Current Status of Cell Culture Drug Resistance Testing [Functional Tumor Cell Profiling]
1.) Larry M Weisenthal MD PhD and 2.) Peter
Nygren MD PhD
1). Weisenthal Cancer Group,
2.) Departments of Clinical Pharmacology and
Oncology,
Abstract
Cell culture drug resistance testing (CCDRT)
[aka Functional Tumor Cell Profiling] is purported to correlate with
response to chemotherapy and/or with patient survival after chemotherapy.
Advocates of CCDRT maintain that this information is of value in clinical
drug selection, particularly in situations where there is a choice to be
made between more than one acceptable drug regimen. Assays based on a cell
proliferation or DNA synthesis endpoint were largely studied in the early to
mid-1980s and are currently advocated chiefly for the identification of
inactive drugs. Assays based on cell death as an endpoint were the subject
of increasing study during the late 1980s and throughout the 1990s. An
extensive, diverse, and consistent literature documents the ability of cell
death assays to identify forms of chemotherapy which are associated with
both favorable and unfavorable prognoses. CCDRT should be much more widely
utilized in clinical oncology practice and as an integral component of
ongoing and future clinical trials.
Introduction
Cell culture drug resistance [Functional
Tumor Cell Profiling] tests are laboratory tests in which fresh biopsy
specimens of human tumors are cultured in the presence and absence of
anticancer drugs. At the conclusion of the cell culture, measurements are
made to determine whether or not the drugs were effective in either killing
the tumor cells or in preventing the growth of the tumor cells. Proponents
of these tests maintain that this information correlates with drug effects
in the patient and can therefore be used to assist the clinical oncologist
in selecting the most appropriate drugs to be used in the treatment of
individual patients. This paper will review the data relevant to this point
of view.
To begin with, there has been an unfortunate
proliferation of names/terms applying to this testing. It should be noted
that the terms "chemosensitivity assay," "chemoresistance assay," "drug
resistance assay," and "drug response assay" can be used interchangeably.
Likewise, the terms "in vitro assay" and "ex vivo assay" can be used
interchangeably in this context. Some authors have tried to draw a
distinction between assays which are geared and/or used more for the
identification of inactive drugs versus active drugs. These are, however,
purely semantic distinctions. Depending on where cut-off lines are drawn,
all assays will have differing specificities and sensitivities for
identifying inactive drugs and active drugs. It is much more useful to
describe the specificity and sensitivity of an assay than to arbitrarily
label the assay to be either a "chemoresistance" or "chemosensitivity"
assay. The generic term "cell culture drug resistance testing" (CCDRT)
describes laboratory tests in which gradations of drug resistance are
determined by measuring drug effects on short term cultures of viable cells.
Depending on the conditions of the assays, they will have greater and lesser
specificities and sensitivities for identifying inactive drugs and active
drugs.
One must begin by understanding that there
is a clear divide between CCDRT [Functional Tumor Cell Profiling] based on
cell proliferation as an endpoint and CCDRT based on cell death as an
endpoint. Historically, the cell proliferation endpoint received great
attention, as a result of studies by Salmon, Von Hoff, and others during the
late 1970s and early 1980s [1,2]. These studies occurred during the heyday
of the oncogene discovery period in cancer research, where oncogene products
were frequently found to be associated with cell growth, and where cancer
was most prominently considered to be a disease of disordered cell growth.
In contrast, the concept of apoptosis (programmed cell death) had yet to
become widely recognized. Also unrecognized were the concepts that cancer
may be a disease of disordered apoptosis/cell death and that the mechanisms
of action of most if not all available anticancer drugs may be mediated
through apoptosis [3-5]. When problems with proliferation-based assays
emerged [6,7], there was little enthusiasm for studying cell death as an
alternative endpoint. These factors explain the abandonment of research into
CCDRT by American universities and cancer centers by the mid-80s. However,
clinical laboratories began to offer CCDRT as a service to patients in the
Chapter 1: Cell Proliferation Assays
During the last dozen years, the cell
proliferation assay which has been most heavily promoted and provided as a
service to patients in the USA is the radioactive thymidine incorporation
assay originally described by Tanigawa and Kern [8]. In this assay, applied
only to solid tumors and not to hematologic neoplasms, tumor cells suspended
in soft agarose are cultured for 4 - 6 days in the continuous presence of
antineoplastic drugs. At the end of the culture period, radioactive
thymidine is introduced and differences in putative thymidine incorporation
into DNA are compared between control and drug-treated cultures. Kern and
Weisenthal analyzed the clinical correlation data and defined the concept of
"extreme drug resistance," or EDR [9]. This was defined as an assay result
which was one standard deviation more resistant than the median result for
comparison, database assays. Patients treated with single agents showing EDR
in the assay virtually never enjoyed a partial or complete response. Kern
and Weisenthal also defined "low drug resistance" (LDR) as a result less
resistant than the median and "intermediate drug resistance" (IDR) as a
result more resistant than the median but less resistant than EDR (in other
words, between the median and one standard deviation more resistant than the
median).
The principles and clinical correlation data
with the thymidine "EDR" assay were reviewed in this journal 10 years ago
[10]. There have been only a few follow-up studies published since this
time. One such study showed that EDR to one or more of the single agents
used in a two drug combination is not apparently associated with a lower
probability of response to the two drug combination in the setting of
intraperitoneal chemotherapy of appendiceal and colon cancers [11]. It is,
however, possible that response to the high drug concentrations achievable
with intraperitoneal chemotherapy may be more closely associated with drug
penetration to the tumor than to intrinsic drug resistance of the tumor
cells. It was also shown that EDR to paclitaxel does not appear to be a
prognostic factor in ovarian cancer patients or in patients with primary
peritoneal carcinoma treated with paclitaxel plus platinum [12,13]. However,
it was recently reported that EDR to platinum in ovarian cancer may have
prognostic implications (Fruehauf,J., et al Proc ASCO,v.20,Abs 2529, 2001).
[Note added in proof]: It was also reported that previously-untreated breast
cancer patients with tumors showing LDR (defined above) had superior times
to progression and overall survivals than patients with tumors showing
either IDR or EDR (Mehta,R.S., et al, Breast cancer survival and in vitro
tumor response in the extreme drug resistance assay. Breast Cancer Res Treat
66:225-37, 2001).
Currently in the
A second form of cell proliferation assay
currently provided as a service to patients (NuOncolology Labs, Houston, TX)
is the adhesive tumor cell culture system, based on comparing monolayer
growth of cells over a proprietary "cell adhesive matrix" [14]. Positive
clinical correlations were described with this system in 1987 [14], but
confirmatory and follow-up studies have not been reported.
Chapter 2: Total Cell Kill/Cell Death Assays
As opposed to measuring cell proliferation,
there is a closely-related family of assays based on the concept of total
cell kill, or, in other words, cell death occurring in the entire population
of tumor cells (as opposed to only in a small fraction of the tumor cells,
such as the proliferating fraction or clonogenic fraction) [15-18]. The
concepts underlying cell death assays are relatively simple, even though the
technical features and data interpretation can be very complex. There has
been considerable work based on these assays reported during the past 15
years. This body of work is not currently well appreciated among clinical
oncologists, and the remainder of this review will focus on the cell death
assays.
The basic technology concepts are
straightforward. A fresh specimen is obtained from a viable neoplasm. The
specimen is most often a surgical specimen from a viable solid tumor. Less
often, it is a malignant effusion, bone marrow, or peripheral blood specimen
containing "tumor" cells (a word used to describe cells from either a solid
or hematologic neoplasm). These cells are isolated and then cultured in the
continuous presence or absence of drugs, most often for 3 to 7 days. At the
end of the culture period, a measurement is made of cell injury, which
correlates directly with cell death. There is evidence that the majority of
available anticancer drugs may work through a mechanism of causing
sufficient damage to trigger so-called programmed cell death, or apoptosis
[3,4].
Although there are methods for specifically
measuring apoptosis, per se, there are practical difficulties in applying
these methods to mixed (and clumpy) populations of tumor cells and normal
cells. Thus, more general measurements of cell death have been applied.
These include: (1) delayed loss of cell membrane integrity (which has been
found to be a useful surrogate for apoptosis), as measured by differential
staining in the DISC assay method, which allows selective drug effects
against tumor cells to be recognized in a mixed population of tumor and
normal cells [10,19], (2) loss of mitochondrial Krebs cycle activity, as
measured in the MTT assay [20], (3) loss of cellular ATP, as measured in the
ATP assay [21-23], and (4) loss of cell membrane esterase activity and cell
membrane integrity, as measured by the fluorescein diacetate assay [24-26].
It is very important to realize that all of
the above 4 endpoints can and do, in most cases, produce valid and reliable
measurements of cell death, which correlate very well with each other on
direct comparisons of the different methods [20,25-36]. This should not be
surprising, any more than should the fact that auscultating heart sounds,
observing spontaneous breathing, palpating a carotid pulse, measuring core
body temperature, and recording an electroencelphalogram or
electrocardiogram are all good and reliable methods of determining patient
death.
We have performed direct correlations
between the DISC and MTT assays in approximately 4,000 fresh human tumor
specimens, testing an average of 15 drugs per specimen at two different
concentrations. Thus, we have approximately 120,000 direct comparisons
between DISC (membrane integrity) and MTT (mitochondrial Krebs cycle
activity) endpoints in fresh human tumor specimens. The overall correlation
coefficient between these endpoints in specimens containing > 60% tumor
cells is 0.85 (These data do not include assays on 5FU, which, for
biological reasons, may be tested in the MTT assay but not the DISC assay.
These data also do not include assays for paclitaxel and docetaxel, which,
for different biological reasons, may be tested in the DISC assay but not
the MTT assay).
The above studies, demonstrating the
comparability of results with the 4 different cell death endpoints, are
important for the following reason. For perfectly understandable reasons,
clinical studies correlating assay results with clinical outcome are very
difficult to perform. The literature in this field may be characterized as
including a great many small studies, but no big studies. Additionally,
different investigators have favored different cell death endpoints,
depending on the laboratory and clinical situation.
For example, the DISC assay is extremely
labor intensive, and requires expertise in recognizing and counting tumor
cells using a microscope, but it may be applied to specimens containing a
heterogeneous mixture of tumor cells and normal cells. MTT, ATP, and FDA
endpoints use semi-automated instrument readouts, but can only be applied to
specimens which are relatively homogeneous for tumor cells. In addition,
there are a number of additional reasons why one type of cell death endpoint
may be advantageous in a given tumor specimen and why laboratories may apply
several different cell death endpoints in the testing of a single specimen.
It should be noted that, historically, the
DISC assay studies of the early 1980s provided the prototype for later
studies of the other cell death endpoints. When the MTT endpoint was first
introduced in the late 1980s, the first published studies compared the MTT
results to the DISC results, with culture conditions and drug exposures
being otherwise identical [20,27,29,31]. Many laboratories have preferred
the MTT endpoint (and later the ATP and FDA endpoints), because of the
difficulty in manually scoring the DISC assay microscope slides. But what is
important is that each of the above cell death endpoints do give essentially
the same results (except in the case of isolated drugs, such as taxanes and
5FU). Thus, it is entirely reasonable and proper to consider as a whole the
clinical validation data which has been published using the above 4
endpoints.
The second point to understand is that cell
death assays are not intended to be scale models of chemotherapy in the
patient. The DISC assay was designed to address the major practical problems
with the popular clonogenic assays of the late-70s/early-80s. Chief among
these problems were (1) low evaluability rates and (2) uncertainty of what
was being measured in individual assays (true tumor cell colonies, arising
from clonogenic cell growth versus artifactual colonies arising from cell
aggregation). Unlike the case with the clonogenic assays, there was no
attempt to model in vivo pharmacokinetics (i.e. no attempt to utilize
clinically-achievable drug concentrations or to determine something
analogous to an anti-bacterial minimal inhibitory concentration). Instead,
the assay conditions were rigorously fixed, with respect to culture media
and drug exposure time (the latter being, most typically, 96 hours). Drugs
were first tested in training set assays to determine the drug concentration
which gave the widest scatter of results (mathematically defined as the
greatest standard deviation). The hypothesis to be tested with clinical
correlations was a very simple one - that above-average drug effects in the
assays would correlate with above-average drug effects in the patient, as
measured by both response rates and patient survival.
Chapter 3: Correlations between cell death assay results and chemotherapy
response
The hypothesis to be tested with clinical
correlations was a very simple one - that above-average drug effects in the
assays would correlate with above-average drug effects in the patient, as
measured by both response rates and patient survival.
The tables and figures described below show
that the above hypothesis has been confirmed to be true in every single
study of these assays ever carried out. Table 1 (page 1) and Table 1 (page
2) show the raw published data from which the results were taken, with
literature references. Figure 1 shows the results of each individual study,
arrayed in order of increasing response rates in the total patient
population studied. In every single case, without exception, assay
"sensitive" patients were more likely to respond than the total patient
population as a whole and assay "resistant" patients were much less likely
to respond than the patient population as a whole. In every case, patients
treated with assay "resistant" drugs were considerably less likely to
respond than patients treated with assay "sensitive" drugs. This should not
be a surprising finding. Intuitively, tumors relatively resistant to drugs
in vitro would seem, on the whole, to be less likely to respond to the same
drugs in vivo. This is precisely what the published data show.
Figure 2 shows the correlations between
assay results and treatment, broken down as to histopathologic diagnosis.
These are also arrayed in order of increasing overall response rates of the
patient populations under study. In each case, assay "sensitive" patients
were more likely to respond than the overall patient population and assay
"resistant" patients were less likely to respond. In every case, patients
treated with assay "sensitive" drugs were more likely to respond than
patients treated with assay "resistant drugs." The only "near exception" to
this point was in the case of head and neck cancer, in which results were
available only from a single study, in only a handful of patients. It may
further be concluded from Figure 2 that cell death assays are broadly
applicable to a broad range of neoplasms. This does not prove, for example,
that the assays are clinically valid for a given rare tumor, such as
esthesioneuroblastoma, but there is no reason to expect that the cell death
assays should not be valid in any given type of neoplasm.
Chapter 4: Assay Results in the Context
of Bayes' Theorem
The absolute predictive accuracy of the
tests varies according to the overall response rate in the patient
population being studied, in accordance with Bayesian principles [76].
Figure 3 is of greatest importance, and is
well worth considering. If one understands this figure, one goes a long way
to understanding how the results of these assays should be used in patient
management. The solid and dashed lines in this figure show the theoretical
expectations for the cell death assays, based on Bayes' Theorem, applied to
assays with an overall specificity for drug resistance of 0.92 and an
overall sensitivity of 0.72, which represent the overall findings from the
studies included in the meta-analysis. The circles show the actual response
rates of patients with different types of neoplasms, given that either
"sensitive" or "resistant" results were obtained. It may be seen that, in
every case, the actual performance of the assays in each type of tumor
precisely matched predictions made from Bayes' Theorem, projected from the
overall assay sensitivity and specificity.
The findings in Figure 3 show conclusively
that the cell death assays are broadly applicable to a wide range of human
neoplasms, ranging from low response rate tumors, such as pancreatic cancer
and cholangiocarcinoma (group 1, the non-colon, non-stomach GI
adenocarcinomas) to acute lymphoblastic leukemia (group 11), and including
breast cancer and ovarian cancer.
Of equal importance, this figure shows how
the assay may be best applied to patient management decisions. It is obvious
that, in high response rate neoplasms, there will be many "false negative"
predictions. No one should ever use these assays to deny chemotherapy to
such patients, if chemotherapy is otherwise indicated, any more than one
should deny antibiotics in an infection with an in vitro drug resistant
bacterium. In cases where there is one particular drug regimen which has
been shown to produce a very high cure rate and this regimen is widely
accepted as being superior to all other regimens (e.g. testicular cancer,
where dose-intense cisplatin/etoposide/bleomycin has been shown to produce
the highest cure rate), it would be most unwise to forgo this regimen solely
on the basis of today's available cell culture assays.
On the other hand, the assays could be
appropriately used to identify patients with above and below-average
clinical prognoses if treated with given drugs. In cases where more than one
acceptable regimen exists, the physician could select the regimen containing
the most favorable drugs and avoid the regimen containing the most
unfavorable drugs. This would apply to clinical decisions at all points
along the curve. Thus, the absolute probability of response with assay
"sensitive" and "resistant" drugs varies according to the overall prior
response probability in the patient population studies, but, at all points,
assay "resistant" patients have a below average probability of response and
assay "sensitive" patients have an above average probability of response and
treatment with assay "sensitive" drug(s) is more likely to be associated
with a favorable outcome than treatment with assay "resistant" drugs.
Chapter 5: Specific Diseases/Hematologic
Neoplasms
The preceding was an overview of the forest
of the literature supporting the hypothesis that above-average drug effects
in cell death assays correlate with above-average clinical efficacy in the
patient, and below-average drug effects in the assays correlate with
below-average clinical efficacy in the patient. These (remarkably
consistent) data supported the correlation between in vitro and clinical
drug effects for a wide range of neoplasms.
We will now consider several individual
"trees," or disease types, which have received the greatest amount of study.
The diseases considered are (1) lymphatic neoplasms (CLL, ALL, and
non-Hodgkin's lymphoma), (2) acute non-lymphocytic leukemia, (3) stomach and
colorectal cancer, (4) ovarian cancer, and (5) breast cancer.
(Studies in hematologic neoplasms will be
described below, and studies in GI neoplasms, ovarian cancer, and breast
cancer will be described in the following Chapter 6)
Lymphatic Neoplasms and ANLL
Considering first only correlations between
assay results and clinical response (defined as a complete response in the
case of acute leukemia and as a partial or complete response for CLL and
NHL), Table 1 (page 1) and Table 1 (page 2) show the following correlations:
Acute Lymphoblastic Leukemia: n = 275
published correlations between assay results and response. Overall response
rate for patients studied = 76%. Response rates for patients treated with
drugs with good activity in the assays = 87%. Response rates for patients
treated with drugs with poor activity in the assays = 37%.
CLL: n = 157. Overall response rate = 43%.
Response rate with good assay activity drugs = 74%. Response rate with poor
assay activity drugs = 6%.
NHL: n = 77. Overall response rate = 55%.
Response rate with good assay activity drugs = 71%. Response rate with poor
assay activity drugs = 14%.
ANLL: n = 318. Overall response rate = 67%.
Response rate with good assay activity drugs = 90%. Response rate with poor
assay activity drugs = 23%.
There is a long, extensive, and consistent
body of evidence supporting the clinical relevance of cell death assays in
human hematologic neoplasms. It is very important to consider this evidence
as a whole. One must remember that we are evaluating a laboratory technology
and not a therapy. The issue to be considered is the claim that the cell
death measured in the assays correlates with tumor cell death measured in
the patient. If one considers the CLL and ALL data as a whole, and then also
considers the more limited but also consistent data in non-Hodgkin's
lymphoma, a very powerful case is made to support the clinical relevance of
this testing in human lymphatic neoplasms. If one then goes on to consider
the ANLL data in the context of the lymphatic neoplasm data, a powerful case
is made to support the clinical relevance of this testing in hematologic
neoplasms in general.
The body of literature supporting cell death
assays in lymphatic neoplasms dates to studies in CLL published by Schrek in
the 1960s [77-80]. Schrek measured the in vitro cell death effects of drugs,
heat, and radiation on CLL cells by means of phase contrast microscopy
(undoubtedly measuring what we would today recognize as apoptosis and
undoubtedly being precisely congruent with the DISC assay). Radiation
effects were correlated with clinical outcome [77,80]. Schrek was,
non-incidentally, the investigator who first described the identification of
viable cells by means of dye exclusion [81].
In the late 1970s, Durkin compared in vitro
drug effects in NHL and CLL by means of trypan blue dye exclusion with
clinical drug effects and reported good correlations in a small study [82].
Independently, the DISC assay was developed as an improved variation of the
trypan blue test, in which suspension cultures of cells were first exposed
to trypan blue, spun down onto Cytospin slides, and then counterstained with
either Hematoxylin/Eosin or Wright/Giemsa (to identify the non-trypan
blue-stained cells with respect to whether these surviving cells were tumor
cells or normal cells). With further improvement (substitution of fast green
stain for trypan blue and the addition of acetaldehyde-fixed duck
erythrocytes as an internal standard to aid in scoring the Cytospin slides),
clinical correlations in CLL and other neoplasms were first reported in
abstract form and at meetings in the US and Europe in 1981 [83,84].
The first full journal publication of
clinical correlations with the DISC assay occurred in 1983 and 1984, which
included studies of the activity of glucocorticoids and standard cytotoxic
agents correlated with prior therapy and with clinical outcome in ALL and
CLL [15,19,85]. This was followed, in 1986, with a study showing the
clinical relevance of the DISC assay in CLL, ALL, and NHL using several
clinical endpoints: (1) correlations with known disease-specific activity
profiles, (2) individual patient correlations with clinical response, (3)
greater resistance of specimens from previously-treated patients versus
previously-untreated patients, and (4) a shift to significantly greater drug
resistance in metachronous assays in the presence of intervening
chemotherapy, but no shift in the absence of intervening chemotherapy [45].
It should be noted that these findings were subsequently independently
confirmed by other investigators in more comprehensive studies
[26,31,36,39,40,43,51,59,86-91]. Additionally, studies in pediatric ALL
reported that resistance to dexamethasone in the DISC assay predicted for
poor survival [92,93]. These findings were also independently confirmed (see
below).
By the late 80s, a number of other
investigators had begun to look at the DISC assay and related cell death
assays. These began with a head to head comparison of the DISC assay with
the MTT assay in established cell lines by the NCI lung cancer group
[20,27]. These studies established the comparability of these endpoints in
homogeneous cell populations.
A group at the Free University of Amsterdam
carried out a head to head comparison of the DISC endpoint with the MTT
endpoint in acute lymphoblastic leukemia [29,30]. This group showed that the
endpoints were comparable in specimens in which the percentage of leukemia
cells (relative to normal cells in the specimen) was greater than 80
[29,30,94]. This group found the MTT endpoint to be much less labor
intensive. They used the same general conditions originally described for
the DISC assay (including a 96 hour continuous drug exposure, followed by
comparisons between drug exposed and control cultures with the cell death
endpoint). These Dutch authors went on to publish an extensive, elegant, and
ongoing series of rigorous studies which have established that the assay
results correlate with and predict for both response and survival in ALL,
and that the assay results are, in fact, the only factor which independently
predicts for survival in pediatric ALL [87,88,90,95-103]. They have also
extended this work to ANLL [89,104,105]. Taken in the context of the entire
literature, these studies in pediatric ALL provide complementary support for
the validity of complementary studies in CLL (described below).
Other investigators also showed strong
correlations between cell death assay results and clinical outcome (response
and/or survival) in pediatric ALL [40,41,91-93,106], adult ALL and ANLL
[25,31,44,46,47,49,52,53,99,107-113], CLL [58,59,114-116], and adult NHL
[19,36,45,51]. These studies included further confirmation of the
comparability between DISC and MTT endpoints in assays on clinical specimens
and also introduced the fluorescein diacetate cell death endpoint, which,
like the DISC endpoint, measures cell membrane integrity and which
correlates very well with the DISC endpoint in homogeneous cell populations
[26,36].
In 1991, Bosanquet published in Lancet a
relatively large number of correlations between clinical response and DISC
assay results, chiefly in CLL [39]. He showed, furthermore, highly
significant correlations between assay results and patient survival. This
paper also confirmed the relevance of the "EDR" (extreme drug resistance)
endpoint, which is defined as an assay result more than one standard
deviation more resistant than the median of comparison assays. Bosanquet
later described a paradoxical shift toward increased methylprednisolone
sensitivity in previously-treated CLL and used the DISC assay to identify
high dose methylprednisolone as an effective treatment for otherwise
refractory CLL [117,118].
These studies with the DISC and MTT assays
are supported by studies with the fluorescein diacetate (FDA) endpoint.
Fluorescein diacetate is a lipid soluble material which readily penetrates
cell membranes. Viable cells contains a membrane esterase which cleaves the
dye to non-lipid soluble fluorescein, which is concentrated in cells
containing a functionally-intact membrane. Thus, the assay is conceptually
similar to the DISC assay, which measures the ability of cells with
functionally-intact membranes to exclude non-lipid soluble dyes. Delayed
loss of this membrane integrity is a marker of apoptotic cell death [119].
Investigators at
Within the past several years, additional
studies have provided strong support for the clinical relevance of the
information provided by cell death assays in hematologic neoplasms.
Bosanquet and colleagues reported a study in
243 CLL patients [116]. "Standard" first-line chemotherapy in the
Other investigators, as noted, have reported
that assay results are important predictors of patient survival in pediatric
acute lymphoblastic and non-lymphoblastic leukemia [103,123-126].
Similar studies have been reported for adult
acute non-lymphocytic leukemia [47,53,111,127]. Three different groups have
published strong correlations between CCDRT results and survival in ANLL.
Correlations between DISC assay results and patient survival in ANLL were
first published by a Swedish group in 1989 [53]. These results were recently
confirmed and extended by a group at the
The German group followed up with a
presentation at the American Society of Hematology (ASH) meetings in
December, 1999, in which multivariate analysis showed DISC assay results to
be the strongest factor predicting for clinical outcome in adult ANLL [111].
Most recently, a Danish group reported studies correlating MTT assay results
with both overall and relapse-free survival in 85 adult ANLL patients [127].
Assay results remained significantly correlated with survival on
multivariate analysis. This work on ANLL is precisely analogous and
complementary to the studies by the Dutch (
The only "negative" study ever published
concerning total cell kill (cell death) assays in hematologic neoplasms was
an otherwise "positive" study in adult acute non-lymphocytic leukemia, in
which strong correlations between anthracycline activity and survival were
shown, but poor correlations between cytarabine activity and survival were
seen [113], in contradistinction to several other studies in which assay
results with cytarabine were found to be strongly correlated with patient
survival [47,53,104,111]. The "negative" study was the only one to use the
ATP endpoint, which is disadvantageous in hematologic neoplasms, as normal
cells, red blood cells, and platelets all produce an appreciable
"contaminating" ATP signal, in contradistinction to the other cell death
endpoints, which are less affected by such artifacts. The authors of the
"negative" cytarabine study acknowledged that they did not determine the
percentage of leukemic blast cells at the conclusion of the cell culture and
noted the advantages of the DISC assay in being able to discriminate
neoplastic from normal cells.
Thus we have, in hematologic neoplasms, a 35
year history of highly positive studies, published by investigators in the
USA, the United Kingdom, the Netherlands, Germany, Sweden, Canada, Italy,
and Japan all showing consistent, strong correlations between the results of
cell death assays and clinical outcomes. In summary, there is a strong
scientific rationale for these tests and that the clinical relevance of the
information provided by the tests has been documented in a collectively
large and diverse literature in hematologic neoplasms.
Chapter 6: Specific Disease/Solid Tumors
General Considerations
Studies in solid tumors are technically
different than studies in hematologic neoplasms because solid tumor most
commonly are present as three-dimensional aggregates of cohesive cells,
while hematologic neoplasms are almost exclusively discohesive. Studies by
Teicher and Kerbel in murine tumors showed that in vitro drug activity
correlated with in vivo drug activity when tumors were tested in vitro as
three dimensional clusters, but not when they were tested in two dimensional
monolayers [128]. There is now an extensive literature on what has been
labeled "multicellular resistance" [129-131].
All published clinical correlations with
true fresh tumor assays with cell death endpoints have tested the tumor
cells largely in the form of three dimensional clusters. The only exception
to this statement is the non-small cell lung cancer study of the NCI-Navy
medical oncology group, in which subcultured cells (not true fresh tumors)
were tested in monolayer culture [132]. This latter study not surprisingly
showed poor correlations; all of the other cited studies, which used true
fresh (non-subcultured) tumor cells tested largely as three dimensional cell
clusters (and not in monolayer culture), showed good correlations.
The solid tumors which have received the
greatest degree of study are gastrointestinal adenocarcinomas (colon and
gastric adenocarcinoma), breast cancer, and ovarian cancer. There have been
relatively few clinical correlations published in the cases of melanoma,
soft tissue sarcoma, glioblastoma, and squamous cell carcinomas in general.
GI Neoplasms
In the case of gastrointestinal neoplasms,
there have been 129 published correlations between assay results and
clinical response Table 1 (page 1) and Table 1 (page 2) . Overall, patients
treated with drugs having good activity in the assays had a 48% response
rate, while those treated with drugs having poor activity in the assays had
a response rate of less than 1%, in a population of patients who overall had
a 11% response rate. Also reported in many additional patients were positive
associations between assay results and patient survival [64,133,134].
Taken in the broad context of the entire
literature, these studies provide important confirmation of the broad (with
respect to both drugs and tumor types) applicability of cell death assays.
The issue of whether the MTT assay or measurements of thymidylate synthetase
[146] is more accurate in gauging probability of response to specific types
of fluoropyrimidine-based therapy awaits future head to head comparisons.
Ovarian
Cancer
In the case of ovarian cancer, there have
been 328 published correlations between assay results and clinical response
Table 1 (page 2) . Overall, patients treated with drugs having good activity
in the assays had a 77% response rate, while those treated with drugs having
poor activity in the assays had a response rate of 11%, in a population of
patients who overall had a 51% response rate. Also reported were highly
positive associations between assay results and patient survival [147,163].
Kurbacher and colleagues treated 25 patients
with ovarian cancer with ATP-assay-directed chemotherapy and compared
outcomes with 30 non-randomized but clinically well-matched controls [148].
In the control group, there was a response rate of 37% (2 complete
responders), with median progression-free survival of 20 weeks and median
overall survival of 69 weeks. In the assay-directed group, there was a
response rate of 64% (8 complete responders), with a median progression-free
survival of 50 weeks (P2=0.003) and a median overall survival of 97 weeks
(P2=0.145). Assay directed therapy also produced a greater benefit with
respect to both response rate and progression-free survival in the subgroup
of patients with platinum-resistant disease. A current multi-institutional,
international trial is currently in progress to determine if assay-directed
therapy is superior to empiric therapy.
Breast Cancer
In the case of breast cancer, there are a
total of 179 published correlations between assay results and patient
treatment Table 1 (page 1). Patients treated with assay "sensitive" drugs
had an 82% response rate. Patients treated with assay "resistant" drugs had
a 7.7% response rate. The overall response rate for the patients in the
studies was 66%.
Xu and colleagues treated 73 breast cancer
patients on the basis of MTT-assay directed chemotherapy, and compared
outcomes with 73 patients treated with "physician's choice" chemotherapy
[57]. This was also a non-randomized study, but the patients receiving
assay-directed therapy actually had less favorable prognostic factors, such
as having significantly more sites of disease (the author informed me in a
personal communication that the patients at her medical center with
unfavorable disease were more often referred for biopsy and assay-directed
therapy, while patients with more favorable disease were more likely to
receive standard empiric chemotherapy). The response rate of the
assay-directed group was 77%, while the response rate for the empiric
therapy group was 44%. In a small group of 10 patients who received assays
but in which no active drugs were identified, empiric therapy was given with
no responses (0% response rate). One year survivorship for the two groups
was 74% for assay-directed therapy and 67% for empiric therapy. Three year
survivorships were 25% and 19%, respectively. Five year survivorships were
20.5% and 12.3%, respectively.
The above study showed a clear response
advantage to assay-directed therapy and a trend for a survival advantage,
despite less favorable prognostic factors for the group receiving
assay-directed therapy. The lack of statistical significance for survival is
no doubt owing to the small numbers of patients enrolled in the study.
Putting things into perspective, the adjuvant Cancer and Acute Leukemia
Group B study comparing doxorubicin/cyclophosphamide with and without Taxol
required 2,000 patients to show an absolute 2% difference in survival. And
yet triple drug therapy has now become the standard of care in this setting.
It also required a meta-analysis of studies totalling close to 50,000
patients to establish a small survival advantage for adjuvant chemotherapy
of post-menopausal patients.
Chapter 7: Editorial Conclusions
The title of this review is current status of
cell culture drug resistance testing. This review focused on a description
of the technologies and a review of the clinical correlation data, because
there are many misconceptions and much ignorance about both technology and
data.
One must recall the extraordinary difficulty
in proving the efficacy of chemotherapy in general and of specific drug
regimens in particular in studies of non-assay-directed chemotherapy. Only
with extremely large studies (and sometimes only with meta-analyses of
extremely large studies) has it been possible to document that chemotherapy
of any type produces survival advantages compared to no chemotherapy at all,
in many clinical situations. The quite impossible challenge of documenting
the clinical standard of "efficacy" (as opposed to the heretofore
traditional laboratory standard of "accuracy") with these non-proprietary,
public domain technologies was, in fact, pointed out by Dr. Maurie Markman
(a noted critic of Human Tumor Assays) who correctly wrote that "even if it
were possible to establish the efficacy of [the assays] in a particular
situation, this would do nothing at all to establish the efficacy of [the]
assays in any other situation" [135].
The challenge of "validating" a single test
for a single treatment in a single disease is challenging enough (e.g.
estrogen receptor in breast cancer, which has still, after 30 years, only
been shown to correlate with clinical outcome and has yet to be shown to
improve clinical outcome). Now consider the challenge of "validating" a test
for 40 different drugs which can be used in tens of thousands of
combinations in hundreds of diseases. If documented clinical "efficacy" is
the standard to be demanded of non-proprietary laboratory tests, then
clinicians should abandon all tests currently used in their practices. It
will be interesting to see which standard is applied in the future to other
laboratory tests associated with the prediction of drug resistance, such as
tests based on mechanisms of drug resistance (e.g. expression of thymidylate
synthetase [146,150,151]) and Her2/neu expression [152-154].
While evaluating the data discussed here,
please consider that it has taken 20 years to amass this body of evidence in
an environment of continued hostility and non-support by the academic
oncology community toward work in this area and consider also the little
which has been achieved in the area of empiric methods of drug selection,
despite billions of dollars spent on empiric clinical trials
enthusiastically supported by this same academic oncology community.
If one critically evaluates the clinical
trials data in ovarian cancer, for example, one finds that there is no
advantage for platinum-based combination chemotherapy over single agent
alkylator therapy and no advantage for platinum + paclitaxel over single
agent cisplatin or carboplatin [155-157]. But this did not prevent platinum
combination therapy from becoming "standard of care" before the introduction
of paclitaxel and it did not prevent platinum/paclitaxel from becoming
standard of care over single agent carboplatin or cisplatin. In point of
fact, the only thing clearly established after 30 years of clinical trials
is that carboplatin and cisplatin are therapeutically equivalent, albeit
with different toxicity profiles. And there are absolutely no data to
support any of the half dozen or so available drug choices for second and
third line therapy over any other choice. So what is the "risk" in using
currently available assays to help guide these choices?
Only when these assays are widely performed
and used and routinely included as an integral part of clinical trials will
these already promising technologies be improved and only then will their
role in patient management become better defined. But this is true for all
complex laboratory technologies (a good example being immunohistochemical
staining for batteries of cell antigens).
Absent this testing, on what basis are drugs
chosen today for use in the many clinical settings in which a single "best"
empiric regimen has not been well-defined? An objective reviewer would admit
that many oncology practices would base choice of drug regimen, at least in
part, on the profit "spread" between the wholesale cost of the drug(s) and
the reimbursement which the third party payers provide. This is a conflict
of interest as well as a cost-ineffective method for selecting therapy; yet
it is a method which the oncology and insurance communities support every
single day in their treatment and coverage decisions. It is the loss of this
"freedom to choose" and the overzealous dedication to a weak clinical trials
paradigm (identification of the "best" treatment to give to the average
patient) which is largely behind the reluctance to introduce these
technologies as an important component of current clinical trials and as a
part of the process of clinical drug selection in situations where clear
empiric "best regimens" have not been well defined through prior clinical
trials.
The private sector laboratories offering
CCDRT as a patient service (Table 2) have been able to make considerable
progress in improving the assay technologies and in building databases which
improve the interpretation of "raw" assay results. But this progress has
only been possible because insurers and often patients have been willing to
pay for the tests and because clinicians have wanted to have the information
provided by the tests. The progress would have been much faster (and
doubtless even more substantial) had the academic oncology community not
done everything it could to oppose this work at every step of the way.
By raising the bar of acceptance to levels
unprecedented for a laboratory test, in essence a tariff has been erected to
protect the paradigm of the "best" empiric treatment for the average
patient, as identified in appallingly non-productive clinical trials. This
tariff also serves to protect the paradigm of drug selection with
consideration of the spread between wholesale cost and reimbursement.
Finally, the tariff discourages discovery of
new, effective drug regimens through the use of CCDRT to guide drug
selection. Take, for example, the gemcitabine/cisplatin combination. Years
before gemcitabine/cisplatin became a widely used drug regimen, CCDRT
identified this as the most active regimen in a patient with pancreatic
cancer metastatic to kidney, omentum, and liver, despite the poor activity
of gemcitabine and cisplatin tested as single agents. This patient went on
to achieve a complete remission with gemcitabine/cisplatin and remains alive
with an excellent quality of life 5 1/2 years later [158,159]. A second such
patient was an ovarian cancer patient with primary resistance to
paclitaxel/carboplatin who then underwent tandem stem cell transplant/high
dose chemotherapy regimens (at a cost of more than $250,000) without ever
achieving a response. At a time when she had bulky, non-cytoreducible
abdominal and pleural disease, CCDRT confirmed resistance to single agent
cisplatin, carboplatin, and gemcitabine, but good activity for the
gemcitabine/carboplatin combination. She subsequently received
gemcitabine/carboplatin as an outpatient, achieved a durable complete
response, and returned to work full time as an oncology nurse, where she
remained well, for four years [160], until a recent relapse (she has
recently been re-started on assay-directed chemotherapy). Indeed, early
anecdotal results of this type occurring in diseases in which there was no
existing clinical trials literature accelerated clinical trials of this
regimen in diseases in which assay-directed responses had been observed
[161].
With more widespread use of these assays in
clinical oncology, it is very likely that the activity of new drugs and new
regimens would be identified at a much earlier time than with the current
system relying exclusively on usually-empiric, Phase II trials [162].
Why is it so necessary to protect the
patient from the information provided by a perfectly rational laboratory
test, supported by a wealth of entirely consistent, if understandably
incomplete data? If used to assist in the selection of a regimen chosen from
a series of otherwise reasonable alternatives, then patients will never be
harmed and best available evidence strongly indicates that they will often
be helped.
Think of all the objections to this testing.
Now try to design all of the clinical trials which would be needed to meet
all of these objections and think of how much money these would require and
who is going to provide this money and how many years the studies would take
and how many patients will continue to receive ineffective or suboptimum
treatment in the interim. The body of information will never be sufficiently
large and complete and definitive to encompass even a reasonable fraction of
the situations where the information provided by the tests would be helpful.
Now ask the questions: What is the potential risk? What are the potential
benefits? What is the probability that these tests really do provide
information which can improve the drug selection process in individual
clinical situations? What is the potential cost? How does the benefit/risk
ratio balance out? What is the (financial) cost as a percentage of total
costs relating to management of patients on chemotherapy (including the
costs of radiographic and laboratory studies performed only to determine if
a given form of treatment is working or not)? What are the long term costs
if drug selection always remains an empiric, one-size-fits-all, trial and
error process? What would be the impact on improving existing technologies
(through the attraction of more laboratory and clinical investigators into
the field) and developing new technologies should these assays become more
widely used?
If one wishes to see an example of an
entirely rational technology advance, in a human disease crying out for
precisely such a technology advance, supported by an entirely consistent (if
understandably incomplete) body of data, where the advance continues to be
held hostage to a high bar of extraordinarily difficult clinical trials
which the critics have been entirely unwilling to support, in an area
(laboratory testing) for which such trials would be entirely unprecedented,
one need look no further.
Table 2 shows a partial listing of
laboratories from which CCDRT as a clinical service is currently available.
For specific information concerning the practical and technical aspects of
these services, and for cost and reimbursement issues, the director of each
laboratory should be contacted.
Cited Literature
1.
Salmon SE,
Hamburger AW, Soehnlen B, Durie BG, Alberts DS, Moon TE. Quantitation of
differential sensitivity of human-tumor stem cells to anticancer drugs. N
Engl J Med 1978; 298: 1321-1327.
2.
Von Hoff DD,
Casper J, Bradley E, Sandbach J, Jones D, Makuch R. Association between
human tumor colony-forming assay results and response to an individual
patient's tumor to chemotherapy. Am J Med 1981; 70: 1027-1032.
3.
Hickman JA.
Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 1992; 11:
121-139.
4.
Zunino F,
Perego P, Pilotti S, Pratesi G, Supino R, Arcamone F. Role of apoptotic
response in cellular resistance to cytotoxic agents. Pharmacol Ther 1997;
76: 177-185.
5.
Jaffrezou JP,
Bettaieb A, Levade T, Laurent G. Antitumor agent-induced apoptosis in
myeloid leukemia cells: a controlled suicide. Leuk Lymphoma 1998; 29:
453-463.
6.
Selby P,
Buick RN, Tannock I. A critical appraisal of the "human tumor stem cell
assay". New Engl J Med 1983; 308: 129-134.
7.
Lieber MM,
Kovach JS. Soft agar colony formation assay for chemotherapeutic sensitivity
of human solid tumors. Mayo Clin Proc 1982; 57: 527-528.
8.
Tanigawa N,
Kern DH, Kikasa Y, Morton DL. Rapid assay for evaluating the
chemosensitivity of human tumors in soft agar culture. Cancer Res 1982; 42:
2159-2164.
9.
Kern DH,
Weisenthal LM. Highly specific prediction of antineoplastic drug resistance
with an in vitro assay using suprapharmacologic drug exposures. J Natl
Cancer Inst 1990; 82: 582-588.
10.
Weisenthal
LM, Kern DH. Prediction of drug resistance in cancer chemotherapy: the Kern
and DISC assays. Oncology (U S A ) 1992; 5: 93-103;.
11.
Fernandez-Trigo V, Shamsa F, Vidal-Jove J, Kern DH, Sugarbaker PH.
Prognostic implications of chemoresistance-sensitivity assays for colorectal
and appendiceal cancer. Am J Clin Oncol 1995; 18: 454-460.
12.
Eltabbakh GH,
Piver MS, Hempling RE, et al. Correlation between extreme drug resistance
assay and response to primary paclitaxel and cisplatin in patients with
epithelial ovarian cancer. Gynecol Oncol 1998; 70: 392-397.
13.
Eltabbakh GH.
Extreme drug resistance assay and response to chemotherapy in patients with
primary peritoneal carcinoma. J Surg Oncol 2000; 73: 148-152.
14.
Ajani JA,
Baker FL, Spitzer G, et al. Comparison between clinical response and in
vitro drug sensitivity of primary human tumors in the adhesive tumor cell
culture system. J Clin Oncol 1987;5: 1912-1921.
15.
Weisenthal
LM, Shoemaker RH, Marsden JA, Dill PL, Baker JA, Moran EM. In vitro
chemosensitivity assay based on the concept of total tumor cell kill. Recent
Results Cancer Res 1984; 94: 161-173.
16.
Weisenthal
LM, Lippman ME. Clonogenic and nonclonogenic in vitro chemosensitivity
assays. Cancer Treat Rep 1985; 69: 615-632.
17.
Weisenthal
LM. Cell culture assays for hematologic neoplasms based on the concept of
total tumor cell kill. In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal
LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma.
18.
Weisenthal
LM. Clinical correlations for cell culture assays based on the concept of
total tumor cell kill. Contrib Gynecol Obstet 1994; 19: 82-90.
19.
Weisenthal
LM, Marsden JA,
20.
21.
Kangas L,
Gronroos M, Nieminen AL. Bioluminescence of cellular ATP: a new method for
evaluating cytotoxic agents in vitro. Med Biol 1984; 62: 338-343.
22.
Garewal HS,
Ahmann FR, Schifman RB, Celniker A. ATP assay: ability to distinguish
cytostatic from cytocidal anticancer drug effects. J Natl Cancer Inst 1986;
77: 1039-1045.
23.
Sevin B-U,
Peng ZL, Perras JP, Ganjei P, Penalver M, Averette HE. Application of an
ATP-bioluminescence assay in human tumor chemosensitivity testing. Gynecol
Oncol 1988; 31: 191-204.
24.
Rotman B,
Teplitz C, Dickinson K, Cozzolino JP. Individual human tumors in short-term
micro-organ cultures: Chemosensitivity testing by fluorescent cytoprinting.
In Vitro Cell Dev Biol 1988; 24: 1137-1138.
25.
Larsson R,
Nygren P, Ekberg M, Slater L. Chemotherapeutic drug sensitivity testing of
human leukemia cells in vitro using a semiautomated fluorometric assay.
Leukemia 1990; 4: 567-571.
26.
Nygren P,
Kristensen J, Jonsson B, et al. Feasibility of the fluorometric microculture
cytotoxicity assay (FMCA) for cytotoxic drug sensitivity testing of tumor
cells from patients with acute lymphoblastic leukemia. Leukemia 1992; 6:
1121-1128.
27.
28.
Twentyman PR,
Fox NE, Rees JKH. Chemosensitivity testing of fresh leukaemia cells using
the MTT colorimetric assay. Br J Haematol 1989; 71: 19-24.
29.
Pieters R,
30.
Pieters R,
31.
Kirkpatrick
DL, Duke M, Goh TS. Chemosensitivity testing of fresh human leukemia cells
using both a dye exclusion assay and a tetrazolium dye (MTT) assay. Leuk Res
1990; 14: 459-466.
32.
Tsai CM, Ihde
DC, Kadoyama C, Venzon D, Gazdar AF. Correlation of in vitro drug
sensitivity testing of long-term small cell lung cancer cell lines with
response and survival. Eur J Cancer 1990; 26: 1148-1152.
33.
Dmitrovsky E,
Seifter EJ, Gazdar AF, et al. A phase II trial of carboplatin (CBDCA) in
small-cell and non-small-cell lung cancer with correlation to in vitro
analysis of cytotoxicity. Am J Clin Oncol 1990; 13: 285-289.
34.
Hanson JA,
Bentley DP, Bean EA, Nute SR,
35.
Rhedin AS,
Tidefelt U, J”nsson K, Lundin A, Paul C. Comparison of a bioluminescence
assay with differential staining cytotoxicity for cytostatic drug testing in
vitro in human leukemic cells. Leuk Res 1993; 17: 271-276.
36.
Nygren P,
Hagberg H, Glimelius B, et al. In vitro drug sensitivity testing of tumor
cells from patients with non-Hodgkin's lymphoma using the fluorometric
microculture cytotoxicity assay. Ann Oncol 1994; 5 Suppl 1: S127-S131.
37.
Leone LA,
Meitner PA, Myers TJ, et al. Predictive value of the fluorescent cytoprint
assay (FCA): A retrospective correlation study of in vitro chemosensitivity
and individual responses to chemotherapy. Cancer Invest 1991; 9: 491-503.
38.
Beksac M,
Kansu E,
39.
Bosanquet AG.
Correlations between therapeutic response of leukaemias and in-vitro
drug-sensitivity assay. Lancet 1991; 337: 711-716.
40.
Hongo T,
Fujii Y, Igarashi Y. An in vitro chemosensitivity test for the screening of
anti-cancer drugs in childhood leukemia. Cancer 1990; 65: 1263-1272.
41.
Hongo T,
Fujii Y, Yajima S. In vitro chemosensitivity of childhood leukemic cells and
the clinical value of assay directed chemotherapy. In: Kaspers GJL, Pieters
R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug resistance in
leukemia and lymphoma.
42.
Kaspers GJL,
Pieters R, Van Zantwijk CH, De Waal FC, Van Wering ER, Veerman AJP. Is
resistance to prednisolone in vitro related to the response to
predniso(lo)ne in vivo at initial diagnosis in childhood acute lymphoblastic
leukemia? - a preliminary analysis. In: Kaspers GJL, Pieters R, Twentyman
PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and
Lymphoma. Chur: Harwood Academic Publishers, 1993: 321-328.
43.
Larsson R,
Kristensen J, Sandberg C, Nygren P. Laboratory determination of
chemotherapeutic drug resistance in tumor cells from patients with leukemia,
using a fluorometric microculture cytotoxicity assay (FMCA). Int J Cancer
1992; 50: 177-185.
44.
Lathan B, von
Tettau M, Verpoort K, Diehl V. Pretherapeutic drug testing in acute
leukemias for prediction of individual prognosis. Haematol Blood Transfus
1990; 33: 295-298.
45.
Weisenthal
LM,
46.
47.
Staib P,
Lathan B, Schinkothe T, et al. . Prognosis in Adult AML is precisely
predicted by the DISC-assay using the chemosensitivity-index C1. Adv Exper
Med Biol 1999; 457: 437-444.
48.
Santini V,
Bernabei PA, Dal Pozzo O, Santini S, Rossi Ferrini P. Acute myeloid leukemia
(AML) sensitivity to antiblastics is predictable by INT assay. In: Kaspers
GJ, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug
Resistance in Leukemia and Lymphoma. Chur: Harwood Academic Publishers,
1993: 365-368.
49.
Sargent JM,
50.
Stute N,
Kohler T, Lehmann L. Drug resistance testing in acute myeloid leukemia. Adv
Exper Med Biol 1999; 457: 445-452.
51.
Larsson R,
Jonsson B, Kristensen J, et al. Drug sensitivity testing of tumor cells from
patients with acute leukemia and non-Hodgkin's lymphoma using a fluorometric
microculture cytotoxicity assay. In: Kaspers GJL, Pieters R, Twentyman PR,
Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma.
Chur: Harwood Academic Publishers, 1993: 399-407.
52.
Santini V,
Bernabei PA, Silvestro L, et al. In vitro chemosensitivity testing of
leukemic cells: prediction of response to chemotherapy in patients with
acute non-lymphocytic leukemia. Hematol Oncol 1989; 7: 287-93.
53.
Tidefelt U,
Sundman-Engberg B,
54.
Blackman KE,
Fingert HJ, Fuller AF, Meitner PA. The Fluorescent Cytoprint Assay in
gynecologic malignancies and breast cancer: methodology and results. In:
Koechli OR, Sevin B-U, Haller U, eds. Chemosensitivity testing in
gynecologic malignancies and breast cancer.
55.
Cree IA,
Kurbacher CM, Untch M, et al. Correlation of the clinical response to
chemotherapy in breast cancer with ex vivo chemosensitivity. Anticancer
Drugs 1996; 7: 630-635.
56.
Koechli OR,
Avner BP, Sevin B-U, et al. Application of the adenosine triphosphate-cell
viability assay in human breast cancer chemosensitivity testing: a report on
the first results. J Surg Oncol 1993; 54: 119-125.
57.
Xu J-M, Song
S-T, Tang Z-M, et al. . Predictive chemotherapy of advanced breast cancer
directed by MTT assay in vitro. Breast Cancer Res Treat 1999; 53: 77-85.
58.
Bosanquet AG,
Copplestone JA, Johnson SA, et al. Response to cladribine in previously
treated patients with chronic lymphocytic leukaemia identified by ex vivo
assessment of drug sensitivity by DiSC assay. Br J Haematol 1999; 106:
474-476.
59.
Silber R,
Degar B, Costin D, et al. Chemosensitivity of lymphocytes from patients with
B-cell chronic lymphocytic leukemia to chlorambucil, fludarabine, and
camptothecin analogs. Blood 1994; 84: 3440-3446.
60.
Bosanquet AG.
The DiSC assay - 10 years and 2000 tests further on. In: Kaspers GJL,
Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in
Leukemia and Lymphoma. Chur: Harwood Academic Publishers, 1993: 373-383.
61.
Asanuma F,
Yamada Y, Kawamura E, et al. [Comparison between clinical response and in
vitro chemosensitivity of solid tumors in the succinic dehydrogenase
inhibition test]. Gan To Kagaku Ryoho 1992; 19: 95-101.
62.
Furukawa T,
Kubota T, Hoffman RM. Clinical applications of the histoculture drug
response assay. Clin Cancer Res 1995; 1: 305-311.
63.
Imai S.
Comparative study of histopathological effects of preoperative chemotherapy
using UFT and in vitro MTT assay of colonoscopy specimens from patients with
colorectal cancer. Jpn J Cancer Chemother 1999; 26: 1289-1293.
64.
Furukawa T,
Kubota T, Suto A, et al. Clinical usefulness of chemosensitivity testing
using the MTT assay. J Surg Oncol 1991; 48: 188-193.
65.
Meitner PA.
The fluorescent cytoprint assay: A new approach to in vitro chemosensitivity
testing. Oncology (U S A ) 1991; 5: 75-82.
66.
Smit EF, De
Vries EGE, Meijer C, Mulder NH, Postmus PE. Limitations of the fast green
assay for chemosensitivity testing in human lung cancer. Chest 1991; 100:
1358-1363.
67.
Wilbur DW,
Camacho ES, Hilliard DA,
68.
Carstensen H,
Tholander B. Chemosensitivity of ovarian carcinoma: In vitro/in vivo
correlations using the dye exclusion assay of Weisenthal (meeting abstract).
Proceedings: 3rd European Conference on Clinical Oncology 1985;
69.
Ng TY, Ngan
HYS, Cheng DKL, Wong LC. Clinical applicability of the ATP cell viability
assay as a predictor of chemoresponse in platinum-resistant epithelial
ovarian cancer using nonsurgical tumor cell samples. Gynec Oncol 2000; 76:
405-408.
70.
Konecny G,
Crohns C, Pegram M, et al. . Correlation of drug response with the ATP
tumorchemosensitivity assay in priamry FIGO stage III ovarian cancer.
Gynecol Oncol 2000; 77: 258-263.
71.
Ohie S,
Udagawa Y, Kozu A, et al. Cisplatin sensitivity of ovarian cancer in the
histoculture drug response assay correlates to clinical response to
combination chemotherapy with cisplatin, doxorubicin and cyclophosphamide.
Anticancer Res 2000; 20: 2049-2054.
72.
Sargent J,
Elgie A,
73.
Sevin B-U,
Perras JP, Averette HE, Donato DM, Penalver M. Chemosensitivity testing in
ovarian cancer. Cancer 1993; 71(Suppl): 1613-1620.
74.
Sevin B-U,
Perras JP, Koechli OR. Current status and future directions of
chemosensitivity testing. In: Koechli OR, Sevin B-U, Haller U, eds.
Chemosensitivity testing in gynecologic malignancies and breast cancer.
75.
76.
Weisenthal
LM. Predictive assays for drug and radiation resistance. In: Masters JM, ed.
Cancer in Primary Culture: A Handbook (Developments in Oncology, V. 64).
77.
Schrek R.
Differences between responsive and intractable chronic lymphocytic leukemia.
Med Hypotheses 1990; 31: 81-82.
78.
Schrek R,
Dolowy WC, Ammeraal RN. L-asparaginase: toxicity to normal and leukemic
human lymphocytes. Science 1967; 155: 329-330.
79.
Schrek R.
Sensitivity of normal and leukemic lymphocytes and leukemic myeloblasts
heat. J Natl Cancer Inst 1966; 37: 649-654.
80.
Schrek R,
Leithold SL,
81.
Schrek R. A
method for counting the viable cells in normal and in malignant cell
suspensions. Am J Cancer 1936; 28: 389-392.
82.
Durkin WJ,
Ghanta VK, Balch CM,
83.
Weisenthal
LM, Marsden JA. A novel dye exclusion assay for predicting response to
cancer chemotherapy. Proc Am Assoc Cancer Res 1981; 22: 155.(Abstract)
84.
Weisenthal
LM, Marsden JA, Malefatto J, Dill PL. Predicting response to cancer
chemotherapy with a novel dye exclusion assay. Proceedings of XIIth
International Congress of Chemotherapy,
85.
Bosanquet AG,
Bird MC, Price WJ, Gilby ED. An assessment of a short-term tumour
chemosensitivity assay in chronic lymphocytic leukaemia. Br J Cancer 1983;
47: 781-789.
86.
Nygren P,
Fridborg H, Csoka K, et al. Detection of tumor-specific cytotoxic drug
activity in vitro using the fluorometric microculture cytotoxicity assay and
primary cultures of tumor cells from patients. Int J Cancer 1994; 56:
715-720.
87.
Pieters R,
Loonen AH,
88.
Pieters R,
Kaspers GJ, Klumper E, Veerman AJ. Clinical relevance of in vitro drug
resistance testing in childhood acute lymphoblastic leukemia: the state of
the art. Med Pediatr Oncol 1994; 22: 299-308.
89.
Kaspers GJ,
Kardos G, Pieters R, et al. Different cellular drug resistance profiles in
childhood lymphoblastic and non-lymphoblastic leukemia: a preliminary
report. Leukemia 1994; 8: 1224-1229.
90.
Klumper E,
Pieters R, Veerman AJ, et al. In vitro cellular drug resistance in children
with relapsed/refractory acute lymphoblastic leukemia. Blood 1995; 86:
3861-3868.
91.
Hongo T,
Fujii Y. In vitro chemosensitivity of lymphoblasts at relapse in childhood
leukemia using the MTT assay. Int J Hematol 1991; 54: 219-230.
92.
Nagourney RA,
93.
Nagourney RA,
Weisenthal LM. Dexamethasone-induced cell death in primary cultures of
childhood A.L.L. predict survival: a prospective trial with 13 year
follow-up. leukemia 1995; 9: 531.
94.
Kaspers GJL,
Pieters R, Van Zantwijk CH, et al. In vitro drug sensitivity of normal
peripheral blood lymphocytes and childhood leukaemic cells from bone marrow
and peripheral blood. Br J Cancer 1991; 64: 469-474.
95.
Pieters R,
Huismans DR, Loonen AH, et al. Relation of cellular drug resistance to
long-term clinical outcome in childhood acute lymphoblastic leukaemia.
Lancet 1991; 338: 399-403.
96.
Pieters R,
Kaspers GJL, Van Wering ER, et al. Cellular drug resistance profiles that
might explain the prognostic value of immunophenotype and age in childhood
acute lymphoblastic leukemia. Leukemia 1993; 7: 392-397.
97.
Veerman AJ.
Cellular drug resistance in childhood leukemia. Ann Hematol 1994; 69: 31-34.
98.
Kaspers GJL,
Smets LA, Pieters R, Van Zantwijk CH, Van Wering ER, Veerman AJP. Favorable
prognosis of hyperdiploid common acute lymphoblastic leukemia may be
explained by sensitivity to antimetabolites and other drugs: results of an
in vitro study. Blood 1995; 85: 751-756. 99. Styczynski J, Pieters R,
Huismans DR, Schuurhuis GJ, Wysocki M, Veerman AJ. In vitro drug resistance
profiles of adult versus childhood acute lymphoblastic leukaemia. Br J
Haematol 2000; 110: 813-818.
99.
Janka-Schaub
GE, Harms DO, den Boer ML, Veerman AJ, Pieters R. In vitro drug resistance
as independent prognostic factor in the study COALL-O5-92 Treatment of
childhood acute lymphoblastic leukemia; two- tiered classification of
treatments based on accepted risk criteria and drug sensitivity profiles in
study COALL-06-97. Klin Padiatr 1999; 211: 233-238.
100.
Pieters R,
den Boer ML, Durian M, et al. Relation between age, immunophenotype and in
vitro drug resistance in 395 children with acute lymphoblastic
leukemia--implications for treatment of infants. Leukemia 1998; 12:
1344-1348.
101.
Kaspers GJ,
Pieters R, Van Zantwijk CH, Van Wering ER, Van der Does-van den Berg A,
Veerman AJ. Prednisolone resistance in childhood acute lymphoblastic
leukemia: vitro-vivo correlations and cross-resistance to other drugs. Blood
1998; 92: 259-266.
102.
Kaspers GJ,
Veerman AJ, Pieters R, et al. In vitro cellular drug resistance and
prognosis in newly diagnosed childhood acute lymphoblastic leukemia. Blood
1997; 90: 2723-2729.
103.
Klumper E,
Ossenkoppele GJ, Pieters R, et al. In vitro resistance to cytosine
arabinoside, not to daunorubicin, is associated with the risk of relapse in
de novo acute myeloid leukaemia. Br J Haematol 1996; 93: 903-910.
104.
Zwaan CM,
Kaspers GJ, Pieters R, et al. Cellular drug resistance profiles in childhood
acute myeloid leukemia: differences between FAB types and comparison with
acute lymphoblastic leukemia. Blood 2000; 96: 2879-2886.
105.
Nygren P,
Kristensen J, Jonsson B, et al. Feasibility of the fluorometric microculture
cytotoxicity assay (FMCA) for cytotoxic drug sensitivity testing of tumor
cells from patients with acute lymphoblastic leukemia. Leukemia 1992; 6:
1121-1128.
106.
Kristensen J,
Jonsson B, Sundstrom C, Nygren P, Larsson R. In vitro analysis of drug
resistance in tumor cells from patients with acute myelocytic leukemia. Med
Oncol Tumor Pharmacother 1992; 9: 1-9.
107.
Larsson R,
Nygren P. Prediction of individual patient response to chemotherapy by the
fluorometric microculture cytotoxicity assay (FMCA) using drug specific
cut-off limits and a Bayesian model. Anticancer Res 1993; 13: 1825-1829.
108.
Kristensen J,
Jonsson B, Sundstrom C, Nygren P, Larsson R. In vitro analysis of drug
resistance in tumor cells from patients with acute myelocytic leukemia
[published erratum appears in Med Oncol Tumor Pharmacother 1992;9(3):157].
Med Oncol Tumor Pharmacother 1992; 9: 65-74.
109.
Larsson R,
Fridborg H, Kristensen J, Sundstrom C, Nygren P. In vitro testing of
chemotherapeutic drug combinations in acute myelocytic leukaemia using the
fluorometric microculture cytotoxicity assay (FMCA). Br J Cancer 1993; 67:
969-974.
110.
Staib P,
Schinkothe T, Henrichs T, et al. In vitro drug resistance testing may
provide an independent prognostic factor for adult patients with AML. Proc
Am Soc Hematol (ASH) Annual Meeting 1999; Abs # 269: (Abstract)
111.
Hwang WS,
Chen LM, Huang SH, Wang CC, Tseng MT. Prediction of chemotherapy response in
human leukemia using in vitro chemosensitivity test. Leuk Res 1993; 17:
685-688.
112.
Mollgard L,
Tidefelt U, Sundman-Engberg B, Lofgren C, Paul C. In vitro chemosensitivity
testing in acute non lymphocytic leukemia using the bioluminescence ATP
assay. Leuk Res 2000; 24: 445-452.
113.
Bird MC,
Bosanquet AG, Forskitt S, Gilby ED. Long-term comparison of results of a
drug sensitivity assay in vitro with patient response in lymphatic
neoplasms. Cancer 1988; 61: 1104-1109.
114.
Bosanquet AG.
Correlations between therapeutic response of leukaemias and in-vitro
drug-sensitivity assay. Lancet 1991; 337: 711-714.
115.
Bosanquet AG,
Johnson SA, Richards SM. Prognosis for fludarabine therapy of chronic
lymphocytic leukaemia based on ex vivo drug response by DiSC assay. Br J
Haematol 1999; 106: 71-77.
116.
Bosanquet AG,
McCann SR, Crotty GM, Mills MJ, Catovsky D. Methylprednisolone in advanced
chronic lymphocytic leukaemia: rationale for, and effectiveness of treatment
suggested by DISC assay. Acta Haematologica 1995; 93: 73-79.
117.
Thornton PD,
Hamblin M, Treleaven JG, Matutes E,
118.
119.
Larsson R,
Nygren P. Laboratory prediction of clinical chemotherapeutic drug
resistance: a working model exemplified by acute leukaemia. Eur J Cancer
1993; 29A: 1208-1212.
120.
Fridborg H,
Jonsson E, Nygren P, Larsson R. Relationship between diagnosis-specific
activity of cytotoxic drugs in fresh human tumour cells ex vivo and in the
clinic. Eur J Cancer 1999; 35: 424-432.
121.
Mason JM,
Drummond MF, Bosanquet AG, Sheldon TA. The DiSC assay. A cost-effective
guide to treatment for chronic lymphocytic leukemia? Int J Technol Assess
Health Care 1999; 15: 173-184.
122.
Pieters R,
Huismans DR, Loonen AH, et al. Relation of cellular drug resistance to
long-term clinical outcome in childhood acute lymphoblastic leukaemia.
Lancet 1991; 338: 399-403.
123.
Kaspers GJ,
Veerman AJ, Pieters R, Van Zantwijk CH, Van Wering ER, Van der Does-van den
Berg A. In vitro drug resistance profile: strongest prognostic factor in
childhood acute lymphoblastic leukemia (ALL) (meeting abstract). Blood 1994;
84: 516a.
124.
Veerman AJ,
Kaspers GJ, Pieters R. Cellular drug resistance in childhood leukemia. Ann
Hematol 1994; 69 Suppl 1: S31-S34.
125.
Klumper E,
Ossenkoppele GJ, Pieters R, et al. In vitro resistance to cytosine
arabinoside, not to daunorubicin, is associated with the risk of relapse in
de novo acute myeloid leukaemia. Br J Haematol 1996; 93: 903-910.
126.
Norgaard JM,
Langkjer ST, Palshof T, Pedersen B, Hokland P. Pretreatment leukaemia cell
drug resistance is correlated to clinical outcome in acute myeloid
leukaemia. Eur J Haematol 2001; 66: 160-167.
127.
Kobayashi H,
Man S, Graham CH, Kapitain SJ, Teicher BA, Kerbel RS. Acquired
multicellular-mediated resistance to alkylating agents in cancer. Proc Natl
Acad Sci U S A 1993; 90: 3294-3298.
128.
Kerbel RS.
Impact of multicellular resistance on the survival of solid tumors,
including micrometastases. Invasion Metastasis 1994; 14: 50-60.
129.
Desoize B,
Gimonet D, Jardiller JC. Cell culture as spheroids: an approach to
multicellular resistance. Anticancer Res 1998; 18: 4147-4158.
130.
Desoize B,
Jardillier J. Multicellular resistance: a paradigm for clinical resistance?
Crit Rev Oncol Hematol 2000; 36: 193-207.
131.
Shaw GL,
Gazdar AF, Phelps R, et al. Individualized chemotherapy for patients with
non-small cell lung cancer determined by prospective identification of
neuroendocrine markers and in vitro drug sensitivity testing. Cancer Res
1993; 53: 5181-5187.
132.
Furukawa T,
Kubota T, Hoffman RM. Clinical applications of the histoculture drug
response assay. Clin Cancer Res 1995; 1: 305-311.
133.
Baba H,
Takeuchi H, Inutsuka S, et al. Clinical value of SDI test for predicting
effect of postoperative chemotherapy for patients with gastric cancer. Semin
Surg Oncol 1994; 10: 140-144. 135. Markman M. Chemosensitivity and
chemoresistance assays: are they clinically relevant? J Cancer Res Clin
Oncol 1995; 121: 441-442.
134.
Suto A,
Kubota T, Shimoyama Y, Ishibiki K, Abe O. MTT assay with reference to the
clinical effect of chemotherapy. J Surg Oncol 1989; 42: 28-32.
135.
Saikawa Y,
Kubota T, Furukawa T, et al. Single-cell suspension assay with an MTT end
point is useful for evaluating the optimal adjuvant chemotherapy for
advanced gastric cancer. Jpn J Cancer Res 1994; 85: 762-765.
136.
Kurihara N,
Kubota T, Furukawa T, et al. Chemosensitivity testing of primary tumor cells
from gastric cancer patients with liver metastasis can identify effective
antitumor drugs. Anticancer Res 1999; 19: 5155-5158.
137.
Abe S, Kubota
T, Matsuzaki SW, et al. Chemosensitivity test is useful in evaluating the
appropriate adjuvant cancer chemotherapy for stage III non-scirrhous and
scirrhous gastric cancers. Anticancer Res 1999; 19: 4581-4586.
138.
Fujita K,
Kubota T, Matsuzaki SW, et al. Further evidence for the value of the
chemosensitivity test in deciding appropriate chemotherapy for advanced
gastric cancer. Anticancer Res 1998; 18: 1973-1978.
139.
Yamaue H,
Tanimura H, Tsunoda T, et al. Chemosensitivity testing with highly purified
fresh human tumour cells with the MTT colorimetric assay. Eur J Cancer 1991;
27: 1258-1263.
140.
Yamaue H,
Tanimura H, Noguchi K, et al. Chemosensitivity testing of fresh human
gastric cancer with highly purified tumour cells using the MTT assay. Br J
Cancer 1992; 66: 794-799.
141.
Yamaue H,
Tanimura H, Nakamori M, et al. Clinical evaluation of chemosensitivity
testing for patients with colorectal cancer using MTT assay. Dis
142.
Kimura H,
Yonemura Y, Ohyama S, et al. The succinate dehydrogenase inhibition test for
evaluating biopsy specimens and resected tumors of advanced gastric cancer.
Surg Today 1992; 22: 508-511.
143.
Vescio RA,
Redfern CH, Nelson TJ, Ugoretz S, Stern PH, Hoffman RM. In vivo-like drug
responses of human tumors growing in three-dimensional gel-supported primary
culture. Proc Natl Acad Sci U S A 1987; 84: 5029-5033.
144.
Salonga D,
Danenberg KD, Johnson M, et al. Colorectal tumors responding to
5-fluorouracil have low gene expression levels of dihydropyrimidine
dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin
Cancer Res 2000; 6: 1322-1327.
145.
Konecny G,
Crohns C, Pegram M, et al. Correlation of drug response with the ATP tumor
chemosensitivity assay in primary FIGO stage III ovarian cancer. Gynecol
Oncol 2000; 77: 258-263.
146.
Kurbacher CM,
Cree IA, Bruckner HW, et al. Use of an ex vivo ATP luminescence assay to
direct chemotherapy for recurrent ovarian cancer. Anticancer Drugs 1998; 9:
51-57.
147.
Cortazar P,
Johnson BE. Review of the efficacy of individualized chemotherapy selected
by in vitro drug sensitivity testing for patients with cancer. J Clin Oncol
1999; 17: 1625-1631.
148.
Link KH,
Kornmann M, Butzer U, et al. Thymidylate synthase quantitation and in vitro
chemosensitivity testing predicts responses and survival of patients with
isolated nonresectable liver tumors receiving hepatic arterial infusion
chemotherapy. Cancer 2000; 89: 288-296.
149.
Lenz HJ,
Leichman CG, Danenberg KD, et al. Thymidylate synthase mRNA level in
adenocarcinoma of the stomach: a predictor for primary tumor response and
overall survival. J Clin Oncol 1996; 14: 176-182.
150.
McNeil C.
Using HER2 to choose chemotherapy in breast cancer: is it ready for the
clinic? J Natl Cancer Inst 1999; 91: 110-112.
151.
Paik S,
Bryant J, Tan-Chiu E, et al. HER2 and choice of adjuvant chemotherapy for
invasive breast cancer: National Surgical Adjuvant Breast and Bowel Project
Protocol B-15. J Natl Cancer Inst 2000; 92: 1991-1998.
152.
Nelson NJ.
Can HER2 status predict response to cancer therapy? J Natl Cancer Inst 2000;
92: 366-367.
153.
Aabo K, Adams
M, Adnitt P, et al. Chemotherapy in advanced ovarian cancer: four systematic
meta-analyses of individual patient data from 37 randomized trials. Advanced
Ovarian Cancer Trialists' Group. Br J Cancer 1998; 78: 1479-1487.
154.
155.
Muggia FM,
Braly PS, Brady MF, et al. Phase III randomized study of cisplatin versus
paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage
III or IV ovarian cancer: a gynecologic oncology group study. J Clin Oncol
2000; 18: 106-115.
156.
Strauss E.
Pretesting Tumors. Sci Am 1999; 280: 19-20.
157.
Stein R.
Official U.S. Government transcript of Health Care Finance Administration
technology assessment committee meeting on Human Tumor Assay Systems,
Baltimore, MD, November 15, 1999, Volume 1, pp. 92-99. Available on the
internet: http://cms.hhs.gov/coverage/8b1-h1.asp Backup link:
http://weisenthal.org/hcfa_1.htm
158.
Nalick R.
Official U.S. Government transcript of Health Care Finance Administration
technology assessment meeting on Human Tumor Assay Systems, Baltimore, MD,
Nov. 15, 1999, Volume 1, pp. 106-107. Available on the internet:
http://cms.hhs.gov/coverage/8b1-h1.asp Backup
link:http://weisenthal.org/hcfa_1.htm
159.
Nagourney RA,
Link JS, Blitzer JB, Forsthoff C, Evans SS. Gemcitabine plus cisplatin
repeating doublet therapy in previously treated, relapsed breast cancer
patients. J Clin Oncol 2000; 18: 2245-2249.
160.
Weisenthal
LM. Antineoplastic drug screening belongs in the laboratory, not in the
clinic [editorial]. J Natl Cancer Inst 1992; 84: 466-469.
161.
Taylor CG,
Sargent JM, Elgie AW, Williamson CJ, Lewandowicz GM, Chappatte O, and Hill
JG. Chemosensitivity testing predicts survival in ovarian cancer. Eur J
Gynaecol Oncol 2001; 22:278-82
Click here to view abstract
Please
use the “back” button on your browser to return to your previous location