Download Circulating Tumor Cells, Disease Progression, and Survival in

Circulating Tumor Cells, Disease
Progression, and Survival in Metastatic Breast Cancer
Massimo Cristofanilli
Metastatic breast cancer (MBC) is considered incurable; therefore, palliative treatment is
the only option. The biologic heterogeneity of the disease is reflected in its somewhat
unpredictable clinical behavior. The presence of circulating tumor cells (CTCs) in patients
with MBC about to start a new line of treatment has been shown to predict progression-free
and overall survival. This prognostic value is independent of the line of therapy (eg, first or
second line). Moreover, a multivariate analysis has shown the prognostic value of CTCs to
be superior to that of site of metastasis, type of therapy, and length of time to recurrence
after definitive primary surgery. These data suggest that the presence of CTCs may be used
to modify the staging system for advanced disease. Larger studies are needed to confirm these
data and evaluate the use of CTC detection in monitoring treatment and furthering our
understanding of breast cancer biology when combined with other diagnostic technologies.
Semin Oncol 33(suppl 9):S9-S14 © 2006 Elsevier Inc. All rights reserved.
B
reast cancer is one of the most frequently diagnosed types
of cancer and a leading cause of cancer death in women.1
The vast majority of these deaths are caused by recurrent
metastatic disease. Occult dissemination of tumor cells is the
main cause of recurrent metastatic breast cancer (MBC) in
patients who have undergone resection of their primary tumor.2 Approximately 5% of patients with breast cancer have
clinically detectable metastases at the time of initial diagnosis.
A further 30% to 40% of patients who appear clinically free of
metastases harbor occult metastases.3,4
The formation of metastatic colonies is a continuous process, commencing early during the growth of the primary
tumor. Metastasis occurs through a cascade of linked sequential steps involving multiple host-tumor interactions. To successfully create a metastatic deposit, a cell or group of cells
must be able to leave the primary tumor, invade the local host
tissue, and survive to proliferate. This complex process requires the cells to enter the circulation, arrest at the distant
vascular bed, extravasate into the organ interstitium and parenchyma, and proliferate as a secondary colony. Several experimental studies suggest that during each stage of the process only the most fit tumor cells survive.2 A very small
percentage (less than .01%) of circulating tumor cells (CTCs)
ultimately initiate successful metastatic colonies. Thus, me-
tastasis is a highly selective competition favoring the survival
of a minor subpopulation of metastatic tumor cells. These
findings suggest that the use of novel, sophisticated diagnostic technologies can allow early identification of micrometastatic foci, providing an opportunity for early intervention in
patients at high risk, and a better risk stratification in patients
with metastatic disease, leading to appropriate treatment development and patient selection.
The detection of microscopic disease in breast cancer has
been evaluated in lymph nodes, bone marrow (primary
breast cancer), and peripheral blood (metastatic disease).3-6
Most of these studies have shown that the detection of microscopic disease in patients with breast cancer contributes
prognostic information and, in selected cases, can predict the
efficacy of treatments.6 In primary breast cancer, the detection of microscopic disease in lymph nodes and bone marrow
has led to a better understanding of the role of minimal residual disease. The detection of tumor cells in the locoregional lymph nodes of women with early breast cancer using
immunohistochemical analysis is technically feasible, and the
presence of these cells has been shown to have a negative
effect on long-term prognosis.7 In this review, we specifically
discuss the detection of microscopic disease in the peripheral
blood, and the prognostic implication and clinical use of
determining the presence of CTCs.
Department of Breast Medical Oncology, The University of Texas M. D.
Anderson Cancer Center, Houston, TX.
Address reprint requests to Massimo Cristofanilli, MD, FACP, Department
of Breast Medical Oncology, Unit 1354, The University of Texas M. D.
Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 770304009. E-mail: [email protected]
Detection of CTCs in MBC
0093-7754/06/$-see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1053/j.seminoncol.2006.03.016
The first report on tumor cells in the peripheral circulation
was attributed to Ashworth in 1869.8 Since then, the existence, origin, and clinical significance of CTCs have been
S9
M. Cristofanilli
S10
debated. The introduction of more sensitive and specific immunohistochemical techniques in the late 1970s led to renewed interest in the detection of CTCs and their possible
association with minimal residual disease in solid malignancies.9,10 But, despite evidence of the prognostic value of CTCs
in some studies, the detection of micrometastases was never
incorporated into cancer staging protocols or considered a
valuable tool for clinical use. This may be the result of a
combination of factors, such as variable antigen expression in
poorly differentiated tumors and reports of cytokeratin and
epithelial membrane antigen positivity in nonepithelial cells,
which showed a need for more sensitive and specific methods
of detection than were available at the time. This need was
fulfilled to some degree by more sensitive polymerase chain
reaction (PCR) techniques in the late 1980s, which greatly
facilitated the detection of occult tumor cells through the use
of nucleic acid analysis.11-13 In the past decade, a few studies
have shown that the detection of occult disease by PCR has
prognostic significance in some solid tumors.14 However, because these PCR-based assays for the detection of occult tumor cells have limitations, particularly contamination of
samples, sensitivity and specificity of the assay, and inability
to quantify tumor cells, they cannot be used to perform functional assays. These factors have precluded the widespread
use of PCR in in vitro diagnostic applications.
Over the past few years, immunomagnetic separation technology, with its higher level of sensitivity and specificity, has
been used to improve the detection of CTCs.15-17 In this technique, the specimen is incubated with magnetic beads coated
with antibodies directed against epithelial cells. The epithelial
cells are then isolated using a powerful magnet. The magnetic
fraction can be used for downstream reverse-transcriptase PCR,
flow cytometry, or immunocytochemical analysis.18,19 Using
this approach, Austrup et al reported the prognostic significance of genomic alterations (eg, c-erbB2 overexpression)
present in circulating cells purified from the blood of patients
with breast cancer.20 The authors investigated genomic imbalances such as mutation, amplification, and loss of heterozygosity of 13 tumor suppressor genes and two protooncogenes using DNA from isolated minimal residual cancer
cells. The presence and number of genomic imbalances measured in disseminated tumor cells were significantly associated
with worse prognosis.20 More recently, advances in technology
have facilitated the detection of extremely rare CTCs. The
CellSearch system (Veridex, LLC, Warren, NJ) was designed
to detect circulating epithelial cells in whole blood. This system is based on the principle of immunomagnetic isolation of
epithelial cells, with subsequent counting provided by immunofluorescent analysis of cytokeratin expression.21,22
Prognostic Role of CTCs
The predictive and prognostic roles of CTCs were recently
investigated in a prospective multicenter clinical trial led by
researchers at The University of Texas M. D. Anderson Cancer Center (Houston, TX).6 In this study, CellSearch was used
to prospectively determine the prognostic and predictive
value of CTCs in patients with MBC who were about to start
a new systemic treatment. The 177 enrolled patients underwent blood collection at monthly intervals for up to 6 months
after enrollment (Table 1). A cutoff of 5 CTCs/7.5 mL was
used to stratify patients into positive and negative groups
(positive, ⱖ5 CTCs/7.5 mL; negative, ⬍5 CTCs/7.5 mL).
Patients classified as positive had shorter progression-free
survival times (2.7 v 7.0 months; P ⫽ .0001) and shorter
overall survival times (10.9 v 21.9 months; P ⬍.0001) than
did those classified as negative (Fig 1). At first follow-up after
initiation of therapy (3 weeks) there was an increased difference between CTC-positive and -negative patients in terms of
progression-free survival time (2.1 v 7.0 months; P ⬍.0001)
and overall survival time (8.2 v ⬎18 months; P ⬍.0001). On
multivariate Cox hazards regression analysis, CTC levels,
both at baseline and at first follow-up, were the most significant predictors of progression-free survival and overall survival.6
These data suggest several important considerations. First,
and more relevant to clinical practice, is the demonstration
Table 1 Demographics of Patients With Metastatic Breast
Cancer Before Undergoing New Systemic Treatment
N
All patients
Age at baseline (yrs)
Median (yrs)
Race
White
Black
Hispanic
Unknown
ER/PR
ER/PRⴙ
ER/PRⴚ
Unknown
HER2 status
HER2ⴚ (0ⴚ2ⴙ)
HER2ⴙ (3ⴙ) 26
Unknown
Line of chemotherapy for
metastases
1st
2nd
>3rd
Unknown
Site of metastasis
Visceral
Non-visceral
Survival status
Alive
Dead
Average [mean] follow-up
Time alive (mos)
(N ⴝ 74)
Average [mean] dead ⴞ SD
(mos) (N ⴝ 103)
%
177
58.5 ⴞ 13.4
59
152
14
7
4
86
8
4
2
120
55
2
68
31
1
122
15
29
69
83
25
67
2
47
14
38
1
145
32
82
18
16
74
42
103
58
19.5 ⴞ 5.7 (median,
20.9 months)
10.3 ⴞ 6.9 (median,
8.8 months)
Abbreviations: ER, estrogen receptor; HER, human epidermal growth
factor receptor; PR, progesterone receptor; SD, standard deviation.
CTCs, disease progression, and survival
that detection of CTCs is superior to known factors, such as
site of metastasis (visceral versus non-visceral) and estrogenreceptor status, that have been previously considered important variables for prognostic evaluation. Furthermore, these
results showed that CTCs are detectable in MBC irrespective
of the site of metastasis, the line of therapy (eg, patients who
are newly diagnosed versus those undergoing second- or
third-line therapy), and, more importantly, initial hormone
receptor status. Of note was the fact that the proportion of
patients with newly diagnosed metastatic disease about to
undergo first-line treatment who were CTC-positive (ⱖ5
CTCs/7.5 mL) was similar to the proportion of patients undergoing at least second-line treatment who were CTC-positive (52% v 48%, respectively).6 The detection of CTCs predicted overall survival independently of the number of
previous treatments, but the association was particularly
strong for patients undergoing first-line treatment for metastatic disease (Fig 2).23 The data at first follow-up supported
the predictive value of CTCs. As expected, the CTC detection
rate in the group tested at first follow-up was lower than the
baseline, particularly in patients undergoing first-line treatment (25% v 52%) and those with visceral disease (28% v
S11
Figure 2 Kaplan-Meier plots of overall survival in first-line therapy
MBC patients with ⬍5 CTCs or ⱖ5 CTCs (per 7.5 mL) at baseline.
The analysis includes 94 patients with measurable and evaluable
(bone only) disease. Overall survival was calculated from the time of
the baseline blood draw. Coordinates of dashed lines indicate median survival time. CTCs, circulating tumor cells; CI, confidence
interval; HR, hazard ratio; OS, overall survival.
50%).6 These data indicate that patients with newly diagnosed disease who are about to start first-line therapy may
have detectable CTCs, and the changes in these detection
rates at 3 to 4 weeks may indicate benefit from the use of
systemic treatment (particularly chemotherapy). However,
this short follow-up testing might be less useful in patients
who presumably have more indolent disease (eg, patients
undergoing hormonal treatment). A recent update and reanalysis of the data from this pivotal study, which included
data from an additional 46 patients with bone-only MBC,
confirmed the observation in the initial report indicating that
the detection of CTCs at baseline was associated with a significant cumulative hazard risk of death (53% v 19% with
and without CTCs, respectively; P ⫽ .0001) in patients with
measurable as well as ‘nonmeasurable’ MBC.24
In summary, the detection of CTCs in patients with MBC is
associated with important prognostic implications, allowing
for a defined stratification of risk of death. These findings
suggest that CTC detection can be used for a stage subclassification of MBC that would modify our approach to treatment
planning and, more importantly, allow for more sophisticated design of efficacy trials.
Staging Classification
for MBC Using CTCs
Figure 1 Kaplan-Meier plots of (A) progression-free survival and
(B) overall survival in MBC patients with ⬍5 CTCs or ⱖ5 CTCs (per
7.5 mL) at baseline. Progression-free survival and overall survival
were calculated from the time of the baseline blood draw. Coordinates of dashed lines indicate median survival time. CTCs, circulating tumor cells.
The American Joint Committee on Cancer (AJCC) classification system includes much of the traditional prognostic information used by clinicians when developing a comprehensive treatment plan. This system was based on a schema
developed by the Union Internationale Contre le Cancer
S12
(UICC).25-27 In brief, the AJCC system attempts to define the
disease by incorporating all aspects of cancer distribution in
terms of the primary tumor (T), lymph nodes (N), and distant
metastasis (M). A ‘stage’ group (0, I, II, III, or IV) is then
assigned on the basis of the possible TNM permutations, with
0 reflecting minimal involvement and IV either the most tumor involvement or distant metastasis. This basic TNM staging is then further subdivided according to the time the evaluation is performed: c ⫽ clinical (before surgical treatment)
and p ⫽ pathologic (after surgical specimen analysis).7,28
Over the past 45 years, the AJCC has regularly updated its
staging standards to incorporate advances in prognostic technology. The committee’s current work concerns the development of prognostic indices based on molecular markers. But
until these changes are incorporated, TNM staging quantifies
only the physical extent of disease and, although it includes
the approximately 2% of women who initially present with
primary metastatic disease (stage IV), the prognostic information is only applicable to in situ, local, and regional primary breast cancers. This is somewhat problematic in that
because of the heterogeneity of the disease, the potential for
continued mutation, and the variety of treatment options
available, the information acquired at the time of the primary
diagnosis of breast cancer may not be as relevant to planning
the treatment of the approximately 30% of women with
breast cancer who present with recurrent MBC years after
their initial diagnosis.
The recent demonstration that the presence of CTCs predicted the prognoses of two subgroups of patients with MBC
raises the possibility that this method will allow for a true
‘biologic staging’ of breast cancer (eg, stages IVA and IVB).6
The main limitations of the previous completed study were
the sample size (only 177 patients), the inclusion of measurable-disease-only patients, which did not reflect the real clinical heterogeneity of MBC, the inclusion of patients at different points in treatment, and those who have newly diagnosed
disease versus those undergoing second- or third-line treatment, whose benefit from further therapy could have been
limited by the advanced status of their disease. To overcome
these limitations, a larger international validation trial, the
International Stage IV Stratification Study, has recently begun (Fig 3). The objective of this study is to confirm the
prognostic value of CTC detection in a larger and more homogeneous cohort of patients with newly diagnosed MBC.
The study aims to enroll 660 patients and will enable a more
detailed analysis of the association between CTC detection
and other factors, such as ethnicity (black compared with
white and Hispanic groups) and specific disease sites (visceral v non-visceral disease). Accrual is expected to be completed by November 2006, which will allow completion of
data analysis by late 2007. This study may well help provide
definitive information on the subclassification of stage IV disease
for the revised 7th edition of the AJCC’s Cancer Staging Manual.
Clinical Application of CTCs
Despite years of clinical research, the odds of patients with
MBC achieving a complete response remain extremely low.
M. Cristofanilli
Figure 3 Schematic representation of the prospective International
Stage IV Stratification Study. Six hundred sixty patients with newly
diagnosed MBC will be enrolled and, after having blood drawn at
baseline, will be receiving treatments. Patients will have follow-up
of their condition until death. There is no planned interim analysis
or additional blood evaluation. Dx, diagnosed; MBC, metastatic
breast cancer; Tx, treatment.
Thus, the main goal in the management of this entity is palliation.29,30 Only a few patients who experience a complete
response after chemotherapy remain in this state for prolonged periods, with some remaining in remission for more
than 20 years.30 These long-term survivors are usually young,
have an excellent performance status, and, more importantly,
have limited metastatic disease.31,32 Most patients with metastatic disease respond transiently to conventional therapies
and develop evidence of progressive disease within 12 to 24
months of starting treatment.29 For these patients, systemic
treatment does not result in a significant improvement in
survival time but substantially improves their quality of life.
At the present time, clinicians can use three different
systemic treatment modalities for advanced breast cancer:
endocrine therapy, chemotherapy, and biologic targeted
therapy.33-35 Appropriate selection of patients for these modalities is based mostly on tissue assessment of hormone
receptor status (estrogen and progesterone receptors) and
c-erbB2 status. In patients who lack expression of hormone
receptors or who show no amplification of c-erbB2, cytotoxic
chemotherapy is used. However, no standard therapeutic
regimen has been defined. For example, the optimal schedule
of chemotherapy administration in MBC (ie, concurrent v
sequential) remains controversial, and the decision must be
individualized. Sledge et al36 addressed this issue in a prospective study that included 739 chemotherapy-naive patients who were randomly assigned to receive, at progression,
doxorubicin, paclitaxel, or a combination of both. Although
the response rates and times to treatment failure were improved with the combination regimen, the overall survival
rate was comparable between the groups. Other trials have
shown a survival advantage for combination regimens, but all
of these studies have reported differences in median overall
survival time, suggesting that even with comparable inclusion criteria the heterogeneity typical of MBC cannot be eliminated.37,38 This is indeed one of the major limitations to the
development of more personalized treatments for patients.
Therefore, although the data show that patients can benefit
from combination therapy, they do not clearly identify the
CTCs, disease progression, and survival
subsets of patients who most benefit or who experience only
additional toxicity.
In this context, the use of CTCs to stratify patients at the
time of disease recurrence may be appropriate. CTC detection may allow a more rational selection of treatments for
patients with newly recurred disease, and this approach
could maximize the chance of a particular combination or
single new drug showing clinical benefit and, eventually,
prolonging survival. In essence, it is possible that CTC detection could be used in the design of efficacy trials of different
therapeutic approaches. The efficacy of these treatments will
be more easily assessed when patients have been stratified by
their prognosis, leading to more tailored treatment strategies.
We believe that the challenge for the next generation of clinical trials, and the responsibility for both clinical investigator
and the pharmaceutical industry, will be to incorporate these
concepts into the process of drug development.
S13
tional, with decreased levels of major histocompatibility
complex class II, CD86 (B7) expression, and interleukin-12
secretion.49
The inherent and dynamic phenotypic instability of breast
tumor cells complicates these issues of breast cancer biology.
Because tumors arise endogenously and progress, a continual
dialogue between the tumor phenotype and the immune response ensues, with each influencing the other to evolve.50
Therefore, being able to evaluate both the phenotype of CTCs
and the related immune response is a critical step in understanding the complexity of tumor immunity in patients with
breast cancer, and will enable more effective vaccine strategies to be designed. A recently developed glycan array has
shown the capacity to detect a particular profile of antibodies
directed against cancer-specific carbohydrate antigens.51,52
The further development of this novel technology in combination with CTC detection will represent a new frontier in
breast cancer microdiagnostics.
Future Uses of CTCs
The process of sorting cancer cells from other cellular components (eg, blood and stromal cells) in clinical samples is
fundamentally important for the future of genomic and proteomic analysis.39-42 Indeed, the feasibility of evaluating the
gene expression profiles of cancer cells depends mostly on
the quality of the specimen (relative proportion of cancer
cells) and the amount of nucleic acid that can be extracted
from the appropriate cells to provide mRNA suitable for analysis.41,42 Fine-needle aspiration biopsy of malignant lesions is
frequently used for diagnostic purposes, but the percentage
of malignant cells recovered with this procedure ranges
widely (60% to 90%).41,42 Pusztai et al42 showed that singlepass fine-needle aspiration biopsies can provide samples consisting of approximately 79% cancer cells (range, 25% to
100%).
Collecting representative tissue in the metastatic setting
from solid tumors is more complex and usually requires
more invasive procedures that increase the risk of complications and discomfort. Furthermore, these procedures may
not provide an adequate specimen for detailed analysis and
typically cannot be repeated for a dynamic evaluation of the
biologic changes during treatments. Theoretically, CTC detection would allow specific genes (eg, c-erbB2, EGFR, and
MGB) or more global gene expression to be analyzed while
using specific targeted treatments in metastatic disease.43-46
This information could then be used to design specific treatments that more appropriately reflect the dynamics and heterogeneity of MBC.43
The prognostic value of CTCs in the setting of MBC raises
the possibility that their presence or absence could be indicative of the complex interplay between the host and the disease, providing some indirect information on tumoral immunity. Substantial evidence exists for immune defects in
patients with breast cancer, demonstrated particularly by increased numbers of functionally suppressive CD4⫹CD25⫹
T-regulatory cells in the peripheral blood.47,48 Furthermore,
dendritic cells obtained from the peripheral blood and lymph
nodes of patients with operable breast cancer are dysfunc-
Conclusion
The detection of microscopic disease in the peripheral blood
of patients with MBC is associated with prognostic information. These data will allow appropriate risk stratification and
modification of the current staging system for advanced disease. Moreover, it is expected that investigators and the pharmaceutical industry will derive benefit from these technologies that will contribute to more tailored treatments and
sophisticated trial designs. Using a combination of CTC detection and other diagnostic technologies will help us further
understand the complex and heterogeneous tumor phenotype and its relationship with tumor-related immunity.
References
1. National Cancer Institute: SEER Cancer Statistics Review (1975-2000).
Available at: http://seer.cancer.gov/csr/1975_2000 Accessed September 2005
2. Folkman J: Tumor angiogenesis: Therapeutic implications. N Engl
J Med 285:1182-1186, 1971
3. Clare SE, Sener SF, Wilkens W, et al: Prognostic significance of occult
lymph node metastases in node-negative breast cancer. Ann Surg Oncol
4:447-451, 1997
4. Braun S, Pantel K, Müller P, et al: Cytokeratin-positive cells in the bone
marrow and survival of patients with stage I, II or III breast cancer.
N Engl J Med 342:525-533, 2000
5. Cristofanilli M, Budd GT, Ellis M, et al: Circulating tumor cells, disease
progression, and survival in metastatic breast cancer. N Engl J Med
351:781-791, 2004
6. Reed W, Bohler PJ, Sandstad B, et al: Occult metastases in axillary
lymph nodes as a predictor of survival in node-negative breast carcinoma with long-term follow-up. Breast J 10:174-180, 2004
7. Singletary SE, Allred C, Ashley P, et al: Staging system for breast cancer:
revisions for the 6th edition of the AJCC Cancer Staging Manual. Surg
Clin North Am 83:803-819, 2003
8. Ashworth TR: A case of cancer in which cells similar to those in the
tumours were seen in the blood after death. Aust Med J 14:146-149,
1869
9. Roberts S, Watne A, McGrath R, et al: Technique and results of isolation
of cancer cells from the circulating blood. AMA Arch Surg 76:334-346,
1958
10. Long L, Jonasson O, Roberts S, et al: Cancer cells in blood. Results of
simplified isolation technique. Arch Surg 80:910-919, 1960
S14
11. Bossolasco P, Ricci C, Farina G, et al: Detection of micrometastatic cells
in breast cancer by RT-PCR for the mammaglobin gene. Cancer Detect
Prev 26:60-63, 2002
12. Hawes D, Neville AM, Cote RJ: Occult metastasis. Biomed Pharmacother 55:229-242, 2001
13. Hu XC, Chow LW: Detection of circulating breast cancer cells with
multiple-marker RT-PCR assay. Anticancer Res 21:421-424, 2001
14. An XY, Egami H, Hayashi N, et al: Clinical significance of circulating
cancer cells in peripheral blood detected by reverse transcriptase-polymerase chain reaction in patients with breast cancer. Tohoku J Exp Med
193:153-162, 2001
15. Gross HJ, Verwer B, Houck D, et al: Model study detecting breast
cancer cells in peripheral blood mononuclear cells at frequencies as low
as 10(-7). Proc Natl Acad Sci U S A 92:537-541, 1995
16. Martin KJ, Graner E, Li Y, et al: High-sensitivity array analysis of gene
expression for the early detection of disseminated breast tumor cells in
peripheral blood. Proc Natl Acad Sci U S A 98:2646-2651, 2001
17. Racila E, Euhus D, Weiss AJ, et al: Detection and characterization of
carcinoma cells in the blood. Proc Natl Acad Sci U S A 95:4589-4594,
1998
18. Witzig TE, Bossy B, Kimlinger T, et al: Detection of circulating cytokeratin-positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy. Clin Cancer Res
8:1085-1091, 2002
19. Pachmann K, Heiss P, Demel U, et al: Detection and quantification of
small numbers of circulating tumour cells in peripheral blood using
laser scanning cytometer (LSC). Clin Chem Lab Med 39:811-817, 2001
20. Austrup F, Uciechowski P, Eder C, et al: Prognostic value of genomic
alterations in minimal residual cancer cells purified from the blood of
breast cancer patients. Br J Cancer 83:1664-1673, 2001
21. Kagan M, Howard D, Bendele T, et al: A sample preparation and analysis system for identification of circulating tumor cells. J Clin Ligand
Assay 25:104-110, 2002
22. Terstappen LW, Rao C, Gross S, et al: Peripheral blood tumor cell load
reflects the clinical activity of the disease in patients with carcinoma of
the breast. Int J Oncol 17:573-578, 2000
23. Cristofanilli M, Hayes DF, Budd GT, et al: Circulating tumor cells: A
novel prognostic factor for newly diagnosed metastatic breast cancer.
J Clin Oncol 23:1420-1430, 2005
24. Cristofanilli M, Budd GT, Ellis MJ, et al: Presence of circulating tumor
cells (CTC) in metastatic breast cancer (MBC) predicts rapid progression and poor prognosis. J Clin Oncol 23:16S, 2003 (abstr)
25. Denoix PF, Baclesse F: Proposed clinical classification of breast cancers;
Statement of the International Cancer Union. Bull Assoc Fr Etud Cancer 42:433-440, 1955
26. International Union Against Cancer (UICC): TMN Classification of
Malignant Tumours. Geneva, UICC, 1968
27. Berndt H, Titze U: TNM clinical stage classification of breast cancer. Int
J Cancer 4:837-844, 1969
28. Greene FL, Page DI, Fleming ID, et al: AJCC Cancer Staging Manual.
(ed 6). New York, NY, Springer, 2002
29. Ellis M, Hayes DF, Lippman ME: Treatment of metastatic disease, in
Harris J, Lippman M, Morrow M, et al (eds): Diseases of the Breast.
(ed 2). Philadelphia, PA, Lippincott-Raven, 2000, pp 749-799
30. Greenberg PA, Hortobagyi GN, Smith TL, et al: Long-term follow-up of
patients with complete remission following combination chemotherapy for metastatic breast cancer. J Clin Oncol 14:2197-2205, 1996
31. Nieto Y, Cagnoni PJ, Shpall EJ, et al: Phase II trial of high-dose chemotherapy with autologous stem cell transplant for stage IV breast cancer
with minimal metastatic disease. Clin Cancer Res 5:1731-1737, 1999
32. Rivera E, Holmes FA, Buzdar AU, et al: Fluorouracil, doxorubicin, and
cyclophosphamide followed by tamoxifen as adjuvant treatment for
patients with stage IV breast cancer who have no evidence of disease.
Breast J 8:2-9, 2002
M. Cristofanilli
33. Swenerton KD, Legha SS, Smith L, et al: Prognostic factors in metastatic
breast cancer treated with combination chemotherapy. Cancer Res 39:
1552-1562, 1979
34. Hortobagyi GN, Smith TL, Legha SS, et al: Multivariate analysis of
prognostic factors in metastatic breast cancer. J Clin Oncol 1:776-786,
1983
35. Bernard-Marty C, Cardoso F, Piccart MJ: Facts and controversies in
systemic treatment of metastatic breast cancer. Oncologist 9:617-632,
2004
36. Sledge GW, Neuberg D, Ingle J, et al: Phase III trial of doxorubicin,
paclitaxel and the combination of doxorubicin and paclitaxel as frontline chemotherapy for metastatic breast cancer (MBC): An Intergroup
trial (E1193). J Clin Oncol 21:588-592, 2003
37. O’Shaughnessy J, Miles D, Vukelja S, et al: Superior survival with capecitabine plus docetaxel combination therapy in anthracycline-pretreated patients with advanced breast cancer: Phase III trial results.
J Clin Oncol 20:2812-2823, 2002
38. O’Shaughnessy J, Nag S, Calderillo-Ruiz G, et al: Gemcitabine plus
paclitaxel versus paclitaxel as first-line treatment for anthracycline pretreated metastatic breast cancer: Interim results of a global phase III
study. Proc Am Soc Clin Oncol 22:7, 2003 (abstr)
39. Hancock W, Apffel A, Chakel J, et al: Integrated genomic/proteonomic
analysis. Anal Chem 71:742-748, 1999
40. Marshall A, Hodgson J: DNA chips: An array of possibilities. Nat Biotechnol 16:27-31, 1998
41. Page MJ, Amess B, Townsend RR, et al: Proteonomic definition of
normal human luminal and myoepithelial breast cells purified from
reduction mammoplasties. Proc Natl Acad Sci U S A 96:12589-12594,
1999
42. Pusztai L, Ayers M, Stec J, et al: Gene expression profiles obtained from
fine-needle aspirations of breast cancer reliably identify routine prognostic markers and reveal large-scale molecular differences between
estrogen-negative and estrogen-positive tumors. Clin Cancer Res 9:
2406-2415, 2003
43. Meng S, Tripathy D, Frenkel EP, et al: Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res 10:8152-8162,
2004
44. Lin DW, Holcomb I, Arfman EW, et al: Detection, isolation and initial
characterization of disseminated tumor cells following enrichment in
patients with prostate cancer. Proc Am Assoc Cancer Res 46:97, 2005
(abstr 419)
45. Kimura H, Fukumoto H, Park S, et al: Deletional mutant EGFR detected in circulating tumor-derived DNA from lung cancer patients
treated with gefitinib. Proc Am Assoc Cancer Res 46:112, 2005 (abstr
479)
46. Smirnov DA, Zweitzig DR, Foulk BW, et al: Global gene expression
profiling of circulating tumor cells. Cancer Res 65:4993-4997, 2005
47. Wolf AM, Wolf D, Steurer M, et al: Increase of regulatory T cells in the
peripheral blood of cancer patients. Clin Cancer Res 9:606-612, 2003
48. Liyanage UK, Moore TT, Joo H-G, et al: Prevalence of regulatory T cells
is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 169:27562761, 2002
49. Pockaj BA, Basu GD, Pathangey LB, et al: Reduced T-cell and dendritic
cell function is related to cyclooxygenase-2 overexpression and prostaglandin E2 secretion in patients with breast cancer. Ann Surg Oncol
11:328-339, 2004
50. Dunn GP, Old LJ, Schreiber RD: The three Es of cancer immunoediting.
Annu Rev Immunol 22:329-360, 2004
51. Blixt O, Head S, Mondala T, et al: Printed covalent glycan array for
ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci
U S A 101:17033-17038, 2004
52. Huflejt M, Cristofanilli M, Shaw L, et al: Detection of neoplasia-specific
clusters of anti-glycan antibodies in sera of breast cancer patients using
a novel glycan array. Proc Am Assoc Cancer Res 46:1312, 2005 (abstr
5578)