Abstract
Mesothelin is a cell surface associated antigen expressed on mesothelial cells and in some malignant neoplasms. Mesothelin-targeted therapies are in phase I/II clinical trials. The clinicopathologic and prognostic significance of mesothelin expression in triple negative breast carcinomas (TNBC) has not been fully assessed. We evaluated the expression of mesothelin and of basal markers in tissue microarrays of 226 TNBC and 88 non-TNBC and assessed the clinicopathologic features of mesothelin-expressing breast carcinomas. Furthermore, we investigated the impact of mesothelin expression on the disease-free and overall survival of patients with TNBC. We found that mesothelin expression is significantly more frequent in TNBC than in non-TNBC (36% vs 16%, respectively; p = 0.0006), and is significantly correlated with immunoreactivity for basal keratins, but not for EGFR. Mesothelin-positive and mesothelin-negative TNBC were not significantly different by patients’ race, tumor size, histologic grade, tumor subtype, lymphovascular invasion and lymph node metastases. Patients with mesothelin-positive TNBC were older than patients with mesothelin-negative TNBC, developed more distant metastases with a shorter interval, and had significantly lower overall and disease-free survival. Based on our results, patients with mesothelin-positive TNBC could benefit from mesothelin-targeted therapies.
Introduction
Mesothelin (MSLN) is a 40-kDa glycosylphosphatidylinositol-linked cell surface antigen present in normal mesothelial cells and overexpressed in several human malignancies, including mesothelioma, pancreatobiliriary, ovarian and lung adenocarcinomas [1]–[8]. In mesothelioma MSLN promotes tumor cell invasion by increased MMP-9 secretion [9]. MSLN also binds CA-125/MUC16 with very high affinity and may contribute to the adhesion of tumor cells in peritoneal metastasis [10], [11]. Mesothelin expression increases resistance to TNFα-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 expression in pancreatic carcinoma cell lines [12]. MSLN-overexpressing pancreatic cancer cell lines showed increased cyclin E and cyclin dependent kinase 2 expression, resulting in increased cell proliferation and cell cycle progression [13]. Membrane-bound MSLN is also released into body fluids and its use as a potential serum tumor marker is currently under investigation [14], [15]. MSLN is an attractive target for targeted therapy due to its limited distribution in normal tissues, high immunogenicity, and elevated expression in several human malignancies [16]. Several ongoing clinical trials in patients with ovarian cancer, with pancreatic cancer or with mesothelioma suggest that MSLN-specific T-cell responses have a beneficial effect [16]–[22].
Triple negative breast carcinomas (TNBC) are invasive breast carcinomas that lack expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). They constitute approximately 10–17% of all invasive breast carcinomas and tend to be more common in young women [23]–[28], and often of African-American or Hispanic ethnicity [27], [29], [30]. Patients with TNBC have an aggressive clinical course [23], [26]–[29], [31] characterized by short survival after the first metastatic event [26], [29] and death within 5 years of the initial diagnosis [26], [28]. Approximately 71–80% of TNBC are basal carcinomas by gene expression profiling [32]–[36]. Basal TNBC tend to have more aggressive clinical course than non-basal TNBC, with even earlier disease recurrence, often times with lung and/or brain metastases [31], [37]–[40], shorter disease free survival and breast cancer specific survival [41]. At present no effective targeted therapy is available for treatment of TNBC [42] and significant efforts are currently focused on the identification of novel therapeutic targets for these tumors.
In this study, we assessed the expression of MSLN in a large cohort of TNBC and non-TNBC. We also correlated MSLN overexpression with clinicopathologic features and basal-like immunophenotype of TNBC [39], [43]. Furthermore, we evaluated MSLN as a potential prognostic marker in TNBC by correlating its expression with clinical outcome.
Materials and Methods
Tissue microarrays
Tissue microarrays (TMAs) containing 226 TNBC and 88 non-TNBC were used in this study. A breast carcinoma was defined as TNBC if nuclear staining for ER and PR was detected in less than 1% of the tumor cells, and HER2 was negative (0 or 1+) by immunohistochemistry (IHC) or equivocal (2+) by IHC and showed no HER2 gene amplification by fluorescence in situ hybridization (FISH) [44], [45]. The TNBC cases were obtained from consecutive patients who underwent surgical excision of the primary breast carcinoma at our center between 2002 and 2006 and for which slides and blocks were available for the study. A TMA of non-TNBC from consecutive patients treated at our institution in 2004 was used for reference. Triplicate 0.6-mm diameter cores from formalin- fixed, paraffin-embedded blocks were used to construct the TMAs. Only carcinomas spanning 0.5 cm or larger were used for the TMAs, to ensure the availability of residual carcinoma for possible future clinical use. Tumor size, grade and the presence or absence of lymphovascular invasion (LVI) were extracted from the original pathology reports. Collection or study of existing data - application for exemption from IRB/PB review (including waiver of HIPAA authorization and informed consent) was reviewed and approved by the Institutional Review Board (IRB) and Human Biospecimen Utilization Committee.
Immunohistochemistry
We assessed MSLN expression on TMAs by IHC (Vector Lab, USA, clone 5B2, 1∶50, mouse monoclonal Ab, lgG1). Mesothelioma tissue was used as positive control. Immunohistochemical stains for ER (Ventana Medical Systems, Inc. USA, clone 6F11, rabbit monoclonal Ab, lgG1), PR (Ventana Medical Systems, Inc. USA, clone 1E2, rabbit monoclonal Ab, lgG1), HER2 (Ventana Medical Systems, Inc. USA, clone 4B5, rabbit monoclonal Ab lgG1), and basal-like markers, including CK5/6 (Dako, clone D5/16B4, mouse monoclonal antibody, 1∶50 dilution), CK14 (AbCam, clone LL002, mouse monoclonal antibody, 1∶100 dilution), EGFR (Thermoscientific, clone EP38Y, rabbit monoclonal antibody, 1∶50 dilution) were performed with appropriate controls. Carcinomas showing any cytoplasmic staining for CK5/6 and CK14 were regarded as positive for these markers. Scoring of EGFR membranous reactivity followed the ASCO/CAP criteria for HER2 [45]; cases with EGFR staining intensity of 2+ or 3+ were regarded as positive. Two pathologists blinded to the patients’ clinical outcomes separately assessed MSLN expression in all TNBC and non-TNBC and concordant scores were obtained. A previously described [9] semi-quantitative scoring system was used to score MSLN reactivity. Briefly, the percentage of tumor cells with MSLN staining was assigned a score of 0 (<1% tumor cells), 1 (1%–50% tumor cells), or 2 (>50% tumor cells). Staining intensity was scored as 0 (none), 1 (weak), 2 (moderate) or 3 (strong). The final MSLN score was calculated by the sum of the percentage and intensity scores of each tumor. Any case with final MSLN score ≥3 was classified as positive. MSLN expression was correlated with age at diagnosis, tumor size, grade, LVI, regional lymph node involvement, subsequent distant metastases, interval to metastases, site of metastases, and survival.
Statistical analysis
The relationship between MSLN staining, basal-like phenotype, and clinicopathologic features was assessed using Fisher’s exact test. Five-year estimates of overall survival (OS) and disease-free survival (DFS) by MSLN positivity, basal-like phenotype and clinicopathologic features were calculated using Kaplan-Meier methods. Differences between the Kaplan-Meier curves were tested using log-rank test. A p-value <0.05 was considered as statistically significant.
Results
Clinicopathologic features
Patients with TNBC and non-TNBC were similar in age. The mean age at diagnosis of patients with TNBC was 55 years (range, 54–57). The mean age of patients with non-TNBC was 54 years (range, 51–57). Among patients with TNBC, 163 (72%) were White, 48 (21%) were Black, 13 (6%) were Asian, and 2 (1%) were of other races. Among patients with non-TNBC, 75 (85%) were White, 10 (11%) were Black, 1 (1%) was Asian, and 2 (2%) were of other races. There was a higher proportion of White patients in non-TNBC group comparing to TNBC group (85% vs 72%, p = 0.0184). Although not statistically significant, there was a trend of higher proportion of Black patients in TNBC group comparing to non-TNBC group (21% vs 11%, p = 0.0515). The average tumor size of TNBC and non-TNBC was 2.2 cm (range, 0.7–28) and 1.8 cm (range, 0.9–11), respectively. TNBC had significantly larger tumor size and higher histologic and nuclear grade compared to non-TNBC ( Table 1 ). The incidence of LVI was similar in TNBC and non-TNBC, although TNBC had slightly higher rate of axillary lymph node metastases ( Table 1 ). The histologic sub-types of TNBC included: invasive ductal carcinoma not otherwise specified (IDC-NOS) (n = 218), metaplastic carcinoma (n = 5), pleomorphic lobular carcinoma (n = 1) and mixed ductal and pleomorphic lobular carcinoma (n = 2). Among the non-TNBC, 76 were IDC-NOS type. The remaining non-TNBC were invasive lobular carcinoma (n = 8), mixed ductal and lobular carcinoma (n = 3) and invasive mucinous carcinoma (n = 1).
Table 1. Clinicopathologic Features of TNBC versus non-TNBC.
| TNBC (n = 226) | non-TNBC (n = 88) | p value | |
| Mean age (years) [range] | 55 [54–57] | 54 [51–57] | 0.39 |
| Race | |||
| White | 163 (72%) | 75 (85%) | 0.0184 |
| Black | 48 (21%) | 10 (11%) | 0.0515 |
| Asian | 13 (6%) | 1 (1%) | 0.1236 |
| Other | 2 (1%) | 2 (2%) | 0.3132 |
| Mean tumor size (cm) [range] | 2.2 [0.7–28] | 1.8 [0.9–11] | 0.04 |
| Histologic grade | <0.00001 | ||
| 1 | 0 (0%) | 0 (0%) | |
| 2 | 9 (4%) | 25 (28%) | |
| 3 | 217 (96%) | 62 (71%) | |
| Nuclear grade | <0.00001 | ||
| 1 | 0 (0%) | 0 (0%) | |
| 2 | 11 (5%) | 46 (52%) | |
| 3 | 215 (95%) | 42 (48%) | |
| Lymphovascular invasion | 83 (37%) | 32 (36%) | 0.53 |
| Lymph node metastasis | 129 (57%) | 39 (44%) | 0.044 |
Abbreviations: TNBC = triple negative breast carcinoma.
Immunohistochemical analysis of MSLN expression
Immunohistochemical stain for MSLN yielded uniform, strong cytoplasmic and membranous reactivity in the mesothelioma tumor cells used as positive control. Minimal background staining was detected in inflammatory cells or benign stroma. In the breast cancer specimens, MSLN showed variable staining intensity and percentage in the tumors cells ( Fig. 1 , Table 2 ). A significantly higher proportion of TNBC (82/226, 36%) showed MSLN score ≥3 than non-TNBC (14/88, 16%) (Fisher’s exact test, p = 0.0006) ( Tables 2 and 3 ).
Figure 1. Examples of immunohistochemical staining for MSLN in TNBC.
A and B) MSLN negative. A) No staining. B) Percentage score = 1 (1–50%), intensity score = 1 (weak), final score = 2. C–F) MSLN positive. C) Percentage score = 1 (1–50%), intensity score = 2 (moderate), final score = 3. D) Percentage score = 2 (>50%), intensity score = 1 (weak), final score = 3. E) Percentage score = 2 (>50%), intensity score = 2 (moderate), final score = 4. F) Percentage score = 2 (>50%), intensity score = 3 (strong), final score = 5.
Table 2. Distribution of MSLN scores in TNBC versus non-TNBC.
| MSLN Score | 0 | 1 | 2 | 3 | 4 | 5 |
| TNBC (n = 226) | 77 (34%) | 28 (13%) | 39 (17%) | 43 (19%) | 27 (12%) | 12 (5%) |
| Non-TNBC (n = 88) | 48 (55%) | 15 (17%) | 11 (13%) | 6 (7%) | 8 (9%) | 0 (0%) |
Abbreviations: MSLN = mesothelin; TNBC = triple negative breast carcinomas.
Table 3. MSLN correlates significantly with TNBC status.
| TNBC (n = 226) | Non-TNBC (n = 88) | ER/PR(+)/HER2(−) | ER/PR(+)/HER2(+) | p-value | |
| MSLN (+) | 82 (36%) | 14 (16%) | 10 (11%) | 4 (5%) | *0.0006 |
| MSLN (−) | 144 (64%) | 74 (84%) | 57 (65%) | 17 (19%) |
Abbreviations: MSLN = mesothelin; TNBC = triple negative breast carcinomas. *comparing TNBC with non-TNBC.
Among TNBC, MSLN was positive in 80 of 218 IDC-NOS and 2 out of 5 metaplastic carcinomas. MSLN expression correlated with slightly older age at diagnosis, but not with race, tumor size, histologic or nuclear grade, LVI or lymph node metastases ( Table 4 ). MSLN positivity also significantly correlated with basal keratins CK5/6 (56/80, 70%; p<0.00001) and CK14 (29/72, 40%; p = 0.017), but not with EGFR (57/78, 73%; p = 0.87) ( Table 5 ).
Table 4. MSLN expression and clinicopathological characteristics of TNBC.
| MSLN(+) (n = 82) | MSLN(−) (n = 144) | p value | |
| Mean patient age (years) | 58.0 | 54.0 | 0.043 |
| Race | |||
| White | 58 (71%) | 105 (73%) | 0.7588 |
| Black | 21 (26%) | 27 (19%) | 0.2398 |
| Asian | 3 (4%) | 10 (7%) | 0.3845 |
| Other | 0 | 2 (1%) | 0.5356 |
| Mean tumor size (cm) | 2.3 | 2.2 | 0.70 |
| Histologic grade | 0.49 | ||
| 1 | 0 (0%) | 0 (0%) | |
| 2 | 2 (2%) | 7 (5%) | |
| 3 | 80 (98%) | 137 (95%) | |
| Nuclear grade | 0.20 | ||
| 1 | 0 (0%) | 0 (0%) | |
| 2 | 2 (2%) | 9 (6%) | |
| 3 | 80 (98%) | 135 (94%) | |
| Lymphovascular invasion | 36 (44%) | 47 (33%) | 0.11 |
| Lymph node(+) | 40 (49%) | 89 (62%) | 0.07 |
Abbreviations: MSLN = mesothelin; TNBC = triple negative breast carcinoma.
Table 5. MSLN and basal markers expression in TNBC.
| Basal marker | TNBC MSLN(+) (n = 82) | TNBC MSLN(−) (n = 144) | p value |
| CK5/6(+) | 56/81 (69%) | 50/142 (35%) | <0.0001 |
| CK14(+) | 29/72 (40%) | 33/137 (24%) | 0.017 |
| CK5/6 and/or CK14(+) | 63/76 (83%) | 63/137 (46%) | <0.0001 |
| EGFR(+) | 57/78 (73%) | 102/139 (73%) | 0.88 |
| CK5/6 and EGFR(+) | 43/74 (58%) | 53/134 (39%) | 0.013 |
| CK5/6 and/or EGFR(+) | 57/78 (73%) | 102/137 (75%) | 0.82 |
Abbreviations: MSLN = mesothelin; TNBC = triple negative breast carcinomas. *CK5/6 status unknown for 3 cases, CK14 status unknown for 17 cases, EGFR status unknown for 9 cases.
Among non-TNBC, 67 cases were ER and/or PR positive and HER2 negative, 21 were ER and/or PR positive and HER2 positive. MSLN was positive in 13 out of 76 IDC-NOS and one mixed ductal and lobular carcinoma. No statistically difference in the prevalence of MSLN expression was observed between ER/PR positive, HER2 negative and ER/PR positive, HER2 positive tumors ( Table 3 ).
Correlation with clinical outcome
The median follow-up time was 5.3 years (range 0.7–8.2). We observed a general trend towards increased frequency of distant metastases in patients with MSLN-positive TNBC, compared to patients with MSLN-negative TNBC and non-TNBC. Patients with MSLN-positive TNBC also had shorter interval to metastases and showed a greater propensity to develop brain metastasis ( Table 6 ).
Table 6. Correlation of MSLN expression and Distant Metastasis.
| TNBC MSLN(+) | TNBC MSLN(−) | |
| Patients distant metastasis | 16/70* (23%) | 12/128* (9%) |
| Median interval to metastasis (month) [95% CI] | 19.2 [13.5–24.9] | 35.2 [23.8–46.6] |
| Site of metastases | ||
| Bone | 2 (13%) | 2 (17%) |
| Brain | 10 (63%) | 4 (33%) |
| Liver | 2 (13%) | 2 (17%) |
| Lung | 8 (50%) | 4 (33%) |
| Multiple | 6 (38%) | 3 (25%) |
Abbreviations: MSLN = mesothelin; TNBC = triple negative breast carcinomas. *Distant metastasis status unknown in 28 TNBC cases.
The 5-year Kaplan-Meier survival estimates confirmed that TNBC had significantly shorter overall probability of survival (0.82; 95% Cl: 0.75–0.87), compared to non-TNBC (0.959; 95% Cl: 0.895–0.984) ( Fig. 2 ). Nonetheless, among patients with TNBC, MSLN positivity correlated with significantly shorter OS (0.659; 95% Cl: 0.515–0.770 versus 0.913; 95%CI: 0.838–0.954) ( Fig. 3 ), and DFS (0.665; 95%CI: 0.536–0.766 versus 0.865; 95%CI: 0.785–0.916) ( Fig. 4 ). Node-positive and MSLN-positive TNBC fared the worst, with 5-year OS of 0.564 (95%CI: 0.348–0.733), compared to node-positive and MSLN-negative TNBC (0.865; 95%CI: 0.699–0.943), and to node-negative and MSLN-positive TNBC (0.758; 95%CI: 0.572–0.871) ( Fig. 5 ). Analysis of the survival data by log-rank test suggests that the negative survival impact of MSLN is independent of lymph node status (log rank test, p = 0.0003). Basal-keratin (CK5/6 and/or CK14) positive and MSLN-positive cases had significantly lower 5-year OS (0.617; 95%CI: 0.449–0.747) compared to all of the other groups ( Fig. 6 ). The survival impact of MSLN also appears independent of basal-like phenotype.
Figure 2. Decreased 5-Year overall survival in TNBC compared to non-TNBC (n = 314, p = 0.0001).
Figure 3. MSLN expression in TNBC correlates with significantly decreased overall survival (p<0.0001, n = 198).
Figure 4. MSLN expression in TNBC correlates with significantly decreased disease-free survival (p = 0.0003, n = 198).
*Distant metastasis (DM) status unknown in 28 TNBC cases.
Figure 5. The difference in overall survival for MSLN(+) TNBC is independent of lymph node (LN) status (log rank p = 0.0003, n = 188).
Figure 6. The difference in overall survival for MSLN(+) TNBC is independent of basal marker expression (log rank p = 0.0002, n = 188).
Basal-keratin(+): CK5/6 and/or CK14 positive; basal-keratin(−): CK5/6(−) and CK14(−).
Discussion
Most TNBC have an aggressive clinical course and do not respond to current therapies targeting ER, PR, and HER2. MSLN is a cell-surface antigen overexpressed in several human malignancies and constitutes a promising immunotherapy target [1]–[8], which could provide a much needed therapeutic option for patients with TNBC.
Several published studies have evaluated MSLN expression in different tumor types, using various scoring systems to quantify the immunohistochemical expression of MSLN. Argani et al [3] categorized any tumor with ≥1% staining of any intensity as “positive” for MSLN and any staining between 1%–25% as “focal”. Swierczyncki et al [4] also used similar cut-off percentage, but required “moderate to strong” labeling intensity. In their study, cases with “focal” staining and “positive” cases were combined together for statistical analysis [4]. Ho et al [6] did not explicitly specify an immunohistochemical cut-off score in their analysis. Frierson et al [8] scored MSLN immunoreactivity as absent, 1+ (<10% positive cells), 2+ (10%–50% positive cells), or 3+ (>50% positive cells). Tchou et al [46] evaluated 99 primary breast cancer samples by IHC and regarded as “positive” cases with MSLN staining in greater than 1% of tumor cells. The median proportion of cells positive for MSLN was 10% (range of 5–80%), with varied staining intensity [46]. Using these criteria, the authors reported MSLN overexpression in 67% (29/43) of TNBC, compared to only 3% and 4% of ER-positive and HER2-positive tumors respectively [46].
Table 7 summarizes studies evaluating the prognostic significance of mesothelin expression by immunohistochemistry in a variety of adenocarcinomas [47]–[55]. Although there is no consensus on the scoring criteria of immunohistochemical staining for mesothelin, “high level” of mesothelin expression was significantly associated with worse outcome [48], [50], [52], [53], [55].
Table 7. Summary of published studies of MSLN immunohistochemical studies and correlation with survival.
| Study | MSLN antibodyand dilution | Scoring methods | TMA vs wholetissue section | Cancer type | Correlation withsurvival |
| Yenet al [47] | clone 5B2,Promega, 1∶500 | 0: <5%; 1+: 5% to 50%; 2+: 51%to 75%; 3+: 76% to 95%; 4+: >95% | Wholetissue section | Ovarian serouscarcinoma | Related to favorableoverall survival |
| Einamaet al [48] | clone 5B2,Novocastra, 1∶50 | High levels: >50% with any intensity,or moderate to strong intensity ofany percentage. Low level: <50%with weak intensity or absent | Wholetissue section | Pancreaticcancer | Co-expression ofmesothelin and CA-125(high levels for both) isassociated with poor prognosis |
| Babaet al [49] | clone HBME1, DAKO, 1∶50 | 0: <5%; 1∶5–50%; 2∶51–100% | Wholetissue section | Gastriccancer | Correlated withprolonged survival |
| Shimizuet al [50] | clone 5B2,Novocastra, 1∶20 | Score = Staining intensity (0, 1, 2,3)×percentage. Cut-off was setat the medial score. High: >median. Low: <median | Wholetissue section | Pancreatic ductadenocarcinomas | Co-expression ofmesothelin and MUC16(high levels for both) isassociated with poorprognosis |
| Winteret al [51] | Vector Labs,1∶100 | 0: <10%; 1+: 11–25%; 2+:26–75%; 3+: >75% | Tissuemicroarray | Pancreatic adenocarcinomas | Significant predictors ofearly cancer-specific mortality |
| Kawamataet al [52] | clone 5B2,Novocastra, 1∶50 | High levels: >50% with any intensity,or moderate to strong intensity ofany percentage. Low level: <50%with weak intensity or absent | Wholetissue section | Extrahepatic bile duct cancer | High-level expressionwas correlated with liver metastasis andpoor patient outcome |
| Einamaet al [53] | clone 5B2,Novocastra, 1∶50 | Positive: >50% with any intensity,or moderate to strong intensity ofany percentage. Negative: <50%with weak intensity or absent | Wholetissue section | Gastriccancer | Poor prognosticfactor |
| Parinyanitikulet al [54] | clone 5B2, Novocastra, 1∶30 | H score = staining intensity (0, 1, 2,3) ×percentage. Positive: H score >10 | Tissuemicroarray | Triple negativebreast cancer | Mesothelin expression did notcorrelate with survival outcomes |
| Kachala et al[55] | clone 5B2,Vector lab, 1∶200 | Sum score = Intensity (0, 1, 2, 3)+percentage(0: staining absent; 1∶1%–50%; or2∶51%–100%). High: sum score5. Low: sum score 0–4 | Tissuemicroarray | Lung adenocarcinoma | High expressioncorrelates withworse survival |
| Currentstudy | clone 5B2,Vector lab, 1∶50 | Sum score = Intensity (0, 1, 2,3)+percentage (0: staining absent;1∶1%–50%; or 2∶51%–100%).Positive: sum score >3 | Tissuemicroarray | Triple negativebreast cancer | Correlates withworse survival |
In our study of 226 TNBC treated at our institution we found MSLN to be overexpressed in 36% of cases. The rate of MSLN-positive TNBC in our study may appear relatively low compared to that reported by Tchou et al [46], because we regarded as MSLN-positive in only cases that showed substantial MSLN reactivity, including at least moderate staining intensity or if weak intensity in more than 50% of the tumor cells. Our scoring criteria is similar to that used in previous studies [48], [52], [53] demonstrating prognostic significance of mesothelin expression by immunohistochemistry in pancreatobiliary and gastric carcinomas. The use of a high cutoff of MSLN positivity, albeit arbitrarily selected, identifies cases that are more likely to be targetable to treatment with anti-MSLN. Interestingly, in our series, the use of strict criteria of MSLN- positivity identified carcinomas with significantly worse clinical behavior.
A recent study by Parinyanitikul and colleagues showed no correlation between MSLN expression and survival outcomes in triple negative breast carcinomas [54]. MSLN staining was quantified using an H-score that combined the percentage (0–100%) and intensity (1+, 2+, 3+). The H-score was calculated by multiplying the percentage of positive cells by a factor representing the intensity of immunoreactivity, with final score ranging between 0 and 300. An H-score of 10 was chosen as the threshold for MSLN positivity [54]. Using this scoring system, 37 (34%) of 109 TNBC were deemed MSLN positive [54], but MSLN expression in TNBC did not show prognostic significance. In contrast, we found that MSLN positive TNBC had significant worse prognosis. The difference between the study by Parinyanitikul et al and ours probably stems from the different criteria used for MSLN positivity. In addition, in the study by Parinyanitikul et al [54], TNBC was defined as ER and PR ≤5%, HER2 negative by IHC and or FISH. The different criteria used to assign ER and/or PR positivity in the study by Parinyanitikul et al [54] (<5%) and in our own (<1%) [44] may also account in part for the different results.
Over two-thirds of TNBC in our series demonstrated at least focal MSLN staining in at least 1% of tumor cells ( Table 2 ), a percentage similar to that reported by Tchou et al [46], but we documented higher rates of MSLN reactivity in non-TNBC (45% showing at least focal staining, and 16% with substantial MSLN expression in our study versus only 3% in a prior study [46]). However, in our series significantly more TNBC than non-TNBC were strongly MSLN-positive (82/226, 36% vs 14/88, 16%; p = 0.0006). Differences in proportion and intensity of staining could potentially be explained by differences in the choice of MSLN antibody dilution utilized, and definitive characterization of MSLN expression in non-TNBC requires further evaluation.
To the best of our knowledge, our series is the largest to date to assess MLSN expression in TNBC, allowing to further evaluate its correlation, or lack thereof, with basal immunophenotype. In our study, MSLN expression in TNBC correlated with basal cytokeratin expression but not with that of EGFR. Furthermore, MSLN expression was a predictor of worse outcome independent of basal immunophenotype.
In conclusion, MSLN, a cell surface antigen overexpressed in several malignancies, shows substantial expression in TNBC. Among TNBC, MSLN appears to be an independent prognostic marker associated with distant metastasis and worse survival. Patients with MSLN-positive TNBC could benefit from MSLN-targeted immuno therapies currently in development.
Data Availability
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding Statement
This work was supported by the Department of Defense (DOC) Breast Cancer Research Program (BC132124). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
- 1. Chang K, Pastan I, Willingham MC (1992) Isolation and characterization of a monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium. Int J Cancer 50:373–381. [DOI] [PubMed] [Google Scholar]
- 2. Chang K, Pastan I (1996) Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci U S A 93:136–140. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Argani P, Iacobuzio-Donahue C, Ryu B, Rosty C, Goggins M, et al. (2001) Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE). Clin Cancer Res 7:3862–3868. [PubMed] [Google Scholar]
- 4. Swierczynski SL, Maitra A, Abraham SC, Iacobuzio-Donahue CA, Ashfaq R, et al. (2004) Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays. Hum Pathol 35:357–366. [DOI] [PubMed] [Google Scholar]
- 5. Glass JP, Parasher G, Arias-Pulido H, Donohue R, Prossnitz ER, et al. (2011) Mesothelin and GPR30 staining among a spectrum of pancreatic epithelial neoplasms. Int J Surg Pathol 19:588–596. [DOI] [PubMed] [Google Scholar]
- 6. Ho M, Bera TK, Willingham MC, Onda M, Hassan R, et al. (2007) Mesothelin expression in human lung cancer. Clin Cancer Res 13:1571–1575. [DOI] [PubMed] [Google Scholar]
- 7. Fan D, Yano S, Shinohara H, Solorzano C, Van Arsdall M, et al. (2002) Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin. Mol Cancer Ther 1:595–600. [PubMed] [Google Scholar]
- 8. Frierson HF Jr, Moskaluk CA, Powell SM, Zhang H, Cerilli LA, et al. (2003) Large-scale molecular and tissue microarray analysis of mesothelin expression in common human carcinomas. Hum Pathol 34:605–609. [DOI] [PubMed] [Google Scholar]
- 9. Servais EL, Colovos C, Rodriguez L, Bograd AJ, Nitadori J, et al. (2012) Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin Cancer Res 18:2478–2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Rump A, Morikawa Y, Tanaka M, Minami S, Umesaki N, et al. (2004) Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J Biol Chem 279:9190–9198. [DOI] [PubMed] [Google Scholar]
- 11. Gubbels JA, Belisle J, Onda M, Rancourt C, Migneault M, et al. (2006) Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors. Mol Cancer 5:50. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q (2011) Mesothelin confers pancreatic cancer cell resistance to TNF-alpha-induced apoptosis through Akt/PI3K/NF-kappaB activation and IL-6/Mcl-1 overexpression. Mol Cancer 10:106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Bharadwaj U, Li M, Chen C, Yao Q (2008) Mesothelin-induced pancreatic cancer cell proliferation involves alteration of cyclin E via activation of signal transducer and activator of transcription protein 3. Mol Cancer Res 6:1755–1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Hellstrom I, Raycraft J, Kanan S, Sardesai NY, Verch T, et al. (2006) Mesothelin variant 1 is released from tumor cells as a diagnostic marker. Cancer Epidemiol Biomarkers Prev 15:1014–1020. [DOI] [PubMed] [Google Scholar]
- 15. Rizk NP, Servais EL, Tang LH, Sima CS, Gerdes H, et al. (2012) Tissue and serum mesothelin are potential markers of neoplastic progression in Barrett’s associated esophageal adenocarcinoma. Cancer Epidemiol Biomarkers Prev 21:482–486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Hassan R, Ho M (2008) Mesothelin targeted cancer immunotherapy. Eur J Cancer 44:46–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal M, et al. (2009) Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci U S A 106:3360–3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Moon EK, Carpenito C, Sun J, Wang LC, Kapoor V, et al. (2011) Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor. Clin Cancer Res 17:4719–4730. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Kelly RJ, Sharon E, Pastan I, Hassan R (2012) Mesothelin-targeted agents in clinical trials and in preclinical development. Mol Cancer Ther 11:517–525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Hassan R, Cohen SJ, Phillips M, Pastan I, Sharon E, et al. (2010) Phase I clinical trial of the chimeric anti-mesothelin monoclonal antibody MORAb-009 in patients with mesothelin-expressing cancers. Clin Cancer Res 16:6132–6138. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Kreitman RJ, Hassan R, Fitzgerald DJ, Pastan I (2009) Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P. Clin Cancer Res 15:5274–5279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Le DT, Brockstedt DG, Nir-Paz R, Hampl J, Mathur S, et al. (2012) A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase I studies of safety and immune induction. Clin Cancer Res 18:858–868. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Haffty BG, Yang Q, Reiss M, Kearney T, Higgins SA, et al. (2006) Locoregional relapse and distant metastasis in conservatively managed triple negative early-stage breast cancer. J Clin Oncol 24:5652–5657. [DOI] [PubMed] [Google Scholar]
- 24. Thike AA, Cheok PY, Jara-Lazaro AR, Tan B, Tan P, et al. (2010) Triple-negative breast cancer: clinicopathological characteristics and relationship with basal-like breast cancer. Mod Pathol 23:123–133. [DOI] [PubMed] [Google Scholar]
- 25. Thike AA, Iqbal J, Cheok PY, Chong AP, Tse GM, et al. (2010) Triple negative breast cancer: outcome correlation with immunohistochemical detection of basal markers. Am J Surg Pathol 34:956–964. [DOI] [PubMed] [Google Scholar]
- 26. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, et al. (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13:4429–4434. [DOI] [PubMed] [Google Scholar]
- 27. Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V (2007) Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Registry. Cancer 109:1721–1728. [DOI] [PubMed] [Google Scholar]
- 28. Tischkowitz M, Brunet JS, Begin LR, Huntsman DG, Cheang MC, et al. (2007) Use of immunohistochemical markers can refine prognosis in triple negative breast cancer. BMC Cancer 7:134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Harris LN, Broadwater G, Lin NU, Miron A, Schnitt SJ, et al. (2006) Molecular subtypes of breast cancer in relation to paclitaxel response and outcomes in women with metastatic disease: results from CALGB 9342. Breast Cancer Res 8:R66. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Morris GJ, Naidu S, Topham AK, Guiles F, Xu Y, et al. (2007) Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s Surveillance, Epidemiology, and End Results database. Cancer 110:876–884. [DOI] [PubMed] [Google Scholar]
- 31. Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, et al. (2007) The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res 13:2329–2334. [DOI] [PubMed] [Google Scholar]
- 32. Bertucci F, Finetti P, Cervera N, Esterni B, Hermitte F, et al. (2008) How basal are triple-negative breast cancers? Int J Cancer 123:236–240. [DOI] [PubMed] [Google Scholar]
- 33. Cancer Genome Atlas N (2012) Comprehensive molecular portraits of human breast tumours. Nature 490:61–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Prat A, Perou CM (2011) Deconstructing the molecular portraits of breast cancer. Mol Oncol 5:5–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Perou CM (2011) Molecular stratification of triple-negative breast cancers. Oncologist 16 Suppl 161–70. [DOI] [PubMed] [Google Scholar]
- 36. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, et al. (2000) Molecular portraits of human breast tumours. Nature 406:747–752. [DOI] [PubMed] [Google Scholar]
- 37. Rakha EA, El-Sayed ME, Green AR, Lee AH, Robertson JF, et al. (2007) Prognostic markers in triple-negative breast cancer. Cancer 109:25–32. [DOI] [PubMed] [Google Scholar]
- 38. Rakha EA, Ellis IO (2009) Triple-negative/basal-like breast cancer: review. Pathology 41:40–47. [DOI] [PubMed] [Google Scholar]
- 39. Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, et al. (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10:5367–5374. [DOI] [PubMed] [Google Scholar]
- 40. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, et al. (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98:10869–10874. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41. Rakha EA, Elsheikh SE, Aleskandarany MA, Habashi HO, Green AR, et al. (2009) Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin Cancer Res 15:2302–2310. [DOI] [PubMed] [Google Scholar]
- 42. Hudis CA, Gianni L (2011) Triple-negative breast cancer: an unmet medical need. Oncologist 16 Suppl 11–11. [DOI] [PubMed] [Google Scholar]
- 43. Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, et al. (2006) Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 19:264–271. [DOI] [PubMed] [Google Scholar]
- 44. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, et al. (2010) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Arch Pathol Lab Med 134:907–922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, et al. (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 131:18–43. [DOI] [PubMed] [Google Scholar]
- 46. Tchou J, Wang LC, Selven B, Zhang H, Conejo-Garcia J, et al. (2012) Mesothelin, a novel immunotherapy target for triple negative breast cancer. Breast Cancer Res Treat 133:799–804. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47. Yen MJ, Hsu CY, Mao TL, Wu TC, Roden R, et al. (2006) Diffuse mesothelin expression correlates with prolonged patient survival in ovarian serous carcinoma. Clin Cancer Res 12:827–831. [DOI] [PubMed] [Google Scholar]
- 48. Einama T, Kamachi H, Nishihara H, Homma S, Kanno H, et al. (2011) Co-expression of mesothelin and CA125 correlates with unfavorable patient outcome in pancreatic ductal adenocarcinoma. Pancreas 40:1276–1282. [DOI] [PubMed] [Google Scholar]
- 49. Baba K, Ishigami S, Arigami T, Uenosono Y, Okumura H, et al. (2012) Mesothelin expression correlates with prolonged patient survival in gastric cancer. J Surg Oncol 105:195–199. [DOI] [PubMed] [Google Scholar]
- 50. Shimizu A, Hirono S, Tani M, Kawai M, Okada K, et al. (2012) Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma. Cancer Sci 103:739–746. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51. Winter JM, Tang LH, Klimstra DS, Brennan MF, Brody JR, et al. (2012) A novel survival-based tissue microarray of pancreatic cancer validates MUC1 and mesothelin as biomarkers. PLoS One 7:e40157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52. Kawamata F, Kamachi H, Einama T, Homma S, Tahara M, et al. (2012) Intracellular localization of mesothelin predicts patient prognosis of extrahepatic bile duct cancer. Int J Oncol 41:2109–2118. [DOI] [PubMed] [Google Scholar]
- 53. Einama T, Homma S, Kamachi H, Kawamata F, Takahashi K, et al. (2012) Luminal membrane expression of mesothelin is a prominent poor prognostic factor for gastric cancer. Br J Cancer 107:137–142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54. Parinyanitikul N, Blumenschein GR, Wu Y, Lei X, Chavez-Macgregor M, et al. (2013) Mesothelin expression and survival outcomes in triple receptor negative breast cancer. Clin Breast Cancer 13:378–384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55. Kachala SS, Bograd AJ, Villena-Vargas J, Suzuki K, Servais EL, et al. (2014) Mesothelin overexpression is a marker of tumor aggressiveness and is associated with reduced recurrence-free and overall survival in early-stage lung adenocarcinoma. Clin Cancer Res 20:1020–1028. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.