| Abstract|| |
Background: Myeloid cell leukemia-1 (Mcl-1) is a member of the B-cell lymphoma 2 family known to play a significant role in the regulation of apoptosis. Mcl-1 expression has been studied in nonsmall cell lung cancer (NSCLC) cell lines but has not been previously evaluated as a prognostic factor in clinical samples. Materials and Methods: Formalin-fixed, paraffin-embedded sections from 119 NSCLC, including 33 squamous cell carcinomas (SCC), 55 adenocarcinomas (AC), and 31 either pure adenocarcinoma in situ (AIS) or AC with lepidic features were immunostained by an automated method with rabbit polyclonal Mcl-1. Cytoplasmic Mcl-1 (cMcl-1) immunoreactivity was scored based on intensity and percentage of positive tumor cells in both tumor and adjacent benign epithelium in each case. MCL1 amplification was determined by hybrid capture-based comprehensive genomic profiling (CGP) on a separate cohort of 9393 NSCLC samples. Results: Intense diffuse cMcl-1 overexpression was noted in 35/119 (29%) tumors overall and correlated with tumor type (52% AIS vs. 31% AC vs. 6% SCC, P < 0.0001), tumor grade (48% grade 1 vs. 14% grade 2 vs. 31% grade 3, P = 0.007), small tumor size (36% ≤3.0 cm vs. 16% >3.0 cm, P = 0.016), and lengthened survival within the AIS subgroup (100% alive vs. 42% expired, P = 0.018) while showing a trend toward correlation with nonrecurrent disease overall (32% nonrecurrent vs. 11% recurrent, P = 0.072) and within the AC subgroup (33% nonrecurrent vs. 0% recurrent, P = 0.092). MCL1 amplification was identified in 569 (6%) of 9393 NSCLC by CGP. Conclusions: cMcl-1 overexpression appears to occur independently from MCL1 gene amplification in NSCLC and correlates with AIS histologic type, lower tumor grade, smaller tumor size, nonrecurrent disease, and increased survival.
Keywords: Adenocarcinoma, myeloid cell leukemia-1 amplification, myeloid cell leukemia-1 immunohistochemistry, nonsmall cell lung carcinoma
|How to cite this article:|
El Jabbour T, Dalvi SD, Kim S, Sheehan C, Ross JS. Myeloid cell leukemia-1 protein expression and myeloid cell leukemia-1 gene amplification in non small cell lung cancer. Indian J Pathol Microbiol 2018;61:27-30
|How to cite this URL:|
El Jabbour T, Dalvi SD, Kim S, Sheehan C, Ross JS. Myeloid cell leukemia-1 protein expression and myeloid cell leukemia-1 gene amplification in non small cell lung cancer. Indian J Pathol Microbiol [serial online] 2018 [cited 2020 Aug 8];61:27-30. Available from: http://www.ijpmonline.org/text.asp?2018/61/1/27/228190
| Introduction|| |
Myeloid cell leukemia-1 (Mcl-1) is one of the members of the B-cell lymphoma 2 (Bcl-2) family of antiapoptotic proteins. Originally identified in differentiating myeloid cells, Mcl-1 heterodimerizes with other members of the Bcl-2 family, namely, Bim, Bid, Bik, Noxa, Puma and Bak.,, Depletion of Mcl-1 or preventing its interaction with Bak leads to apoptosis following a cytotoxic stimulus.
A balance of Mcl-1 is of critical importance for the maintenance of cellular homeostasis. Mcl-1 deficiency, for example, leads to bone marrow failure due to increased apoptosis of hematopoietic precursors, and excess of Mcl-1 has been associated with the development of hematologic malignancies in transgenic mice engineered to have constitutive Mcl-1 expression. To maintain this intricate balance between increased apoptosis and increased cell survival and proliferation, the levels of Mcl-1 are tightly regulated at multiple levels.
On a transcriptional level, the mRNA for Mcl-1 is upregulated by interleukin-3 (IL-3), IL-5, IL-6, IL-15, IL-22, interferon-alpha, granulocyte/macrophage colony-stimulating factor, and epidermal growth factor receptor activation. On a translational level, Mcl-1 is negatively regulated by specific micro-RNAs such as MiR-29, MiR-101, and MiR-133B., The half-life of Mcl-1 protein is 2–4 h, relatively short as compared with other antiapoptotic members of the Bcl-2 family, making it an attractive target for molecular targeted therapies in cancer. Proteasomal degradation of Mcl-1 protein is brought about by E3 ligase.
Mcl-1 overexpression has been reported in a variety of solid tumors including cholangiocarcinoma, hepatocellular carcinoma, nonsmall cell lung carcinoma (NSCLC), Merkel cell carcinoma, and melanoma  but has not been widely studied as a prognostic factor or therapy target in these solid tumors.
| Materials and Methods|| |
The study participants included patients whose tissues were sent for molecular genetic analysis to Foundation Medicine, Inc., Cambridge, Massachusetts, USA, from different institutions across the United States as well as international locations.
All of the immunohistochemical and molecular testing performed was done in an ethical manner, after due informed consent from the patients, which was taken at the different institutions, at the time of initial diagnosis.
A total of 119 cases of nonsmall cell lung carcinoma were studied for expression of Mcl-1 protein by immunohistochemistry that included 33 squamous cell carcinomas (SCC), 55 adenocarcinomas (AC), and 31 either pure adenocarcinoma in situ (AIS; formerly called bronchioloalveolar carcinoma [BAC]) or AC with BAC features. A separate cohort of 9393 NSCLC cases was studied from the tumor bank at Foundation Medicine, Inc., Cambridge, Massachusetts, USA, for MCL-1 gene amplification by hybrid capture-based comprehensive genomic profiling (CGP).
Formalin-fixed, paraffin-embedded tissue sections were cut from a representative block in each of the 119 cases of NSCLC. Immunohistochemical staining for the Mcl-1 protein was performed by an automated method on the Ventana BenchMark XT instrument (Ventana Medical Systems, Inc., Tucson, AZ, USA), utilizing rabbit polyclonal Mcl-1 (clone S-19, Santa Cruz) and ultraView Universal DAB Detection kit – an indirect biotin-free detection system. Adequate positive and negative controls were included in each run.
Cytoplasmic Mcl-1 (cMcl-1) immunoreactivity was scored based on intensity and percentage of positive tumor cells in both the tumor and adjacent benign epithelium in each case. Scoring was based on staining intensity (weak, moderate, intense) and percentage of positive cells (focal ≤10%, regional 11%–50%, diffuse >50%). Scoring was performed by two pathologists separately, in a blinded fashion. Of the 119 cases, a consensus was not reached in only 7 cases. These cases were studied together by the two pathologists, and a consensus was reached.
Statistical comparisons were carried out using STATA software (Stata Corporation, College Station, TX, USA). Chi-square test was used to determine the significance of the associations between protein expression and prognostic variables. Multivariate analysis including clinicopathologic parameters and Mcl-1 protein expression was performed using the Cox proportional hazards model. The level of significance was set at P < 0.05.
MCL1 amplification was determined by hybrid capture-based CGP on the separate cohort of 9393 NSCLC samples, as outlined above, using the Illumina HiSeq DNA sequencing system (Foundation Medicine, Inc., Cambridge, MA, USA). A minimum of 6 copies per cell was required for the presence of MCL-1 amplification.
All of the patients were staged and followed with a whole body computed tomography (CT) scan or with a chest CT along with a positron emission tomography scan. All positive scans were followed with a tissue diagnosis for confirmation.
The prognostic variables studied were tumor type, tumor grade, and tumor size and were correlated with disease recurrence and overall 10-year survival.
| Results|| |
Clinical features of the patients are tabulated in [Table 1]. Intense diffuse cMcl-1 overexpression was noted in 35/119 (29%) tumors overall and correlated with tumor type (52% AIS vs. 31% AC vs. 6% SCC, P < 0.0001), tumor grade (48% grade 1 vs. 14% grade 2 vs. 31% grade 3, P = 0.007), small tumor size (36% ≤3.0 cm vs. 16% >3.0 cm, P = 0.016), and lengthened survival within the AIS subgroup (100% alive vs. 42% expired, P = 0.018) while showing a trend toward correlation with nonrecurrent disease overall (32% nonrecurrent vs. 11% recurrent, P = 0.072) and within the AC subgroup (33% nonrecurrent vs. 0% recurrent, P = 0.092). MCL1 amplification was identified in 569 (6%) of 9393 NSCLC by CGP. On multivariate analysis, positive lymph node status (P< 0.0001) independently predicted overall survival.
| Discussion|| |
Nonsmall cell lung carcinoma (NSCLC) is the second most common malignancy in the United States after prostate cancer, with an estimated 224,390 new cases in 2016 and the leading cause of cancer deaths among both males and females with an estimated 153,080 deaths in 2016. The common subtypes of NSCLC include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.
Overexpression of Mcl-1 protein has been reported in a wide variety of solid tumors including a subset of NSCLC, where it promotes and enhances survival of lung cancer cells. The same study also showed that depletion of Mcl-1 leads to increased apoptosis of lung carcinoma cells in response to chemotherapy and radiation.
Many studies have correlated overexpression of Mcl-1 with poor clinical outcomes in a variety of cancers. Specifically, in a study of NSCLC by Luo et al., increased Mcl-1 expression correlated with a higher pathological grade and greater incidence of lymph node metastases.
Boucher et al. showed that Mcl-1 was one of the important antiapoptotic proteins that protected pancreatic tumor cells from apoptosis. Booy et al. showed that Mcl-1 mRNA and Mcl-1 protein levels are elevated in human breast tumors, where they confer a survival advantage by inhibiting apoptosis.
Mcl-1 is highly upregulated in gastric cancer and correlates with a poorer prognosis in these patients. In this study by Likui et al., high Mcl-1 expression significantly correlated with higher tumor stage, higher clinical stage, and a higher incidence of venous invasion and metastases.
In a study by Ding et al., overexpression of Mcl-1 in breast carcinoma was correlated with higher tumor grade and poor survival of patients.
A study by Zhang et al. showed overexpression of Mcl-1 in human cervical carcinoma cells as compared with normal human cervical cells. This overexpression was also significantly associated with larger tumor size, higher grade, and increased incidence of lymph node metastasis.
Mcl-1 expression has also been studied in hematologic malignancies. Wuillème-Toumi et al. showed that Mcl-1 overexpression in multiple myeloma correlated with disease relapse and shorter patient survival. Mcl-1 expression has also been extensively studied in chronic lymphocytic leukemia (CLL). Pepper et al. showed that Mcl-1 expression significantly correlated with stage of disease, VH gene mutation status, and coexpression of CD38 and ZAP-70; two markers that have poor prognosis in patients with CLL. Increased expression of Mcl-1 in this study also correlated with in vitro resistance to fludarabine. A similar study by Kitada et al. correlated elevated levels of Mcl-1 with failure to achieve complete remission after single-agent chemotherapy for CLL.
The current study assessed 119 cases of NSCLC, including 33 SCC, 55 invasive AC, and 31 pure or mixed AIS. We observed intense, diffuse nuclear, and cytoplasmic overexpression of Mcl-1 in 35 cases (29%) [Figure 1] and [Figure 2]. This was a significantly higher proportion of cases as compared with our separate assessment of a different cohort of patients which featured MCL1 gene amplification in 569 of 9393 cases (6%) only. Our study found that Mcl-1 protein overexpression appears to occur independently from MCL1 gene amplification. Our study also found that Mcl-1 was overexpressed in a significantly higher proportion of AIS as compared with SCC or AC. Furthermore, as seen in [Figure 3], the survival rates were significantly higher in the subgroup that overexpressed cMcl-1 (P = 0.018).
|Figure 1: Lung, adenocarcinoma in situ: Intense, diffuse cytoplasmic overexpression of myeloid cell leukemia-1 (×10)|
Click here to view
|Figure 2: Lung, adenocarcinoma in situ: Intense, diffuse cytoplasmic overexpression of myeloid cell leukemia-1 (×40)|
Click here to view
|Figure 3: Kaplan–Meier survival estimates showed significantly better survival in the subgroup overexpressing myeloid cell leukemia-1|
Click here to view
Reports of MCL1 amplification as a therapy target in solid tumors are extremely limited. In one recent study,MCL1 amplified triple-negative metastatic breast cancer responded to a combination of targeted therapy with chemotherapy backbone.
| Conclusions|| |
The current study is the first to correlate Mcl-1 overexpression with long-term patient outcomes in NSCLC. Surprisingly, in contrast with previous studies, in our study, the overexpression of Mcl-1 protein appears to be a good prognostic indicator and correlated with lower tumor grade, smaller tumor size, lengthened overall survival, and absence of disease recurrence. One of the reasons for this discrepancy could be the fact that most of the earlier studies on lung cancer were done either on cancer cells lines or on patients where the subtype of NSCLC was not independently evaluated. In the current study, there is a specific correlation between expression of Mcl-1 and AIS. In addition, we show that Mcl-1 overexpression appears to occur independently from MCL1 gene amplification. Based on these observations, further study of Mcl-1 expression in NSCLC and its potential role as a prognostic factor for the disease appears warranted.
Financial support and sponsorship
Conflicts of interest
Th authors have no conflicts of interest.
| References|| |
Michels J, Johnson PW, Packham G. Molecules in focus: Mcl-1. Int J Biochem Cell Biol 2005;37:267-71.
Mott JL, Kobayashi S, Bronk SF, Gores GJ. Mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene 2007;26:6133-40.
Thomas LW, Lam C, Edwards SW. Mcl-1; the molecular regulation of protein function. FEBS Lett 2010;584:2981-9.
Perciavalle RM, Opferman JT. Delving deeper: MCL-1's contributions to normal and cancer biology. Trends Cell Biol 2013;23:22-9.
Crawford M, Batte K, Yu L, Wu X, Nuovo GJ, Marsh CB, et al.
MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer. Biochem Biophys Res Commun 2009;388:483-9.
Akgul C. Mcl-1 is a potential therapeutic target in multiple types of cancer. Cell Mol Life Sci 2009;66:1326-36.
Sieghart W, Losert D, Strommer S, Cejka D, Schmid K, Rasoul-Rockenschaub S, et al.
Mcl-1 overexpression in hepatocellular carcinoma: A potential target for antisense therapy. J Hepatol 2006;44:151-7.
Song L, Coppola D, Livingston S, Cress D, Haura EB. Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells. Cancer Biol Ther 2005;4:267-76.
Brunner M, Thurnher D, Pammer J, Geleff S, Heiduschka G, Reinisch CM, et al.
Expression of VEGF-A/C, VEGF-R2, PDGF-alpha/beta, c-kit, EGFR, Her-2/Neu, Mcl-1 and Bmi-1 in Merkel cell carcinoma. Mod Pathol 2008;21:876-84.
Zhuang L, Lee CS, Scolyer RA, McCarthy SW, Zhang XD, Thompson JF, et al.
Mcl-1, Bcl-XL and stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of melanoma. Mod Pathol 2007;20:416-26.
Boucher MJ, Morisset J, Vachon PH, Reed JC, Lainé J, Rivard N, et al.
MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000;79:355-69.
Booy EP, Henson ES, Gibson SB. Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer. Oncogene 2011;30:2367-78.
Likui W, Qun L, Wanqing Z, Haifeng S, Fangqiu L, Xiaojun L, et al.
Prognostic role of myeloid cell leukemia-1 protein (Mcl-1) expression in human gastric cancer. J Surg Oncol 2009;100:396-400.
Ding Q, He X, Xia W, Hsu JM, Chen CT, Li LY, et al.
Myeloid cell leukemia-1 inversely correlates with glycogen synthase kinase-3beta activity and associates with poor prognosis in human breast cancer. Cancer Res 2007;67:4564-71.
Zhang T, Zhao C, Luo L, Zhao H, Cheng J, Xu F, et al.
The expression of Mcl-1 in human cervical cancer and its clinical significance. Med Oncol 2012;29:1985-91.
Wuillème-Toumi S, Robillard N, Gomez P, Moreau P, Le Gouill S, Avet-Loiseau H, et al.
Mcl-1 is overexpressed in multiple myeloma and associated with relapse and shorter survival. Leukemia 2005;19:1248-52.
Pepper C, Lin TT, Pratt G, Hewamana S, Brennan P, Hiller L, et al.
Mcl-1 expression has in vitro
and in vivo
significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers. Blood 2008;112:3807-17.
Kitada S, Andersen J, Akar S, Zapata JM, Takayama S, Krajewski S, et al.
Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: Correlations with in vitro
and in vivo
chemoresponses. Blood 1998;91:3379-89.
Ali SM, Watson J, Wang K, Chung JH, McMahon C, Ross JS, et al.
Acombination of targeted therapy with chemotherapy backbone induces response in a treatment-resistant triple-negative MCL1-amplified metastatic breast cancer patient. Case Rep Oncol 2016;9:112-8.
Siddhartha Dilip Dalvi
Department of Pathology, Albany Medical Center, 43 New Scotland Avenue, Albany, New York 12208
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]