Chemotherapy and Immunological Aspects

Author: Kornel Kusznieruk

There are four main uses of chemotherapy, these are:

a) Induction. This refers to using the therapy as a frontline treatment for a cancer in
which no other alternative exists.

b) Adjunct or Adjuvant. This involves using chemotherapy to prevent reoccurrence after
another primary treatment was applied. Usually, adjunct therapy is used after surgery or
radiation.

c) Neoadjuvant. This treatment is performed before an intervention such as radiation or
surgery in order to increase efficacy.

d) Site-directed perfusions. This treatment involves place slow release chemotherapy
doses or pumps in a position proximal to the tumor.

Alkylating Agents

Modern day chemotherapy has its roots in the observations that exposure of soldiers to
mustard gas in World War II led to bone marrow hypoplasia (1). Therapeutic use of
mustard gas, and its less toxic derivative nitrogen mustard, for leukemias was able to
induce remission even in advanced stages (2). The mechanism of action of nitrogen
mustard, an alkylating agent, is by crosslinking DNA through the formation of covalent
bonds, mainly on the 7 nitrogen atom of guanine. Many other alkylating agents are in use
today, these include cyclophosphamide, busulfan and melphalan. Alkylating agents kill
cells the best when they are in the S phase of cell cycle, although their cytotoxicity is
generally considered to be cell cycle phase independent but proliferation dependent.

Antimetabolites

The origin of this type of drug came from Farber who discovered that leukemia cells
proliferate in the presence of folic acid. In 1948, using the folic acid antagonist
aminopterin, he treated 16 children with acute leukemia and was able to induce remission
(3). Although aminopterin administration is associated with toxicity, other folic acid
antagonists which were more clinically useful were developed, one of these being
methotrexate. Mechanism of action for this type of drug is that reduction of folate levels
causes cessation of thymidylate and purine biosynthesis, while at the same time blocking
protein synthesis (4). 5-Fluorouracil (5-FU) is another antimetabolite but in contrast to
methotrexate, it works via its metabolites: a) directly binding RNA causing shut-down of
protein synthesis and b) blocking the activity of the enzyme thymidylate synthetase, an
enzyme needed for making the DNA base thymidine (5). Other antimetabolites which
are used clinically include cytosine arabinoside (Ara C), gemcitabine, and hydroxyurea.
Antineoplastic Antibiotics
This drug class inhibits cancer cells in part by blocking activity of topoisomerase, the
enzyme involved in unwinding DNA during cell proliferation. These antibiotics were
originally discovered from a culture of Streptomyces bacteria which yielded the clinically
useful drugs doxorubicin and daunorubicin (6). Other drugs belonging to this class
include mitomycin, bleomycin, and idarubicin. Etoposide and campothecin are inhibitors
of topoisomerase but are not antibiotics.

Microtubule Inhibitors

During mitosis microtubules align the chromosomes on the metaphase plate in order for
chromosomes to be properly distributed after the cell divides. Microtubules are
comprised of the subunit tubilin. Drugs such as vincristine, vinblastin, and taxol block
formation of microtubules and therefore block cell division.

Platinum Based Drugs

In 1961 Rosenberg et al were assessing the effects of electric current on bacterial growth.
Interestingly, the treated bacteria started to display significant structural abnormalities.
Further analysis revealed that the platinum in the electrodes were altering bacterial
growth. It was the cis-isomer of platinum that only had this activity (7). Subsequently,
administration of cis-platinum to tumor-bearing mice was shown to reduce burden (8).
The mechanism of its antitumor activity is via formation of intrastrand crosslink between
neighboring guanines. Several platinum based drugs are in use today including, cisplatin
and carboplatin.

Immune Suppressing Effects of Chemotherapy

All of the drugs mentioned above target proliferating cells. The hematopoietic
compartment is always proliferating, this explains why for many chemotherapeutic
interventions the dose-limiting toxicity is hematological. In addition, specific immune
system cells need to replicate in order to perform their functions. T cells and B cells need
to clonally expand to recognize antigens and mount a sufficient response. Let us examine
the immunological effects of these chemotherapeutic drugs with hope that combinations
of drugs, or sequential administrations can be devised to reduce tumor burden without
overtly damaging the immunological compartment.

In general patients receiving high dose chemotherapy (doses used for induction therapy)
have suppressed lymphocyte proliferation in response to mitogens, as well as, suppressed
production of anti-tumor cytokines such as IFN-γ. These effects were recently reviewed
by Mackall (9). Antimetabolites such as purine analogues given to patients induce an
over 90% reduction in circulating T cells (10). Immunosuppressive activities of these
analogues have even prompted their use in autoimmunity (11). Methotrexate, the folate
antagonist, also possesses potent immune cell suppressive activity which includes
blockade of CD8 proliferation as well as macrophage activation (12,13). For this reason
methotrexate is clinically used in the treatment of autoimmune arthritis (14). TNF-α is
secreted by macrophages and T cells during a cell mediated response. In a mouse model
methotrexate administration suppressed production of this cytokine (15). Furthermore,
there is some evidence from arthritic patients that methotrexate therapy is associated with
increased production of the immune suppressive cytokine IL-10 (16). In contrast, there is
some evidence, at least in vitro, that methotrexate may have immunoenhancing effects by
stimulating activity of natural killer cells (17). Nevertheless, has can be imagined, the
immune suppressive effects of antimetabolite therapies often result in infections and
other complication (18).

But it is not only the antimetabolites which have immune suppressive properties.
Alkylating agents (19), anti-neoplastic antibiotics (20), microtubule inhibitors (21) and
platinum compounds (22) all have immune suppressive activity. These activities are not
always associated with suppression of cell cycle progression. For example, patients
taking cyclophosphamide have a more profound depletion of naïve CD45RA T cells, than
cells with the memory phenotype, CD45RO (23). This is paradoxical since memory T
cells are proliferating, in the basal state, much more frequently than naïve T cells (24). It
appears that in different settings, different types of chemotherapy may bring about
different types of immune modulation. Mackall et al noticed that in children receiving
various intensive chemotherapy regimes of cyclophosphamide, vincristine, and
doxorubicin, recovering T cells contained rapidly expanding CD8+ CD28-, and CD8+
CD57+ populations. It took more than a year for the normal, CD28+ T cells to repopulate
these children (25). CD28- and CD57+ T cell phenotypes have been previously
associated with suppressor T cells (26,27), thus in this context chemotherapy may have a
dominant negative role on immune function. On this note, there is a quick point that
needs to be made. Chemotherapy depletes T cells and the hematopoietic compartment
needs to replete them. When the new T cells are made they still need to mature in the
thymus. However, after puberty the thymus involutes. Furthermore, glucocorticoids,
which also decrease thymic size are sometimes used in chemotherapeutic protocols. It
therefore seems logical that to get better restoration of the T cell compartment something
must be done to increase thymic function. Several thymic hormones exist and are
clinically available. It will be interesting to see if these agents can accelerate the
reconstitution of the T cell compartment (both in numbers and function) after
chemotherapy.

In conclusion, immune suppression is often associated with chemotherapy, but the
mechanisms of this suppression are situation specific. It is possible to treat arthritis with
methotrexate, but why not with other cell cycle inhibitory drugs? The answer is that
there are many different biochemical and immunological players which contribute to the
clinical effect. This is not as simple as “chemotherapy targets all proliferating cells.”

Immune Enhancing Effects of Chemotherapy

The immune system is extremely complex and sometimes pushing it in one direction will
induce it to pull its self the opposite direction from the one you wanted it to go.
Cyclophosphamide is toxic to immune cells, systemically at the doses used for induction
therapy it causes suppression. But the immune system contains suppressor cells which
regulate function of other immune system cells. So is it perhaps possible that
cyclophosphamide could actually stimulate immune function by destroying suppressor
cells at a higher rate than effector cells? One indication of such an effect is seen in the
non-obese diabetic (NOD) mouse model of diabetes. This strain is genetically
predisposed to develop diabetes at about eleven weeks of age. Treatment of mice at an
earlier age with cyclophosphamide induces diabetes. This effect is believed to be due to
depletion of suppressor cells by the cyclophosphamide (as opposed to islet cell toxicity)
since effector cells from treated mice can transfer disease to untreated mice (28). This
effect of cyclosphosphamide does not seem to be a mouse-specific artifact since clinical
cases exist of cyclophosphamide-treated patients developing diabetes (29).
Can cyclophosphamide increase immune response to cancer cells? Mouse studies have
been performed hoping to address this issue. In 1984, Greenberg and Cheever described
a murine leukemia model in which complete tumor resolution was possible through
concomitant administration of immune T cells together with cyclophosphamide (30).
Other combination therapies involving cyclophosphamide together with immune
stimulants such as IL-2 (31), IL-12 (32) and IL-15 (33) have demonstrated an augmented
anticancer response in mice. Studies have shown that cyclophosphamide can increase
efficacy of vaccination in the cases of: IL-2 modified tumor vaccine (34), MBT-2
vaccine for bladder cancer (35), and autologous tumor vaccine for chemically-induced
sarcoma (36). A possible mechanism for this immune stimulation is that
cyclophosphamide decreases production of the immune suppressive cytokine IL-10 in
tumor-bearing rats, while blocking the tumor-induced suppression of lymphocyte
proliferation (37). Another possible mechanism which has been shown is killing of the
tumor-induced suppressor cells (37a).

The alkylating agent melphalan has been shown to increase expression of CD80 on tumor
cells (38), this is accomplished through increased oxidative stress and NF-κB activation
on the tumor cells (39). In the latter reference it was also demonstrated that γ-irradiation
could also increase expression of costimulatory molecules on tumor cells. Such an
increase in costimulatory molecules could make the cells more immunogenic and thus
assist in stimulation of immune response (39a-c).

Cyclophosphamide has also been used to increase tumor immunity in humans. In 1986
Berd et al demonstrated patients injected with autologous melanoma vaccine had higher
DTH response when the vaccine was given together with cyclophosphamide than in
controls. Two patients receiving cyclophosphamide plus the vaccine had complete
disappearance of skin metastasis and were disease free for 42 and 33 months, while no
responses were seen in the patients receiving vaccine alone (40). Levels of antitumor
antibodies were increased in the cyclophosphamide group a clinical II trial evaluating
efficacy of vaccination to the tumor antigen sialyl-Tn (41). The problem with combined
cyclophosphamide-immune therapy in humans is that some efficacy is shown, but not
much greater than conventional therapies (42).

Conclusion

Chemotherapy is useful due to its ability to substantially decrease tumor burden in
inoperable areas, as well as target systemically metastasized tumors. The problem with it
is the severe side effects and immunosuppressive aspects. Although not mentioned,
chemotherapy is implicated in nutritional deficiencies of many cancer patients. Adequate
levels of many nutrients such as zinc, vitamin C, and selenium are vital for proper
immune function. The psychological impact of many chemotherapeutic regimes can
profoundly influence immune response. But besides these negative aspects, there are
some very interesting properties of many types of chemotherapies, which should be taken
advantage of. Immune modulation by many of these agents has been studied on and off
almost since the inception of chemotherapy. Precise monitoring of immune status in
patients should help devise protocols which are more cytotoxic to the cancer cells and
less immune suppressive. New methods of dealing with toxicity of chemotherapy have
allowed for greater patient access and greater cures. An example of this is the advances
in autologous stem cell support which have allowed usage of chemotherapeutic drugs at
dosages which otherwise would have been lethal.

In the future some of the questions that need to be answered are:

1. What is the impact of various regimes on functions of T cells, B cells, NK cells, and
dendritic cells?

2. Do correlations exist between function of these cell types and clinical outcome?

3. How long does it take for chemo-induced immune alterations to subside?

4. Are there ways to increase immune response after chemotherapy by the use of
immune modulators or thymic hormones?

5. Can other modalities be used together with chemotherapy and immune therapy, for
example hyperthermia?

6. Are there specific enzymatic pathways (DNA arrays + proteomics will tell us) which
suppressor T cells use that can be chemically targeted?

7. Can we induce immune response to epitopes associated with the drug-resistant
phenotype so that the cells that are not killed by the chemotherapy will be killed by
immunotherapy?