Cancer Therapy Vol 3, 101-104, 2005
Avi Khafif1, Elizabeth Gillis2, Jesus E. Medina3,
*
1Department of Otorhinolaryngology the Tel-Aviv
Sourasky Medical Center, Tel-Aviv, Israel,
2Department of Pathology and
3Department
of Otorhinolaryngology, The University of Oklahoma Health Sciences Center
Oklahoma City, Oklahoma
__________________________________________________________________________________
*Correspondence: Avi Khafif, Department of Otorhinolaryngology, Tel
Aviv Sourasky Medical Center, 6 Wiezman street, Tel Aviv; Tel: (9723) 540-0526;
E-mail: khafif@tasmc.health.gov.il
Key
words: G-glyco protein,
hypopharyhgeal cancer, chemotherapy, Immunohistochemistry, Clinical study
Abbreviations: 5-fluorouracil, (5-FU);
diaminobenzidenetertrahydrochloride, (DAB); multidrug resistance gene, (MDRG);
proliferating cell nuclear antigen, (PCNA); P-glycoprotein, (P-GP); squamous
cell carcinoma, (SCC)
Summary
Larynx
preservation protocols using combinations of chemotherapy and radiation are effective
in some patients with advanced resectable head and neck cancer. The purpose of
the current study was to investigate whether P-glycoprotein (P-GP), the product
of the multi-drug resistance gene, correlates with tumor response to
chemotherapy, survival and the rate of laryngeal preservation using a larynx
preservation protocol for advanced squamous cell carcinomas of the larynx and
hypopharynx. Twenty-four patients with advanced cancer of the larynx and
hypopharynx, were prospectively assigned to receive two cycles of cis-platinum
and 5-FU followed by radiation. Immunohistochemistry for P-GP was performed and
staining correlated with response to chemotherapy, survival, and the rate of
laryngeal preservation. Six patients (25%) had tumors that exhibited positive
staining for P-GP. Four of these patients (66%) responded to chemotherapy and
three (50%) are alive at the time of last follow-up. Of the 18 patients with
tumors that exhibited negative staining for P-GP, 15 (83%) responded to
chemotherapy, and 9 (50%) are alive at the time of the last follow-up. The
response to chemotherapy, survival and rate of laryngeal preservation was
similar in the two groups of patients. Positive staining for P-GP did not
correlate with a response to cis-platinum and 5-FU in patients with SCC of the
larynx and hypopharynx, nor did it predict a decreased survival or a lower rate
of laryngeal preservation in these small cohort of patients.
I. Introduction
Larynx
preservation regimens using chemotherapy and radiation are being used
extensively as part of experimental larynx preservation protocols in the
treatment of patients with advanced squamous cell carcinoma (SCC) of the larynx
and hypopharynx. The resistance of tumor cells to chemotherapy, however, is a
major problem, and recent studies have focused on understanding the mechanisms
of this resistance. Bradford et al (Bradford et al, 1999) investigated factors
affecting the response in patients treated as part of the Veterans Affairs
Laryngeal Cancer Cooperative Study. They found that a high T-stage, positive
staining for p-53 and high levels of proliferating cell nuclear antigen (PCNA)
significantly correlated with the rate of laryngeal preservation. However, only
tumor stage correlated with response to chemotherapy and none of these factors
correlated with survival. Glutathione and Glutathione S-Transferase play a
major role in cellular protection against free radicals and oxidative stress
but their levels did not predict tumor response to platinum or 5-FU in patients
with SCC of the Head and Neck (Bier et al, 1996). Etienne et al, (1995) studied
the effect of Dihydropyrimidine dehydrogenase, the enzyme responsible for the
degradation of 5-fluorouracil (5-FU), on response of 62 patients with head and
neck cancer to 5-FU. They concluded that 5-FU catabolism in target cells is
probably a determinant factor for 5-FU responsiveness in cancer patients. This
enhanced catabolism may be mediated by high expression of the multidrug
resistance gene (MDRG). The purpose of this study was to determine whether
response to chemotherapy could be predicted by an immunohistochemistry staining
for P-Glycoprotein (P-GP), the protein product of the MDRG.
II.
Materials and Methods
A.
Clinical study
Patients with advanced, resectable SCC of the larynx
and hypopharynx were enrolled onto the study and were treated with two cycles
of chemotherapy including cis-platinum (100mg/M2) and 5-FU (1gr/M2/24h for 5
days). Patients with cardiac co-morbidity received carboplatin and 5-FU. If the
tumor decreased to less than 50% of its original size, a third cycle of
chemotherapy was usually added and was followed by a full course of radiation.
If less than 50% response was observed, salvage surgery (i.e. total
laryngectomy) was performed.
End points for analysis were death or recurrent
disease and laryngeal preservation. Follow-up data was collected for a minimal
period of two years or until death. The study was approved by the institutional
review board (IRB).
B.
Immunohistochemistry
The histopathology biopsy specimens of the 24
patients included on the study were retrieved. Representative tumor tissue
slide preparations made from the formalin fixed paraffin embedded tissue. These
were immunostained using antibody, NCL- PGLYm (Novocastra Labs) with
specificity for Human P-glycoprotein,C-terminal cytoplasmic domain.
Immunohistochemistry was performed by the avidin-biotin complex method of Hsu
et al*. Following deparaffinization, inhibitor solution was optimized to remove
endogenous peroxidase activity for 4 minutes at 37¡C.The optimized antibody is applied and incubated
for 32 mins. at 37¡C. This was followed by a biotinylated horse
anti-mouse immunoglobulin with 8 mins incubation at 37¡C and Avidin-HRPO for another 8 mins (Ventana,
Tuczon, AR). Chromogen solution consisted of 0.05%
diaminobenzidenetertrahydrochloride (DAB) with 0.07% H202. Sections were
counterstained with hematoxylin and carefully examined. Membrane
immunoreactivity in tumor was scored as follows: positive, trace (< 10% of
tumor cells staining), and negative. Liver was used as the recommended positive
control. Negative controls lacking primary antibody were also evaluated in each
case for background staining. Diffuse weak and diffuse strong staining were
considered positive staining while focal traces or no staining were reported as
a negative staining. Degree of staining was correlated with survival, response
to chemotherapy and the rate of laryngeal preservation. A two-tailed t-test was used to evaluate
the statistical significance of this correlation and a p value equal or smaller
than 0.05 was considered a significant difference.
III.
Results
Twenty-four
patients (18 men and 6 women) were enrolled in the study between the years
1993-1995. All
had locally advanced (T3-T4) primary tumors located in the larynx (n=19) and
hypopharynx (n=5). Nineteen patients had a complete or partial
response and received all three cycles of chemotherapy followed by radiation.
Five patients failed to show more than 50% reduction and had a salvage
laryngectomy. Thirteen patients (54%) had a salvage laryngectomy either for
persistent or recurrent disease. All patients had a minimal follow-up of 2
years. Twelve patients are alive
with no evidence of disease and 12 are dead of disease. The larynx was
preserved in 9 patients and only three patients were salvaged with a total
laryngectomy and were alive with no evidence of disease at the time of last
follow-up.
Altogether,
six patients had tumors that exhibited a positive staining for P-GP while 18
patients had tumors that had negative staining. Four of the patients with
positive staining for P-GP (66.6%) and 15 of the patients with negative
staining (83%) responded (PR or CR) to chemotherapy (p>0.05, table 1). Three
of the six patients with positive staining (50%) died of their disease,
comparable to 9 of the 18 patients (50%) with negative staining for P-GP
(p>0.05). Two of the six patients (33%) that had a positive staining for
P-GP are alive with their larynx preserved compared to 7 of the 18 (38%) that
had negative staining (p > 0.05).
IV.
Discussion
The
MDRG P-GP had been suggested as a reliable predictor of response of tumor cells
to chemotherapy drugs. The results of the present study suggest that
immunohistochemical staining of SCCa of the larynx and hypopharynx does not
correlate with the response of the tumor to chemotherapy with cis-platinum and
5-FU patient survival or rate of larynx preservation. These results are
interesting because the tumors studied represent a relatively homogeneous group
of advanced tumors of only two sites within the head and neck, which were
treated with a single chemotherapeutic regimen and were systematically
evaluated for response to therapy. These results may shed some light on the
controversy existing in the literature about the role of P-GP expression as a
predictor of tumor response to chemotherapy. Recently, Lo-Muzio et al, (2000)
studied tumors from 30 patients with squamous cell carcinomas of the oral
cavity and normal tissues from 6 patients by immunohistochemistry staining to P-GP.
They found that 66% of their normal and 80% of their tumoral tissues showed a
positive staining. Interestingly, in 4 patients the tumor showed strong
staining while the surrounding normal tissues were negative, and in 6 patients
a strong positive staining was observed in areas of higher degree of
differentiation. The authors suggest that activation of the MDRG may occur
early in the carcinogenesis process and may be involved in tumor response to
chemotherapy. This is supported by a recent study (Warnakulasuriya et al, 2000) reporting a
significant correlation of P-GP expression and response to chemotherapy and
radiation in 111 patients with advanced head and neck cancer.
Cordon-Cardo et al, (1990) performed
immunohistochemistry staining for P-GP in various tissues and concluded that
although P-GP expression can be detected in a variety of tumors, further
studies are needed to establish the significance of such findings. Filiptis et
al, (1997) investigated the association between expression of the MDRG and
survival or response to chemotherapy in patients with acute leukemia. Positive
immuno-cytochemistry staining for MDRG had no impact on survival or response to
induction chemotherapy in these patients. Darnton et al. investigated the
response of 27 patients with esophageal cancer to MIC chemotherapy (mitomycin,
ifosfamide, cisplatin) and found that 20/27 (70%) of patients with squamous
cell carcinoma responded to chemotherapy and only one expressed P-GP, whereas
3/10 (30%) of the patients with adenocarcinoma responded to chemotherapy and of
these 7 showed over expression of the P-GP. These concluded that the presence
of P-GP might explain the lower response of adenocarcinoma to MIC chemotherapy;
however, it cannot explain the variable response rates in patients with SCC
since all these tumors showed low expression of P-GP. Rabkin et al, (1995) on
the other hand, reported over expression of P-GP in all squamous cell
carcinomas of the base of the tongue. Analysis of these two later studies
reveals that the rate of expression of P-GP was not different in most patients
with squamous cell carcinomas and the different rates of expression may reflect
various sensitivity for detecting P-GP. The use of P-GP as a predictor of
response in these patients is thus questioned, a fact that is supported in the
current study; P-GP did not predict the outcome after chemotherapy in patients
with advanced Laryngeal and Hypopharyngeal squamous cell carcinomas.
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