International Journal of Gynecological Pathology 33:16–22, Lippincott Williams & Wilkins, Baltimore r 2013 International Society of Gynecological Pathologists
Original Article
Interferon-stimulated Gene, 15 kDa (ISG15) in Ovarian High-grade Serous Carcinoma: Prognostic Impact and Link to NF-kB Pathway Silvia Darb-Esfahani, M.D., Bruno V. Sinn, M.D., Marc Rudl, M.D., Jalid Sehouli, Ioana Braicu, M.D., Manfred Dietel, M.D., and Carsten Denkert, M.D.
M.D.,
Summary: The ubiquitin-like protein interferon-stimulated gene, 15 kDa (ISG15) plays an ambiguous role in the progression and response to chemotherapy of solid cancers. We aimed to investigate the prognostic impact of ISG15 and its link to the nuclear factor kB pathway in ovarian high-grade serous carcinoma. Immunohistochemistry was performed in a cohort of 128 primary ovarian high-grade serous carcinomas treated with standard surgery and adjuvant chemotherapy using tissue microarrays. In addition, 28 matched relapsed carcinomas were investigated. ISG15 protein expression was significantly increased in relapsed carcinomas as compared to primary tumors (P = 0.027). In primary carcinoma, ISG15 was positively associated with total inhibitor of kB a (IkBa) (P = 0.001) as well as nuclear and cytoplasmic phospho-IkBa (p-IkBa) expression (P = 0.039 and P = 0.002, respectively). Patients with ISG15-positive carcinomas had a significantly longer overall survival in univariate analysis (P = 0.002), and in multivariate analysis [hazard ratio = 0.35 (95% confidence interval, 0.14–0.84, P = 0.019)]. ISG15 is a potential prognostic marker in high-grade serous carcinoma of the ovary. Its impact on survival might be explained by its tight link to the nuclear factor kB pathway, and the further evaluation of the interplay between ISGylation machinery and nuclear factor kB, particularly with regard to response to chemotherapy, would be desirable. Key Words: High-grade serous carcinoma— Ovarian—ISG15—NF-kB—Prognosis.
High-grade serous carcinoma of the ovary constitutes the major subtype of all ovarian carcinomas (approximately 70%). Molecularly, it is characterized by a high rate of genomic instability and p53 alterations (1), which on the one hand, account for its highly aggressive behavior, and on the other hand,
explain the high sensitivity to chemotherapy. However, although 75% of ovarian cancer patients initially respond to standard adjuvant platinumbased chemotherapy, which is applied after radical surgery, 75% of these will relapse and ultimately die of their disease (2). Although 5-yr survival rate (approximately 45%) has increased slightly in the last 30 yr (3), overall survival remained unchanged (4). The identification of markers for chemotherapy response and survival would be helpful to define patient groups that are in need for alternative, novel therapeutic approaches. Interferon-stimulated gene, 15 kDa (ISG15) is a member of the ubiquitin-like modifiers family of proteins, and consists of 2 ubiquitin-like domains (5).
From the Institute of Pathology (S.D.-E., B.V.S., M.R., M.D., C.D.); and Department of Gynecology (J.S., I.B.), Charite´ Campus Virchow Klinikum, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany. The authors declare no conflict of interest. Address correspondence and reprint requests to Silvia DarbEsfahani, MD, Institute of Pathology, Charite´ Universita¨tsmedizin Berlin, Charite´platz 1, Berlin 10117, Germany. E-mail: silvia. [email protected].
DOI: 10.1097/PGP.0b013e31827b25a2
16
ISG15 IN OVARIAN HIGH-GRADE SEROUS CARCINOMA It is rapidly induced during stimulation by type 1 interferons (IFN-a and IFN-b) via Janus kinase/ signal transducer and activator of transcription (JAK/STAT) signaling. Nuclear factor kB (NF-kB) has been shown to be involved in ISG15 regulation (6). Similarly to ubiquitin, ISG15 is conjugated to target proteins by activation, conjugating, and ligating enzymes, a process that is called ISGylation. More than 200 target proteins have been identified by high-throughput proteomic approaches to date, and multiple targets seem to be simultaneously ISGylated upon IFN stimulation (5). In contrast to ubiquitination, ISGylation does not generally result in the degradation of target proteins. One major function of ISGylation is the interference with the ubiquitin/26S proteasome pathway (7). A strikingly ambiguous role of ISG15 in cancer has been shown in recent reports. Thus, ISG15 and/or components of the ISGylation machinery are upregulated and correlated with tumor progression in solid cancers such as breast (8) and bladder carcinoma (9), however, coordinated expression of ISG15 has rather tumor suppressive functions by regulating IFN-g and natural killer cells (10,11), and the induction of senescence (5). The role of ISG15 in chemotherapy response is conflictive to date. Thus, ISG15 was a marker of sensitivity to camptothecins in breast cancer (12), whereas in pancreatic cancer a causal relationship between ISG15 and resistance to gemcitabine has been established (13). In this study, we evaluated the prognostic impact of ISG15 protein in high-grade serous carcinoma of the ovary, in which chemotherapy is an essential component of treatment. In addition, we investigated the relationship between the expression of ISG15 and the classic NF-kB pathway that we had described previously (14). METHODS Study Population A total of 128 patients with primary high-grade serous carcinoma, who underwent radical surgery with the aim of total cytoreduction at the Department of Gynecology and Obstetrics (Charite´ Universita¨tsmedizin Berlin) and with informative ISG15 expression data were included. Resection specimens were formalin-fixed and paraffin-embedded for diagnostic purposes at the Institute of Pathology of the Charite´. Hematoxylin-eosin-stained slides were reevaluated according to histology by an experienced gynecopathologist (S.D.E.). Typing as high-grade serous carcinoma was performed as suggested by
17
McCluggage taking into account growth pattern, nuclear polymorphism, and mitotic count (15). As expected from this tumor subtype, most cases were high-stage tumors [Fe´de´ration Internationale de Gyne´cologie et d’Obste´trique (FIGO) Stage III–IV, n = 130, 88.3%]. Data on residual tumor mass after surgery were available for 94 patients (73.4%). The majority of patients had been treated with standard adjuvant carboplatin/paclitaxel (n = 93/117, 79.5%). In addition to carboplatin/paclitaxel, 14 patients (12.0%) had received abagovomab (n = 4), bevacizumab (n = 1), catumaxomab (n = 1), enzastaurine (n = 2), or gemcitabine (n = 6), 4 patients (3.3%) had received another platinum-based therapy (3 carboplatin mono, 1 carboplatin/topotecan), 3 patients (2.6%) had received a non-platinum–based regime (1 melphalan, 1 paclitaxel/gemcitabine, 1 not further specified), and 3 patients (2.6%) had not received adjuvant chemotherapy. Data on overall survival were available for all patients. Median overall survival was 33.5 mo (range, 2.5–162.3 mo), 48 patients died during follow-up (37.5%, 5-yr survival rate 50.6%). Data on progression-free survival were available for 114 patients (89.1%; range, 0.5–127.8; median, 19.0 mo). The distribution of clinicopathologic parameters of the study cohort is shown in Table 1. The study has been approved by the institutional review board of the Charite´ hospital.
Immunohistochemistry Immunohistochemical staining was performed on tissue microarrays (TMA). Data on inhibitor of kB a (IkBa) and/or phospho-IkBa (p-IkBa) expression had been obtained from 44 cases during a previous study; 75 additional cases from the present project were stained and evaluated for total and p-IkBa using the same protocol as previously described (14). An antiIkBa antibody (L35A5, Cell Signalling Technology Inc., Danvers, MA; dilution 1:50), and an anti-pIkBaSer32/36 antibody (Cell Signalling, dilution 1: 200) were used. For detection of ISG15 protein, a rabbit polyclonal antibody was applied in a dilution of 1:100 (Abcam, Cambridge, UK). 3,3’-diaminobenzidine peroxide substrate (DAB+), was used as a chromogen for (p-)IkBa and ISG15 staining. Stained slides were scanned and evaluated using the TMA evaluator software from VM Scope GmbH (Berlin, Germany). Semiquantitative evaluation of the rate of stained tumor cells and the staining intensity was registered as followed for all 3 markers: The percentage of positive cells was scored as 0 (0%), 1 (o10%), 2 (11%–50%), 3 Int J Gynecol Pathol Vol. 33, No. 1, January 2014
18
S. DARB-ESFAHANI ET AL. TABLE 1. Characteristics of the study group n (%)
Total ISG15 expression Negative Positive Age 60 yr or younger Older than 60 yr pT pT1 pT2 pT3 pN pN0 pN1 pNx FIGO stage FIGO I FIGO II FIGO III FIGO IV Residual tumor (cm) 0 40 Missing Chemotherapy Carboplatin/paclitaxel Carboplatin/paclitaxel/other Other platinum-based Other non-platinum based None Missing
128 (100%) 18 (14.1) 110 (85.9)
majority of tumors (Fig. 1). Weak staining intensity was frequently diffuse (n = 34, 26.6%), whereas some carcinomas displayed focal moderate (n = 45, 35.2%) or strong staining intensity (n = 31, 24.2%). Eighteen carcinomas showed no staining (= negative, 14.1%). IRS values ranged from 0 to 9, with quite similar
66 (51.6%) 62 (48.4%) 13 (10.2) 9 (7.0) 106 (82.8)
A
37 (35.9) 66 (64.1) 25 (19.5 of total) 8 7 103 10
(6.3) (5.5) (80.5) (7.8)
62 (66.0) 32 (34.0) 34 (26.6 of total) 93 14 4 3 3 11
(79.5) (12.0) (3.3) (2.6) (2.6)* (8.6% of total)
B
*n = 2 FIGO I, n = 1 FIGO III. FIGO indicates Fe´de´ration Internationale de Gyne´cologie et d’Obste´trique; ISG15, interferon-stimulated gene, 15 kDa.
(51%–80%), or 4 (480%), and the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Multiplication of the percentage of both parameters resulted in the immunoreactivity score (IRS), which ranged from 0 and 12.
C
Statistical Evaluation Statistical analyses were performed with SPSS Statistics 19 (SPSS Inc., Chicago, IL). Associations were tested by Fisher exact, w2, Wilcoxon rank-order test, or Mann-Whitney test, as indicated. Survival analysis was performed using the Kaplan-Meier method or Cox regression. All P values were calculated 2-tailed and P values
Original Article
Interferon-stimulated Gene, 15 kDa (ISG15) in Ovarian High-grade Serous Carcinoma: Prognostic Impact and Link to NF-kB Pathway Silvia Darb-Esfahani, M.D., Bruno V. Sinn, M.D., Marc Rudl, M.D., Jalid Sehouli, Ioana Braicu, M.D., Manfred Dietel, M.D., and Carsten Denkert, M.D.
M.D.,
Summary: The ubiquitin-like protein interferon-stimulated gene, 15 kDa (ISG15) plays an ambiguous role in the progression and response to chemotherapy of solid cancers. We aimed to investigate the prognostic impact of ISG15 and its link to the nuclear factor kB pathway in ovarian high-grade serous carcinoma. Immunohistochemistry was performed in a cohort of 128 primary ovarian high-grade serous carcinomas treated with standard surgery and adjuvant chemotherapy using tissue microarrays. In addition, 28 matched relapsed carcinomas were investigated. ISG15 protein expression was significantly increased in relapsed carcinomas as compared to primary tumors (P = 0.027). In primary carcinoma, ISG15 was positively associated with total inhibitor of kB a (IkBa) (P = 0.001) as well as nuclear and cytoplasmic phospho-IkBa (p-IkBa) expression (P = 0.039 and P = 0.002, respectively). Patients with ISG15-positive carcinomas had a significantly longer overall survival in univariate analysis (P = 0.002), and in multivariate analysis [hazard ratio = 0.35 (95% confidence interval, 0.14–0.84, P = 0.019)]. ISG15 is a potential prognostic marker in high-grade serous carcinoma of the ovary. Its impact on survival might be explained by its tight link to the nuclear factor kB pathway, and the further evaluation of the interplay between ISGylation machinery and nuclear factor kB, particularly with regard to response to chemotherapy, would be desirable. Key Words: High-grade serous carcinoma— Ovarian—ISG15—NF-kB—Prognosis.
High-grade serous carcinoma of the ovary constitutes the major subtype of all ovarian carcinomas (approximately 70%). Molecularly, it is characterized by a high rate of genomic instability and p53 alterations (1), which on the one hand, account for its highly aggressive behavior, and on the other hand,
explain the high sensitivity to chemotherapy. However, although 75% of ovarian cancer patients initially respond to standard adjuvant platinumbased chemotherapy, which is applied after radical surgery, 75% of these will relapse and ultimately die of their disease (2). Although 5-yr survival rate (approximately 45%) has increased slightly in the last 30 yr (3), overall survival remained unchanged (4). The identification of markers for chemotherapy response and survival would be helpful to define patient groups that are in need for alternative, novel therapeutic approaches. Interferon-stimulated gene, 15 kDa (ISG15) is a member of the ubiquitin-like modifiers family of proteins, and consists of 2 ubiquitin-like domains (5).
From the Institute of Pathology (S.D.-E., B.V.S., M.R., M.D., C.D.); and Department of Gynecology (J.S., I.B.), Charite´ Campus Virchow Klinikum, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany. The authors declare no conflict of interest. Address correspondence and reprint requests to Silvia DarbEsfahani, MD, Institute of Pathology, Charite´ Universita¨tsmedizin Berlin, Charite´platz 1, Berlin 10117, Germany. E-mail: silvia. [email protected].
DOI: 10.1097/PGP.0b013e31827b25a2
16
ISG15 IN OVARIAN HIGH-GRADE SEROUS CARCINOMA It is rapidly induced during stimulation by type 1 interferons (IFN-a and IFN-b) via Janus kinase/ signal transducer and activator of transcription (JAK/STAT) signaling. Nuclear factor kB (NF-kB) has been shown to be involved in ISG15 regulation (6). Similarly to ubiquitin, ISG15 is conjugated to target proteins by activation, conjugating, and ligating enzymes, a process that is called ISGylation. More than 200 target proteins have been identified by high-throughput proteomic approaches to date, and multiple targets seem to be simultaneously ISGylated upon IFN stimulation (5). In contrast to ubiquitination, ISGylation does not generally result in the degradation of target proteins. One major function of ISGylation is the interference with the ubiquitin/26S proteasome pathway (7). A strikingly ambiguous role of ISG15 in cancer has been shown in recent reports. Thus, ISG15 and/or components of the ISGylation machinery are upregulated and correlated with tumor progression in solid cancers such as breast (8) and bladder carcinoma (9), however, coordinated expression of ISG15 has rather tumor suppressive functions by regulating IFN-g and natural killer cells (10,11), and the induction of senescence (5). The role of ISG15 in chemotherapy response is conflictive to date. Thus, ISG15 was a marker of sensitivity to camptothecins in breast cancer (12), whereas in pancreatic cancer a causal relationship between ISG15 and resistance to gemcitabine has been established (13). In this study, we evaluated the prognostic impact of ISG15 protein in high-grade serous carcinoma of the ovary, in which chemotherapy is an essential component of treatment. In addition, we investigated the relationship between the expression of ISG15 and the classic NF-kB pathway that we had described previously (14). METHODS Study Population A total of 128 patients with primary high-grade serous carcinoma, who underwent radical surgery with the aim of total cytoreduction at the Department of Gynecology and Obstetrics (Charite´ Universita¨tsmedizin Berlin) and with informative ISG15 expression data were included. Resection specimens were formalin-fixed and paraffin-embedded for diagnostic purposes at the Institute of Pathology of the Charite´. Hematoxylin-eosin-stained slides were reevaluated according to histology by an experienced gynecopathologist (S.D.E.). Typing as high-grade serous carcinoma was performed as suggested by
17
McCluggage taking into account growth pattern, nuclear polymorphism, and mitotic count (15). As expected from this tumor subtype, most cases were high-stage tumors [Fe´de´ration Internationale de Gyne´cologie et d’Obste´trique (FIGO) Stage III–IV, n = 130, 88.3%]. Data on residual tumor mass after surgery were available for 94 patients (73.4%). The majority of patients had been treated with standard adjuvant carboplatin/paclitaxel (n = 93/117, 79.5%). In addition to carboplatin/paclitaxel, 14 patients (12.0%) had received abagovomab (n = 4), bevacizumab (n = 1), catumaxomab (n = 1), enzastaurine (n = 2), or gemcitabine (n = 6), 4 patients (3.3%) had received another platinum-based therapy (3 carboplatin mono, 1 carboplatin/topotecan), 3 patients (2.6%) had received a non-platinum–based regime (1 melphalan, 1 paclitaxel/gemcitabine, 1 not further specified), and 3 patients (2.6%) had not received adjuvant chemotherapy. Data on overall survival were available for all patients. Median overall survival was 33.5 mo (range, 2.5–162.3 mo), 48 patients died during follow-up (37.5%, 5-yr survival rate 50.6%). Data on progression-free survival were available for 114 patients (89.1%; range, 0.5–127.8; median, 19.0 mo). The distribution of clinicopathologic parameters of the study cohort is shown in Table 1. The study has been approved by the institutional review board of the Charite´ hospital.
Immunohistochemistry Immunohistochemical staining was performed on tissue microarrays (TMA). Data on inhibitor of kB a (IkBa) and/or phospho-IkBa (p-IkBa) expression had been obtained from 44 cases during a previous study; 75 additional cases from the present project were stained and evaluated for total and p-IkBa using the same protocol as previously described (14). An antiIkBa antibody (L35A5, Cell Signalling Technology Inc., Danvers, MA; dilution 1:50), and an anti-pIkBaSer32/36 antibody (Cell Signalling, dilution 1: 200) were used. For detection of ISG15 protein, a rabbit polyclonal antibody was applied in a dilution of 1:100 (Abcam, Cambridge, UK). 3,3’-diaminobenzidine peroxide substrate (DAB+), was used as a chromogen for (p-)IkBa and ISG15 staining. Stained slides were scanned and evaluated using the TMA evaluator software from VM Scope GmbH (Berlin, Germany). Semiquantitative evaluation of the rate of stained tumor cells and the staining intensity was registered as followed for all 3 markers: The percentage of positive cells was scored as 0 (0%), 1 (o10%), 2 (11%–50%), 3 Int J Gynecol Pathol Vol. 33, No. 1, January 2014
18
S. DARB-ESFAHANI ET AL. TABLE 1. Characteristics of the study group n (%)
Total ISG15 expression Negative Positive Age 60 yr or younger Older than 60 yr pT pT1 pT2 pT3 pN pN0 pN1 pNx FIGO stage FIGO I FIGO II FIGO III FIGO IV Residual tumor (cm) 0 40 Missing Chemotherapy Carboplatin/paclitaxel Carboplatin/paclitaxel/other Other platinum-based Other non-platinum based None Missing
128 (100%) 18 (14.1) 110 (85.9)
majority of tumors (Fig. 1). Weak staining intensity was frequently diffuse (n = 34, 26.6%), whereas some carcinomas displayed focal moderate (n = 45, 35.2%) or strong staining intensity (n = 31, 24.2%). Eighteen carcinomas showed no staining (= negative, 14.1%). IRS values ranged from 0 to 9, with quite similar
66 (51.6%) 62 (48.4%) 13 (10.2) 9 (7.0) 106 (82.8)
A
37 (35.9) 66 (64.1) 25 (19.5 of total) 8 7 103 10
(6.3) (5.5) (80.5) (7.8)
62 (66.0) 32 (34.0) 34 (26.6 of total) 93 14 4 3 3 11
(79.5) (12.0) (3.3) (2.6) (2.6)* (8.6% of total)
B
*n = 2 FIGO I, n = 1 FIGO III. FIGO indicates Fe´de´ration Internationale de Gyne´cologie et d’Obste´trique; ISG15, interferon-stimulated gene, 15 kDa.
(51%–80%), or 4 (480%), and the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Multiplication of the percentage of both parameters resulted in the immunoreactivity score (IRS), which ranged from 0 and 12.
C
Statistical Evaluation Statistical analyses were performed with SPSS Statistics 19 (SPSS Inc., Chicago, IL). Associations were tested by Fisher exact, w2, Wilcoxon rank-order test, or Mann-Whitney test, as indicated. Survival analysis was performed using the Kaplan-Meier method or Cox regression. All P values were calculated 2-tailed and P values
