Imaging, Diagnosis, Prognosis

Analysis of the MammaPrint Assay in a Predominantly Postmenopausal Cohort Ben S. Wittner,1, 2 Dennis C. Sgroi,1, 3 Paula D. Ryan,1, 2 Ta ko J. B r uin s m a , 4 Annuska M. Glas,4 Anitha Male,1, 3 Sonika Dahiya,1, 3 Karleen Habin,1Rene Bernards,4,5 Daniel A. Haber,1, 2 LauraJ. Van’t Veer,4,6 and Sridhar Ramaswamy1, 2

Abstract Purpose: Most node-negative breast cancer patients are older and postmenopausal and are increasingly being offered adjuvant despite their low overall risk of distant relapse. A molecular diagnostic test with high negative predictive value (NPV) for distant in this subgroup would spare many older breast cancer patients adjuvant treatment. Experimental Design:We determined the NPVand positive predictive value of the MammaPrint assay in breast cancer patients who were consecutively diagnosed and treated at the Massachu- setts General Hospital between 1985 and 1997. Primary tumors from 100 patients with node- negative, invasive breast cancer (median age, 62.5 years; median follow-up, 11.3 years) were subjected to MammaPrint analysis and classified as being at either low or high risk for distant metastasis. Results: The MammaPrint 70-gene signature displayed excellent NPV as in previous studies, correctly identifying 100% of women at low risk for distant metastases at 5 years. However, this assay had a lower positive predictive value (12% at 5 years) than previously observed. Conclusions: The MammaPrint assay was originally designed to identify younger breast cancer patients at low risk for distant metastasis, who might consequently be spared systemic treatment. We show here that the same signature has a very high NPV for distant recurrence after adjuvant treatment in older breast cancer patients.

Breast cancer is clinically heterogeneous. Patients differ widely are imperfect (3, 4). Alarge number of additional factors have with respect to natural history and response to treatment (1). been evaluated for determining likely prognosis or treatment This clinical heterogeneity is probably due to the varying response, but most have limited power and few reflect the mutational spectrum or cell type of origin of tumors and genetics of the disease (5). complex genetic differences among individuals (2). These DNAmicroarrays have recently been used to obtain genome- factors in combination influence the expression of many genes wide views of human tumor gene expression, and these studies involved in tumor growth, invasion, metastasis, and survival. have identified cancer biomarkers with diagnostic, prognostic, Although consensus criteria, based on clinical and histopath- and predictive potential in a wide variety of solid tumors (6). ologic features [age, tumor size, histologic tumor type, Breast cancer has proven particularly fertile ground for micro- pathologic grade, estrogen receptor (ER) status, and axillary array-based biomarker discovery, and recent studies have lymph node status], are currently used to assess risk of distant suggested that the clinical behavior of a patient’s cancer is relapse in patients with breast cancer, these clinical measures encoded in the gene expression profile of their primary tumor (7–9). Members of our group initially reported a 70-gene prognostic Authors’ Affiliations: 1Massachusetts General Hospital Cancer Center; microarray expression signature in primary tumors from Departments of 2Medicine and 3Pathology, Harvard Medical School, Boston, patients with breast cancer who were diagnosed and treated 4 5 Massachusetts and Agendia BV; Divisions of Molecular Carcinogenesis and between 1983 and 1996 at the Netherlands Cancer Institute 6Diagnostic Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands Received 10/24/07; revised 12/24/07; accepted 2/1/08. (NKI; ref. 9). Subsequently, retrospective validation studies Grant support: Avon Foundation (Massachusetts General Hospital Cancer showed that this 70-gene expression signature indeed can be Center) and Dutch National Genomics Initiative ‘‘Cancer Genomics Center’’ used to classify younger patients, with either node-positive or (Netherlands Cancer Institute). node-negative disease, into groups with significantly different The costs of publication of this article were defrayed in part by the payment of page probabilities of remaining metastasis-free (10, 11). Moreover, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the prognostic value of the 70-gene signature is additive to Note: Supplementary data for this article are available at Clinical Cancer Research currently used St. Gallen (3) or NIH (4) consensus criteria for Online (http://clincancerres.aacrjournals.org/). assessing risk of distant relapse. These initial observations Requests for reprints: Sridhar Ramaswamy, Massachusetts General Hospital suggest that the 70-gene signature is a powerful predictor of Cancer Center, 185 Cambridge Street, Boston, MA 02114. Phone: 617-643-3140; Fax: 617-643-3170; E-mail: [email protected]. clinical outcome in younger women with early-stage breast F 2008 American Association for Cancer Research. cancer and that clinically defined high-risk patients with doi:10.1158/1078-0432.CCR-07-4723 microarray-defined ‘‘good prognosis’’ disease might actually

Clin Cancer Res 2008;14(10) May 15, 2008 2988 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2008 American Association for Cancer Research. MammaPrint Breast CancerAssay be at low risk for developing distant metastases. In theory, assessed as absent or present. ER expression was estimated using ER these patients may be spared adjuvant chemotherapy with transcript levels on the microarray for each tumor. Fifteen to thirty 30- A its associated toxicity risk. These issues are currently being m sections were used for RNAisolation. Total RNAwas isolated with addressed in a prospective, multicenter, in RNAzol B and dissolved in RNase-free water. Twenty-five micrograms of total RNAwere treated with DNase using the Qiagen RNase-free DNase Europe (12). kit and RNeasy spin columns for cleanup. RNAwas then dissolved in Most patients with breast cancer, however, are older than the RNase-free water to a final concentration of 0.2 Ag/AL. cohorts used to define and evaluate the 70-gene signature and Microarray expression profiling. RNAlabeling, microarray hybrid- + usually present with smaller, early-stage, ER tumors that tend ization, and scanning were done at Agendia using standardized to metastasize less frequently. In addition, such patients often methods and protocols (14). Briefly, cRNAwas generated by in vitro have comorbid disease that further complicates decisions about transcription with T7 RNApolymerase (Low Input Fluorescent Labeling the use of adjuvant chemotherapy. Despite this concern, many kit, Agilent Technologies) and labeled with Cy3 or Cy5 (Cy Dye, Perkin- older patients are increasingly being offered adjuvant chemo- Elmer). Cy-labeled cRNAfrom one breast cancer tumor was mixed with therapy. Amolecular test that can accurately identify older the same amount of reverse-color Cy-labeled product from a standard patients at low risk of distant metastasis, who could then be ‘‘mamma-reference’’ pooled RNAcontrol. Labeled cRNAswere hybrid- ized to an eight-pack Agendia MammaPrint microarray using standard spared chemotherapy toxicity, might therefore have clinical protocols (Agilent Oligo Microarray kit, Agilent Technologies). This value in selected circumstances. To explore these issues, we did microarray contains eight identical subarrays, each containing 60-mer a retrospective evaluation of the 70-gene MammaPrint assay in probes for the 70 prognosis genes in triplicate. Each sample was 100 older patients with node-negative breast cancer who were hybridized twice to do dye swaps. Fluorescence intensities on scanned initially diagnosed and treated at the Massachusetts General images were quantified, and the values were corrected for the Hospital (MGH) between 1985 and 1997. background level and normalized. Standard quality control measures were applied to determine technically acceptable hybridizations and poor-quality hybridizations were repeated. Materials and Methods MammaPrint tumor classification. Tumor classification was done using the previously reported 70-gene classification model (9). Briefly, Study design. Study design, patient selection, histopathologic for each of the 100 tumors, we calculated the cosine correlation analysis of tumors, clinical annotation, clinical interpretation, RNA coefficient of the level of expression of the 70 prognosis genes with the isolation, microarray profiling, and statistical analysis were carried out previously determined average profile of these genes in breast tumors jointly at the MGH, the NKI, and Agendia. from patients with a good prognosis (9, 14). Apatient with a Patient selection. We first identified those patients for whom frozen correlation coefficient >0.4 (the threshold in the original NKI study primary tumor material was available in the MGH Department of of 78 tumors that resulted in a 10% rate of false-negative results) was Pathology Breast Tumor Bank. We next identified patients who had then assigned to the ‘‘good’’ signature/low-risk group, and all others been consecutively diagnosed and treated for lymph node–negative were assigned to the ‘‘poor’’ signature/high-risk group. (pN0), invasive breast cancer at the MGH between 1985 and 1997 and Adjuvant! Online tumor classification. Analysis of available clinico- for whom histopathologic and clinical information could be retrieved pathologic information for each of the 100 tumors with Adjuvant! 7 from the medical record (through September 2007). This information Online v8.0 yielded population-based estimates of 10-y relapse risk for was obtained under a protocol approved by the MGH Institutional each individual MGH patient. Input variables included age, comorbid- Review Board in accordance with federal human research study ity (set to ‘‘average for age’’), ER status, and tumor grade and size. guidelines. All clinical and histopathologic data were stripped of Statistical analysis. To determine the probability that patients personal identifiers. Information was collected on age and calendar year would remain free of distant metastases, we defined distant metastases of diagnosis, surgery, tumor (size, grade, histologic type, and ER status), as a first event to be a treatment failure; data on all other patients were nodal status, radiation treatment, hormonal therapy or chemotherapy, censored on the date of the last follow-up visit, local or regional disease and clinical follow-up, including local, locoregional, or distant recurrence, the development of a second primary cancer, including recurrences, second primary malignancies, and death or date of last contralateral breast cancer, and death from causes other than breast visit. Patients with prior malignancies (except nonmelanoma skin cancer. cancers) and those with questionable diagnoses about site of tumor Data on patients were analyzed from the date of diagnosis to either origin were excluded. Tumors with >50% tumor cellularity were the time of the first event or the date on which data were censored. subjected to RNAisolation and microarray profiling ( n = 100). Raw Survival data were compared using the log-rank test using the exact microarray data underwent further statistical analysis. form of the Mantel-Haenszel test. Confidence intervals for Kaplan- All patients in this study cohort had been treated with mastectomy or Meier survival curves were constructed using the log method and SE breast-conserving surgery, including axillary lymph node dissection, was calculated by the method of Tsiatis. Confidence intervals for followed by radiotherapy if indicated. Aproportion received adjuvant predictive values were computed by the method of Clopper and systemic therapy consisting of chemotherapy, hormonal therapy, or Pearson. All P values characterizing differences between proportions both. The median duration of follow-up was 11.8 y (range, 1.2-18.5) were determined by Fisher’s exact test. All calculations were done using 8 for the 91 patients without metastasis as a first event and 4.9 y (range, the R statistical package. Clinicopathologic information and Adjuvant! 1.6-13.0) for the 9 patients with metastasis as a first event. The median Online and MammaPrint results are available as Supplementary follow-up among all 100 patients was 11.3 y. Table S1. Histopathologic evaluation and RNA isolation. Tumors had been The NKI lymph node – negative cohort referred to throughout snap frozen in liquid nitrogen within 1 h after surgery and subsequently included 151 consecutive patients originally reported in 2002 (11), stored at -80jC. Histopathologic evaluation of all tumors was done which included 61 lymph node–negative patients originally reported centrally in the Department of Pathology at the MGH (by D.C.S.) separately in 2002 (9). using established and previously published clinical criteria (13). H&E- stained sections of frozen tumors, obtained before and after the sections used for RNAisolation, were evaluated for percent tumor cellularity and samples with <50% were excluded. Histologic grade was assessed 7 http://www.adjuvantonline.com/index.jsp using modified Scarff-Bloom-Richardson criteria. Vascular invasion was 8 http://www.R-project.org

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Table 1. Clinicopathologic features of MGH and node-negative NKI patients

Characteristics MGH, n (%) NKI, n (%) P Age (y) <40 4 (4) 36 (24) <0.001 40-44 6 (6) 42 (28) 45-49 14 (14) 49 (32) 50-54 7 (7) 24 (16) z55 69 (69) 0(0) Tumor size (cm) V2 72 (72) 82 (54) 0.005 >2 28 (28) 69 (46) Histologic grade I 5 (5) 34 (23) <0.001 II 54 (54) 46 (30) III 40(40) 71 (47) Medullary carcinoma 1 (1) 0(0) ER Negative 20 (20) 42 (28) 0.180 Positive 80 (80) 109 (72) Surgery Breast conserving 44 (44) 90 (60) 0.020 Mastectomy 56 (56) 61 (40) Chemotherapy Yes 21 (21) 6 (4) <0.001 No 79 (79) 145 (96) Hormonal therapy Yes 24 (24) 6 (4) <0.001 No 76 (76) 145 (96)

Results the 70-gene microarray test discriminates among tumors with differing metastatic propensity in this cohort despite use of Clinical characteristics of the MGH cohort. The MGH cohort adjuvant treatment (Fig. 2). Despite this trend, and most had a median age of 62.5 years, which was significantly older probably due to the low metastasis event rate in this cohort, than previously studied patients (P < 0.001; Table 1; ref. 11). there was no statistically significant difference in time to distant Statistical analysis revealed additional differences in tumor size, metastasis between patients classified with low-risk or high-risk histologic grade, and treatment with both adjuvant chemo- tumor signatures in the MGH cohort. In contrast, microarray therapy and hormonal therapy between the 100 MGH and 151 classification of tumors from node-negative NKI patients NKI node-negative patients (P < 0.005), but censoring rates in the two cohorts did not differ appreciably. These differences were associated with a strikingly lower rate of distant metastasis as a first event in the MGH cohort compared with the NKI node-negative cohort (P < 0.001; Fig. 1). Notably, the MGH and NKI node-negative cohorts did not differ significantly in overall survival, despite the low metastasis rate in MGH patients. This was due to death from causes other than breast cancer in the older MGH population (data not shown). Primary tumor classification. We next applied the 70-gene predictive model to gene expression profiles from all 100 patients in the MGH cohort. This model classified 27 patients into low-risk group and 73 patients into high-risk group (Table 2). As observed in previous studies, prediction values (i.e., correlations) for these tumors fell along a continuum rather than into discrete pools, suggesting complex, nondiscrete differences in gene expression among individual tumors (data not shown; refs. 9, 11). Patients classified as low risk by tumor gene expression tended to have smaller, lower-grade, ER+ tumors as a group compared with high-risk patients in univariate analysis but, importantly, did not differ significantly in the use of adjuvant treatment. Clinical prediction. Kaplan-Meier analysis for time to Fig. 1. Overall clinical outcome of node-negative MGH versus NKI patients. Kaplan-Meier analysis of time to metastasis as a first event for the MGH and metastasis as a first event revealed nonoverlapping confidence node-negative NKI cohorts. Green, MGH; black, NKI node negative; thin lines, 95% intervals between low- and high-risk patients, suggesting that confidence interval (95% CI); black lines, previously published data (11).

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Table 2. Clinicopathologic features of MGH patients classified as either high or low risk for distant metastasis using the 70-gene signature

Characteristics High-risk signature (n = 73), n (%) Low-risk signature (n = 27), n (%) P Age (y) <40 4 (5) 0 (0) 0.548 40-44 5 (7) 1 (4) 45-49 8 (11) 6 (22) 50-54 5 (7) 2 (7) z55 51 (70) 18 (67) Tumor size (cm) V2 48 (66) 24 (89) 0.025 >2 25 (34) 3 (11) Histologic grade I 2 (3) 3 (11) 0.011 II 35 (48) 19 (70) III 35 (48) 5 (19) Medullary carcinoma 1 (1) 0(0) ER Negative 20 (27) 0 (0) 0.001 Positive 53 (73) 27 (100) Surgery Breast conserving 33 (45) 11 (41) 0.821 Mastectomy 40(55) 16 (59) Chemotherapy Yes 17 (23) 4 (15) 0.420 No 56 (77) 23 (85) Hormonal therapy Yes 18 (25) 6 (22) 1.000 No 55 (75) 21 (78) Radiotherapy Combination 1 (1) 1 (4) 0.221 External beam 30(41) 7 (26) None 42 (58) 19 (70)

previously revealed statistically significant differences between Finally, using the Adjuvant! Online clinical decision-making high- and low-risk signature patients for time to distant tool, we determined population-based estimates of 10-year metastasis (P < 0.001; ref. 11). relapse risk for each individual MGH patient. By applying a Detailed examination of prediction results revealed that the threshold, we defined patients as being at low or high risk for negative predictive value (NPV) of the 70-gene signature was 100% in the MGH cohort, which was comparable with a NPV of 88% in the node-negative NKI cohort (Table 3A). Of 100 patients, 27 were classified as low risk and 73 as high risk by MammaPrint assay. Of the 27 low-risk patients, none had distant metastasis as a first event after initial diagnosis. Notably, the NPV of the 70-gene signature also remained 100% when strictly considering 5- or 10-year distant metastasis-free survival (Table 3B and C). Importantly, all MGH patients who developed distant metastasis as a first event had primary tumors with high-risk 70-gene expression signatures (n = 9). However, 64 additional patients were also classified as poor prognosis by the 70-gene test, which was unexpectedly high given what is generally thought to be the low-risk nature of older breast cancer patients. The positive predictive value (PPV) of the 70-gene signature was therefore only 12%, significantly lower than a previously observed PPV of 52% in node-negative NKI patients. The 70-gene signature was originally optimized for sensitivity by choosing a classification threshold of 0.4 for microarray correlation values, a cutoff that resulted in a 10% false-negative rate for positive prediction of distant metastasis (9). However, reanalysis using a range of alternative cutoffs for the 70-gene Fig. 2. Molecular classification of MGH patients. Kaplan-Meier analysis of time to metastasis as a first event for the MGH cohort based on classification with the predictive model did not reveal any that could yield a high PPV 70-gene signature. Red, MammaPrint high risk; blue, MammaPrint low risk; thin in the MGH cohort (data not shown). lines, 95% CI.

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Table 3. NPV and PPV [overall (A), 5-y (B), and 10-y (C)] for time to distant metastasis in MGH compared with node-negative NKI patients classified as either high or low risk for distant metastasis using the 70-gene signature

A Group Patients Distant metastases Disease-free P Predictive value PPV (95% CI) NPV (95% CI) MGH High-risk signature 73 9 64 0.108 12% (6-22%) 100% (87-100%) Low-risk signature 27 027 NKI lymph node negative High-risk signature 91 47 44 <0.001 52% (41-62%) 88% (77-95%) Low-risk signature 607 53

B

Group Patients Distant metastases Disease-free P 5-y predictive value (V5y) (>5 y) PPV (95% CI) NPV (95% CI)

MGH High-risk signature 60 7 53 0.192 12% (5-23%) 100% (81-100%) Low-risk signature 18 018 NKI lymph node negative High-risk signature 70 36 34 <0.001 51% (39-64%) 93% (82-98%) Low-risk signature 54 4 50

C

Group Patients Distant metastases Disease-free P 10-y predictive value (V10 y) (>10 y) PPV (95% CI) NPV (95% CI)

MGH High-risk signature 49 7 42 0.330 14% (6-27%) 100% (72-100%) Low-risk signature 11 011 NKI lymph node negative High-risk signature 58 42 16 <0.001 72% (59-83%) 75% (55-89%) Low-risk signature 28 7 21

NOTE: In determining 5- and 10-y values, the following were excluded: (a) patients with distant metastasis as a first event (after 5 or 10y), (b) patients without distant metastasis as a first event but with <5 (or 10) y of follow-up, and (c) patients with local or regional disease recurrence or the development of a second primary cancer as a first event. distant relapse. We chose the threshold that simultaneously patients who might be spared adjuvant chemotherapy with its maximized the number of patients classified as low risk while associated toxicity (10, 11). Most patients with breast cancer, maintaining a NPV of 100%. With this threshold, we found however, are older and have comorbid disease that complicates that MammaPrint classified an additional 21 patients (beyond decisions about the use of adjuvant chemotherapy. The Adjuvant! Online) as low risk, and importantly, none of these potential role of the MammaPrint assay in the clinical care of patients developed distant metastasis as a first event (Fig. 3A). these patients remains unclear. Moreover, even when strictly considering 10-year distant We studied the 70-gene signature in an older breast cancer metastasis-free survival, we found that MammaPrint classified cohort who were diagnosed and treated at the MGH, purposely an additional five patients (beyond Adjuvant! Online) as low focusing on patients with lymph node–negative disease like risk, and again, none developed distant metastasis as a first those with whom MammaPrint was initially discovered and event (Fig. 3B). These results suggest that the 70-gene validated (9–11). These MGH patients had a median age of signature might provide useful and additive information to 62.5 years with predominantly small and low-grade tumors. an important clinicopathologic risk model in older breast Forty-five percent of MGH patients also received some form of cancer patients. systemic treatment, reflecting known practice patterns among U.S. oncologists. Consistent with these differences, the MGH Discussion cohort had low distant recurrence rates. The 70-gene signature displayed a remarkably high NPV The 70-gene MammaPrint assay has been retrospectively (100%) in this cohort, which is consistent with earlier findings validated as a prognostic tool, suggesting that younger in younger cohorts (10, 11). Interestingly, MammaPrint- breast cancer patients with intrinsically low-risk disease can based classification also seemed to provide additive informa- be molecularly identified and that this information can be tion to Adjuvant! Online–based risk determination. Although combined with clinical risk profiling to more accurately identify the MammaPrint assay was originally designed to identify

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and clinical outcome could in part be explained by the significant use of adjuvant treatment in these patients. However, the proportional risk reduction for recurrent disease from adjuvant chemotherapy has been estimated to be f20%, and for hormonal therapy f30%, in postmenopausal patients (1). Thus, even in the complete absence of adjuvant treatment, the event rate in the MGH cohort would likely only have doubled to f18%, still far below what might be predicted through molecular classification with the MammaPrint assay. This finding that the 70-gene signature molecularly classifies a significant percentage of older age breast cancer patients as ‘‘high risk,’’ of which few develop metastatic disease, may offer insight into the process of metastases. Following Paget’s hypothesis that metastasis depends both on the ‘‘seed’’ (the cancer cell itself) and the ‘‘soil’’ (the ‘‘host’’), our findings raise the intriguing hypothesis that most breast cancers in older patients are intrinsically high risk but that this intrinsic metastatic capacity does not become manifest due to other (possibly host) factors in these predominantly postmenopausal patients. Because older women with newly diagnosed breast cancer are increasingly being offered adjuvant treatment, in contrast with historical practice patterns, larger studies of the 70-gene signature should be done to determine whether the Mam- Fig. 3. Classification of MGH patients using Adjuvant! Online compared with maPrint assay (with its excellent NPV) is clinically useful for MammaPrint. Adjuvant! Online ^ based relapse risk for individual patients classified as MammaPrint low or high risk for (A) all 100 patients, (B) strictly considering pretreatment determination of intrinsic risk in older breast 10-y distant metastasis-free survival. Solid square, patient with distant metastasis cancer patients. An open question in light of the poor PPV as a first event; circle, patient without distant metastasis as a first event; + in circle, of MammaPrint in older patients is whether there exist addi- patient identified as low risk by MammaPrint beyond Adjuvant! Online ^ based classification; horizontal line, threshold that simultaneously maximizes the number tional, clinically useful gene expression signatures for posi- of patients classified as low risk while maintaining a NPV of 100%. tively predicting distant metastasis risk in the postmenopausal breast cancer population. Clearly, larger studies on postmen- younger breast cancer patients at low risk for distant metastasis opausal breast cancer cohorts will be required to address who might consequently be spared systemic treatment, these this issue. results show that the same signature might identify older breast cancer patients at lower risk for distant recurrence at initial diagnosis. Disclosure of Potential Conflicts of Interest In contrast, the MammaPrint assay had an extremely low PPV L.J.Van’t Veer, A.M. Glass, and T. Bruinsma are employed by Agendia BV. L.J. (12%), significantly lower than that previously observed in Van’t Veer has an ownership interest in MammaPrint. L.J.Van’t Veer and R. Bernards node-negative NKI patients (52%), thus resulting in statistically are named inventors on a patent to use microarray technology to ascertain breast insignificant differences in overall survival between low- and cancer prognosis and hold equity interests in Agendia BV. high-risk patients. Remarkably, 73% of MGH patients were classified as high risk (including all nine patients who eventually developed metastasis), which was strikingly discor- Acknowledgments dant with the low overall metastasis rate in this cohort (9%). We thank Marc van de Vijver for helpful discussions and Arno Floore Because 45% of all patients received some adjuvant and Agendia laboratory staff for excellent technical work in generating micro- treatment, this discrepancy between molecular classification array data.

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Ben S. Wittner, Dennis C. Sgroi, Paula D. Ryan, et al.

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