Introduction
The Malaysian National Cancer Registry Report (2007)
reported colorectal cancer to be the second leading type of cancer
among men as well as among women, with a total of 2,246 diagnosed
cases, including 1,235 affected men and 1,011 women in 2007
(1). With the progression of
colorectal cancer, synchronous occurrence of liver metastases is
identified in ≤50% of the patients, which requires a multimodality
treatment approach (2).
Chemotherapy is currently considered to be the
standard treatment method for stage III colon and stage II rectal
cancer. In patients with terminal colorectal cancer, palliative
care is considered to improve the quality of life (3,4).
The high rate of recurrence and metastasis of
colorectal cancer underlines the need for novel treatment
modalities. The combination of biotherapy with other standard
treatment modalities is increasingly recognised as an effective
method, particularly in cases with advanced-stage cancer (5–8).
Ex vivo natural killer (NK)- and T-cell
expansion has been accepted worldwide as beneficial for the
treatment of metastatic or minimal residual cancer and an improved
prognosis has been reported with this application (9–11). A
number of approaches have been investigated for the growth and
expansion of NK (12–14) and T cells (15,16),
in order to obtain a maximum-fold expansion. Takada et al
(17) and Dewan et al
(18) successfully expanded NK and
T cells by severalfold from a low number of peripheral blood
mononuclear cells (PBMCs) in order to develop a multipronged
approach to cancer management.
Autologous immune enhancement therapy (AIET)
involves the isolation and expansion of NK cells and T lymphocytes
from the patients’ own peripheral blood, followed by re-infusion of
the activated cells to the patients intravenously. AIET may be
combined with currently available treatment methods, in order to
equip the immune system to efficiently recognize and eliminate
tumor cells and may be used in passive or active immunotherapy for
eradicating cancer stem cells (19). In this study, we report our
experience with the application of AIET in 4 patients with stage IV
colorectal cancer.
Patients and methods
Patients characteristics
One male and three female patients who were
diagnosed with stage IV colon carcinoma were enrolled for AIET.
Three patients received a hemicolorectomy, whereas the fourth
patient was inoperable and exhibited poor cardiac function.
Chemotherapy was prescribed to the patients with FOLFOX as
first-line chemotherapy, followed by bevacizumab, cetuximab and
capecitabine as second- and third-line treatment. The median age of
the patients was 56 years. One patient refused to undergo
chemotherapy and received 6 infusions of AIET. The medical history
of the patients who underwent AIET infusions is summarized in
Table I.
 | Table IDetails of the patients who underwent
autologous immune enhancement therapy. |
Table I
Details of the patients who underwent
autologous immune enhancement therapy.
| Cases | Age
(years)/gender | Diagnosis | Surgical
procedure | Cancer stage | Metastatic site | Standard therapy |
|---|
| 1 | 50/F | Advanced colon
CA | Hemicolectomy | IV | Liver to lung | FOLFOXa, FOLFIRIb with bevacizumab |
| 2 | 50/F | Advanced colon
CA | Hemicolectomy,
attempted liver resection | IV | Colon to liver | FOLFOXa, FOLFIRIb with cetuximab |
| 3 | 63/F | Advanced colon
CA | Hemicolectomy | IV | Colon to liver | Cetuximab,
FOLFOXa, capecitabine |
| 4 | 58/M | Advanced colon
CA | Inoperable with poor
cardiac function | IV | - | Refused |
In vitro isolation of PBMCs, activation
and expansion
The peripheral blood collected from the patients was
processed in a Good Manufacturing Practice-certified clean room.
The isolated PBMCs were cultured for 12–15 days using gas-permeable
culture bags as previously described (17,18).
Interleukin-2 and the patient’s own plasma were used as growth
supplements throughout the culture process to enhance the
expansion. The cells were harvested after 12–15 days for
intravenous administration. Pre- and post-expansion
immunophenotyping was performed to determine the initial and final
percentages of NK and T cells from the PBMCs and the expanded cell
population. Multiple intravenous infusions were administered to the
patients, with a maximum of 6 infusions.
Results
Total expanded cell count
The cell counts were determined with the trypan blue
dye exclusion test for the isolated PBMCs and expanded NK cell and
T lymphocyte populations. Cell growth was increased from day 7 or 9
and logarithmic expansion was observed until the cells were
harvested. Table II shows the
average population of cells seeded to respective anti-CD3 and
anti-CD16 coated flasks and the retrieved cell count on the day of
harvest in accordance with the deviation noticed during processing
of each sample. On the day of the harvest, the maximum-fold
increase compared to the initially isolated cell count was found to
be 150.
| Table IIAverage number of cell population pre-
and post-expansion. |
Table II
Average number of cell population pre-
and post-expansion.
| Cases | No. of AIET
infusions | PBMC average
count/collection (mean ± SD) (×106) | Average no. of
cells/infusion (mean ± SD) (×106) | Average initial cell
count for NK-cell culture (×106) | Average final cell
count for NK-cell culture (×106) | Average initial cell
count for T-cell culture (×106) | Average final cell
count for T-cell culture (×106) |
|---|
| 1 | 6 | 52.2±15.5 | 4,731.5±712.6 | 32.7 | 1,444.1 | 19.5 | 3,287.3 |
| 2 | 3 | 38.5±13.0 | 3,017.7±808.9 | 25.7 | 1,170.8 | 12.8 | 1,846.8 |
| 3 | 1 | 33.0 | 859 | 22 | 848 | 11 | 1,100 |
| 4 | 4 | 77.1±41.4 | 2,024.1±737.2 | 45.9 | 1,095.1 | 31.2 | 928.9 |
The pre- and post-expansion immunophenotyping
analysis of various lymphocyte markers revealed a steady increase
in the number of effector cells following expansion. The list of
the analyzed markers, the average percentage of the population and
the corresponding number of infusions administered to each of the
patients, is presented in Table
III, whereas the absolute cell numbers and fold expansions are
presented in Table IV.
| Table IIIResults of immunophenotyping analysis
(average), pre- and post-expansion. |
Table III
Results of immunophenotyping analysis
(average), pre- and post-expansion.
| Cases | No. of
infusions | PBMCs (%) | NK-cell culture
(%) | T-cell culture
(%) |
|---|
|
|
|
|---|
|
CD3+ |
CD3+CD4+ |
CD3+CD8+ |
CD3−CD56+ |
CD3−CD56+ |
CD3−CD16+ |
CD3+ |
CD3+CD4+ |
CD3+CD8+ |
|---|
| 1 | 6 | 77.2 | 40.5 | 36.5 | 7.08 | 39.7 | 21.2 | 80.3 | 33.6 | 71.4 |
| 2 | 3 | 52.9 | 34.9 | 19.4 | 22.5 | 67.8 | 35.6 | 69.6 | 44.5 | 53.9 |
| 3 | 1 | 78.2 | 48.4 | 26.2 | 10.1 | 57.6 | 57.2 | 63.9 | 40.2 | 24.5 |
| 4 | 4 | 86.1 | 63.9 | 21.5 | 6.9 | 78 | 71.9 | 73.8 | 24.1 | 54.8 |
| Table IVAbsolute cell number (average) pre-
and post-expansion. |
Table IV
Absolute cell number (average) pre-
and post-expansion.
| | PBMCs | NK-cell
culture | T-cell culture |
|---|
| |
|
|
|
|---|
| Cases | No. of
infusions | T cells
(×106) | CD4+ T
cels (×106) | CD8+ T
cells (×106) | NK cells before
(×106) | NK cells after
(×106) | NK-cell fold
expansion | T cells
(×106) | CD8+ T
cells (×106) |
CD3+CD8+ T-cell fold
expansion |
|---|
| 1 | 6 | 40.3 | 21.1 | 19.1 | 3.7 | 573.3 | 155 | 2,639.7 | 1,104.5 | 58 |
| 2 | 3 | 20.4 | 13.4 | 7.5 | 8.7 | 793.8 | 91 | 1,285.4 | 821.8 | 110 |
| 3 | 1 | 25.8 | 16.0 | 8.6 | 3.3 | 488.4 | 148 | 702.9 | 442.2 | 51 |
| 4 | 4 | 66.4 | 49.3 | 16.6 | 5.3 | 854.2 | 161 | 685.5 | 223.9 | 13 |
Intravenous infusion and follow-up
The harvested cells were suspended in 100 ml sterile
saline solution and administered intravenously. The patients were
followed up after the AIET infusions. The tumor marker evaluation
revealed a significant decrease, which was accompanied by an
improvement in the patients’ quality of life and a considerable
increase in survival rates. The prognosis of the patients following
administration of AIET is summarized in Table V.
| Table VPrognosis of the colon cancer
patients following AIET. |
Table V
Prognosis of the colon cancer
patients following AIET.
| Cases | Age
(years)/gender | Number of AIET
infusions | Prognosis |
|---|
| 1 | 50/F | 6 | 18 months,
(succumbed to ARDS following radiotherapy to the mediastinum) |
| 2 | 50/F | 3 | 32 months
(succumbed to the disease) |
| 3 | 63/F | 1 | 20 months
(succumbed to the disease) |
| 4 | 58/M | 4 | 12 months (alive,
stable disease, continuing AIET) |
Discussion
Previous studies on the positive outcome of
immunotherapy using in vitro expanded lymphocytes (14,19–21)
reported that this type of treatment may even target cancer stem
cells, which are considered to be a major target of eradication
(22), to achieve a disease-free
survival. Extensive investigation has been focused on expanding the
desired cell population of immune cells to considerable numbers
that may efficiently target the tumor cells (21).
It was suggested that adequate quantities of
clinical grade immune effectors (13,23)
and the safety of multiple infusions as showin in previous
treatments (24–26) remain a major concern in
immunotherapy. In this study, it was substantial to achieve a
maximum 161-fold expansion of NK cells without using any feeder
cells for 2 weeks (14).
There were no adverse reactions following the
administration of the ex vivo expanded lymphocytes. The
infused cells were able to boost the immune response under in
vivo conditions, which was reflected by the improvement in the
quality of life of the patients. The safety of the intravenous
administration of immune cells to cancer patients was previously
reported (14,24,27).
The follow-up of the patients after administration revealed
improved survival in all the patients who underwent immunotherapy.
In fact, the patient who did not undergo any chemotherapy (case 4;
Table I) remains alive with a good
quality of life and is still under follow-up.
One of the most promising anticancer approaches is
the employment of autologous immune cells to eradicate tumor cells.
In that regard, NK cells (28) and
cytotoxic T lymphocytes (9) are
receiving increasing attention worldwide. Various concepts and
strategies were introduced and discussed regarding the adoptive
transfer of various kinds of immune cells, mainly focusing on
cancers such as breast (29),
prostate (30) and ovarian cancer
(31). In addition, adoptive
immunotherapies were further investigated for melanoma (32), pediatric malignancies (33), thyroid carcinoma (34) and renal cell malignancies (35).
In conclusion, our results are encouraging, although
they require further validation regarding the safety of the
expanded population of NK cells and T lymphocytes for advanced
colon cancer patients. With the combined efficiency of chemotherapy
and immediate referrals it may be feasible to generate an optimal
method for the management of the increased number of colon cancer
patients, as this type of cancer is considered to be a major cause
of mortality in Malaysia. The current technology has enabled
maximization of the lower available cell population to a
considerable number that may prove beneficial to cancer patients as
an adjuvant/alternative therapeutic option.
Abbreviations:
|
NK
|
natural killer
|
|
PBMCs
|
peripheral blood mononuclear cells
|
|
AIET
|
autologous immune enhancement
therapy
|
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