology, biotechnology,
immunology, and cytokine biology have resulted in
the availability of many new, promising
immunotherapeutic approaches for gynecologic
cancers.

49. Human Papillomaviruses and Cervical
Neoplasia
 Extensive molecular biologic and epidemiologic
research confirms certain HPV types to be
carcinogenic in humans (13,14,15,79,80,81,82,83,84,85).
 Infections with HPV cause approximately 5% of the
global burden of human cancers and at least 500,000
deaths annually.
 Infection with specific HPV types is necessary for the
development of the vast majority of cervical cancers
(>99.7%) and the immediate precursor lesion (CIN 3) .

50.  The four major steps in the development of cervical
cancer are
(i) infection of the metaplastic epithelium of the
transformation zone with one or more carcinogenic
HPV types.
(ii) viral persistence rather than clearance reflecting the
host immune response.
(iii) clonal progression of persistently infected
epithelium to cervical precancer (CIN 3) and
(iv) invasion.

51. Figure; Major steps in the development of cervical cancer. Incident HPV infection is best
measured by molecular tests. Most HPV infections show no concurrent cytological abnormality.
Approximately 30% of infections produce concurrent cytopathology, usually non-classical
(equivocal) changes. Most HPV infections clear within 2 years. Ten percent persist for 2 years and
are highly linked to development of precancer.

52. Human Papillomavirus and Cervical
Cancer: A Causal or Casual Association
 Although the true prevalence of cervical HPV infection
is unknown, it is the most common sexually
transmitted infection, with most sexually active
women younger than 35 years of age exposed .
 The 2-year cumulative incidence for first time genital
HPV infection for young women is 32% .
 For monogamous women, the risk of acquiring HPV
after sexual debut is 46% at 3 years .
 Smoking, oral contraceptive use, and report of a new
male sexual partner are predictive of incident
infection.

53.  The median time to clearance of HPV infection in an
immunocompetent woman is 6 to 18 months (average
of 12 months) with 90% of women clearing a specific
HPV-type after 2 years of observation .
 Viral clearance is not often associated with
reappearance of the same HPV type.
 Occasionally the same HPV type will reappear .
 It is unclear whether this represents reinfection or
resurgence from a latent state in the basal cells of the
epithelium.

54.  The high rate of HPV infection among immunosuppressed
HIV-infected women supports the concept of latency .
 The longer a specific HPV type persists in the epithelium,
the lower the probability of clearance within a defined
period, and the greater the risk of precancer .
 There is no accepted definition of clinically significant
persistence.
 Follow-up strategies suggest persistence beyond 1 year and
certainly beyond 2 years defines a greater risk to patients .

55.  HPV 16 is highly carcinogenic with an absolute risk of
CIN 3 approaching 40% at 3 to 5 years .
 Women infected with multiple HPV types have a
further increased risk but it is not clear if the risk is
equal to or greater than the cumulative risk associated
with each of the individual HPV types.

56.  Cervical neoplasia can be viewed as the result of a complex
interplay between a “seed,” that is, high-risk HPV
types, and a “soil,” that is, the immature, metaplastic
epithelium of the cervical transformation zone.
 Exposure to specific high-risk HPV types, in the presence
of cofactor activity, may deviate the metaplastic process
along a neoplastic pathway.
 Disease expression begins at the new squamocolumnar
junction.
 The initial abnormality produced is usually a low-grade
cervical lesion.

57.  The most critical step in cervical carcinogenesis is not
acquisition of an HPV infection but progression to CIN
3.
 HPV infection alone is necessary but not sufficient to
induce carcinoma in an immunocompetent host.
 HPV infection with oncogenic viral types is much
more common than cervical neoplasia, indicating the
necessity of cofactors in the process of cervical
carcinogenesis .

58. Cofactor Interaction with Human
Papillomavirus
 Cigarrete smoking
 Infection with other microbial agents - An increased
incidence of other sexually transmitted diseases has been
reported in association with genital HPV infection and
cervical neoplasia.
 Sex hormone influence -Condylomata acuminata
increase rapidly in size and number in pregnancy. This
could suggest that maternal estrogen status is permissive
for HPV replication.CIN and cervical cancer are more
frequently found in women with increased parity and in
women on oral contraceptives independent of sexual
activity .

59.  Exogenous and Endogenous
Immunosuppression-Iatrogenic induction of
immunosuppression in renal transplant recipients
increases the rate of CIN to 16 times that of the general
community .
 Dietary Factors -Dietary deficiencies of vitamin A or
beta-carotene may increase the risk of CIN and
cervical cancer .

60.  The HPV genome consists of approximately 8,000 base
pairs of single-stranded, circular DNA.
 HPV genes are designated as E or L according to their
expression in early or late differentiation stage of the
epithelium: E1, E2, E5, E6, and E7 are expressed early
in the differentiation,
 E4 is expressed throughout
 L1 and L2 are expressed during the final stages of
differentiation.

61.  The viral genome is maintained at the basal layer of
the epithelium, where HPV infection is established.
 Early proteins are expressed at low levels for genome
maintenance (raising the possibility of a latent state)
and cell proliferation.
 As the basal epithelial cells differentiate, the viral life
cycle enters successive stages of genome amplification,
virus assembly and virus release, with a concomitant
shift in expression patterns from early genes to late
genes, including L1 and L2, which assemble into viral
capsid.

63.  The proteins encoded by the E6 and E7 genes of high-risk
HPV types, particularly HPV 16 and 18 , are directly involved
in cellular transformation in the presence of an active
oncogene .
 E6 and E7 are the primary HPV oncoproteins with numerous
cellular targets .
 Both E6 and E7 proteins can immortalize primary
keratinocytes from cervical epithelium and can influence
transcription from viral and cellular promoters .
 The activity of these viral oncoproteins results in genomic
instability, leading to the malignant phenotype.
 E6 proteins of high-risk HPV types bind the tumor
suppressor protein p53 .
 This induces ubiquitination and degradation of
p53, removing the p53-dependent control of the host cell
cycle .

64.  The role of E6 as an antiapoptotic protein is of key
significance in the development of cervical cancer as it
compromises the effectiveness of the cellular DNA damage
response and allows the accumulation of secondary
mutations to go unchecked.
 E6 also increases telomerase activity in keratinocytes
through increased transcription of the telomerase catalytic
subunit gene (hTERT) through induction of c-myc .
 E7 is responsible for inactivation of product of Rb gene
(tumor supressor)
 Degradation of p53 by E6 and the functional inactivation of
pRb by E7 represent the main mechanisms leading to
malignant changes.

65. Human Papillomavirus Vaccines
 The HPV vaccine is a major scientific and public
health advance in the prevention of cervical cancer .
 Most invasive cervical cancers and CIN lesions are
attributable to high-risk HPV infection .
 A prophylactic or therapeutic vaccine against HPV has
the potential to have a substantial impact on HPV
infection and rates of CIN and cervical cancer .
 HPVs cannot be grown in the laboratory so standard
approaches to vaccine development, such as
inactivation of live virus or development of attenuated
virus, are not possible.

66.  Prophylactic vaccines have been produced using
recombinant DNA technology.
 HPV prophylactic vaccines, designed to prevent HPV
infection, are based on viruslike particle (VLP)
technology developed through the pioneering research
of Zhou and Fraser in Brisbane, Australia .

67.  VLPs are three-dimensional structures similar to
papillomavirus particles and produced by expression of L1
and L2 HPV viral capsid proteins.
 These DNA-free VLPs are empty capsids and contain no
oncogenic or infectious materials.
 Viruslike particles resemble the virus immunologically and
induce HPV type-specific antibody on administration .
 The immunogenicity of HPV involves presentation of the
major capsid protein L1 to the immune system.
 L1 VLP vaccines induce strong cell-mediated as well as
humoral immune responses .

68.  Two vaccine products have been developed, a
quadrivalent vaccine incorporating HPVs 16, 18, 6, and
11 (Gardasil; approved by the U.S. Food and Drug
Administration [FDA] in June 2006)
 and a bivalent vaccine incorporating HPVs 16 and 18
(Cervarix; currently under review by FDA).