AJCP / Original Article

Immunohistochemical Reevaluation of Carbonic Anhydrase IX (CA IX) Expression in Tumors and Normal Tissues Adelina Luong-Player, MD,1 Haiyan Liu, MD,1 Hanlin L. Wang, MD, PhD,2 and Fan Lin, MD, PhD1 From the 1Geisinger Medical Center, Danville, PA, and 2UCLA Department of Pathology and Laboratory Medicine, Los Angeles, CA.

CME/SAM

Key Words: Carbonic anhydrase IX; Cholangiocarcinoma; Hepatocellular carcinoma; Renal cell carcinoma; Normal kidney; Clear cell carcinoma of the ovary and uterus DOI: 10.1309/AJCPVJDS28KNYZLD

ABSTRACT Objectives: To immunohistochemically evaluate the carbonic anhydrase IX (CA IX) expression on 1,551 cases of tumors and normal tissues from various organs. Methods: Immunohistochemical evaluation of the expression of CA IX was performed on 1,125 malignant tumors, 69 benign neoplasms, and 322 normal tissues on tissue microarray sections and 18 intrahepatic cholangiocarcinomas (ICCs) and 17 hepatocellular carcinomas (HCCs) on routine sections. Results: There was overexpression of CA IX in clear cell renal cell carcinoma (CRCC) (88%, 68/77). Twenty-six (90%) of 29 ICCs were positive. In contrast, only 5 (15%) of 34 HCCs were focally positive. No staining was seen in chromophobe renal cell carcinoma (ChRCC), oncocytoma, seminoma, or carcinomas of the breast, thyroid, or prostate. All normal renal tubules except one case showed no staining. Conclusions: These data demonstrate the diagnostic utility of CA IX in (1) differentiating CRCC from ChRCC and oncocytomas, (2) distinguishing low-grade CRCC from normal renal tubules in small samples, (3) separating ICC from HCC, and (4) identifying metastatic CRCC from other metastases with clear cell features.

© American Society for Clinical Pathology

Upon completion of this activity you will be able to: • describe the function of carbonic anhydrase (CA) IX in cell physiology and in neoplasms. • summarize the diagnostic sensitivity and specificity of CA IX when evaluating a tumor of unknown origin. • define an effective immunostaining panel to distinguish cholangiocarcinoma from primary hepatocellular carcinoma. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 293. Exam is located at www.ascp.org/ajcpcme.

Carbonic anhydrase IX (CA IX) is a zinc-containing metalloenzyme that regulates cellular proliferation and acidbase balance. Its ability to transport carbon dioxide and maintain cellular homeostasis may play a key role in tumor progression, allowing tumor cells to adapt and survive even under hypoxic conditions. Interestingly, tumor cells cultured in hypoxic environments have been found to upregulate and express CA IX via the hypoxia-inducible factor 1 (HIF-1) pathway, further supporting the role of CA IX in tumor growth and spread.1,2 Carbonic anhydrase IX is a membrane isoenzyme and hence imparts a membrane-predominant staining pattern in immunohistochemistry.2 Studies have shown that staining for CA IX is positive in 85% to 100% of clear cell renal cell carcinomas (CRCCs) and essentially negative in other renal cell carcinoma subtypes (eg, chromophobe renal cell carcinoma [ChRCC]) and benign renal neoplasm (oncocytoma).2-4

219

Am J Clin Pathol 2014;141:219-225 DOI: 10.1309/AJCPVJDS28KNYZLD

219 219

Luong-Player et al / CA IX Expression in Tumors and Normal Tissues

This finding that CA IX is potentially a reliable and promising diagnostic marker for CRCC has laid the groundwork for additional studies to examine the extent of its diagnostic utility in other organs. ❚Table 1❚ Summary of Carbonic Anhydrase IX Immunostaining Results on 1,229 Tumorsa Tumor Type

Total No. of Cases

Total No. (%) Positive

ICC CRCC—low grade CRCC—high grade Endocervical AC Pancreatic AC Squamous cell CA Gastric AC Endometrial CA FIGO II Colonic AC Ovary papillary serous CA Endometrial CA FIGO I Lung AC, mixed type Esophageal AC Infiltrating urothelial CA Papillary RCC Lung neuroendocrine CA Clear cell CA of the uterus Hepatocellular CA Breast invasive ductal CA, G3 Clear cell CA of the ovary Pancreas neuroendocrine CA Testicular tumors/seminomas Breast invasive ductal CA, G2 Thyroid follicular CA ChRCC + oncocytoma Breast invasive ductal CA, G1 Breast invasive lobular CA, G2 Thyroid papillary CA Prostate AC, low-int Prostate foamy gland CA Thyroid follicular adenoma

29 40 37 25 50 49 21 59 43 41 34 61 30 43 20 42 13 34 33 14 17 115 47 37 30 27 31 48 97 11 51

26 (90) 36 (90) 32 (86) 17 (68) 29 (58) 28 (57) 12 (57) 32 (54) 22 (51) 20 (49) 16 (47) 28 (46) 13 (43) 15 (35) 6 (30) 12 (29) 3 (23) 5 (15) 5 (15) 2 (14) 2 (12) 8 (7) 2 (4) 1 (3) 0 0 0 0 0 0 0

AC, adenocarcinoma; CA, carcinoma; ChRCC, chromophobe renal cell carcinoma; CRCC, clear cell renal cell carcinoma; FIGO, International Federation of Gynecologic Oncology; G1, grade 1; G2, grade 2; G3, grade 3; ICC, intrahepatic cholangiocarcinoma; low-int, low grade and intermediate grade; RCC, renal cell carcinoma; TMA, tissue microarray. a Cholangiocarcinoma cases include 11 TMA and 18 routine samples. Hepatocellular carcinoma cases include 17 TMA and 17 routine samples.

Hynninen et al5 showed overexpression of CA IX in endometrial adenocarcinoma compared with normal endometrium. Although normal endometrium did stain focally for CA IX, the extent of staining in the malignant counterpart was higher. A few years ago, Donato et al4 examined the expression of CA IX in tumors of the genitourinary tract and found increased CA IX reactivity in urothelial carcinomas, except those with plasmacytoid and signet ring cell patterns. Unlike these studies, expression of CA IX was more debatable with regard to gastric tumors, with some studies showing elevated expression and others not.6-8 There are also very limited studies in the literature regarding CA IX expression in biliary tumors. Questions also arise about the reactivity of CA IX in normal renal epithelium. Most studies reported the absence of CA IX staining in normal kidney tissue, but a few have noted weak positive staining.2,4 Although some studies in the literature regarding the diagnostic utility of CA IX do sound promising, the sample size from which the conclusions were drawn is limited. In addition, the data on CA IX reactivity in certain tissue types and tumors are not entirely consistent. This study examines the expression of CA IX in various tumor types and normal tissues in the hope to shed more light on the utility of CA IX and clarify its level of expression.

Materials and Methods Construction of Tissue Microarray Blocks This study was approved by the institutional review board at Geisinger Medical Center (Danville, PA). A total of 1,551 cases from various organs (1,160 malignant tumors, 69 benign neoplasms, and 322 normal tissues) from 2004 through 2012 were retrieved from the archives of the Department of Pathology and Laboratory Medicine at Geisinger Medical Center. The details of this sample, including number of cases, tumor type, and site of origin, are summarized in ❚Table 1❚ and ❚Table 2❚. Multiple tissue microarray

❚Table 2❚ Summary of Carbonic Anhydrase IX Immunostaining Results on 322 Normal Tissues

Staining Intensity

Tumor Type

No. of Cases

0

1+

2+

3+

4+

Total No. (%) Positive

Normal liver Normal stomach body Normal stomach antrum Normal duodenum Normal colon Normal ovary Normal kidney Normal lung Normal parotid Normal/benign prostate Normal seminal vesicle

28 18 20 20 86 23 20 18 44 23 22

0 0 0 0 53 21 20 18 44 23 22

0 0 0 0 33s 2s 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

28 BD 18s 20s 20s 0 0 0 0 0 0 0

28 BD (100) 18 (100) 20 (100) 20 (100) 33 (38) 2 (9) 0 0 0 0 0

BD, bile duct; s, strong.

220 220

Am J Clin Pathol 2014;141:219-225 DOI: 10.1309/AJCPVJDS28KNYZLD

© American Society for Clinical Pathology

AJCP / Original Article

(TMA) blocks with two punches of either 1.5 mm or 1.0 mm for each individual case were constructed. All cases were evaluated as TMA sections, except 18 cases of intrahepatic cholangiocarcinoma (ICC) and 17 cases of hepatocellular carcinoma (HCC), which were evaluated on routine sections. Immunohistochemical Stains and Data Analysis Immunohistochemical evaluation of the expression of CA IX (Cell Marque, Rocklin, CA; mouse monoclonal antibody, clone MRQ-54; 1:100 dilution; CCI mild retrieval) using TMA tissue sections was performed on 1,125 malignant tumors, 69 benign neoplasms, and 322 normal tissues. An additional 18 ICCs and 17 HCCs on routine sections were also included. This gives a cumulative total of 1,551 cases. The staining was done using the Ventana staining system (Ventana Medical Systems, Tucson, AZ) based on the previously published protocol.9 Membranous staining was regarded as positive. The staining intensity was graded as weak or strong. The distribution of staining was recorded as negative (75%). The immunostained slides were further evaluated by two surgical pathologists (F.L. and H.L.).

Results CA IX Expression in Tumors The details of the positive staining result for each case are listed in Table 1 and ❚Table 3❚, including the staining intensity and distribution. Thirty-six (90%) of 40 low-grade CRCCs and 32 (86%) of 37 high-grade CRCCs showed overexpression of CA IX. In contrast, all ChRCCs (n = 15) and renal oncocytomas (n = 15) were negative. With regard to biliary/liver tumors, 8 (73%) of 11 cholangiocarcinomas on TMA sections were positive. If including the 18 additional cholangiocarcinomas from routine sections, a total of 26 (90%) of 29 cholangiocarcinomas were positive, with diffuse positivity (3+ or 4+) in 70% of the cases. In comparison, only 5 (15%) of 34 HCCs were focally (1+) and weakly positive. Five (19%) of 27 cases of clear cell carcinoma of the ovary and uterus were positive for CA IX, with two cases showing diffuse positivity (3+). Essentially, all prostate adenocarcinomas, low- to intermediate-grade breast carcinoma, seminomas, and thyroid neoplasms were negative for CA IX. Examples of CA IX expression in CRCC, ChRCC, ICC, and clear cell carcinoma of the ovary are shown in ❚Image 1❚.

❚Table 3❚ Summary of Carbonic Anhydrase IX Staining Intensity and Distribution in 1,229 Tumorsa

Staining Intensity

Tumor Type

No. of Cases

0

1+

2+

3+

4+

CRCC—low grade CRCC—high grade Cholangiocarcinoma, ICC Endocervical AC Pancreatic AC Squamous cell CA Gastric AC Endometrial CA FIGO II Colonic AC Ovary papillary serous CA Endometrial CA FIGO I Lung AC, mixed type Esophageal AC Infiltrating urothelial CA Papillary RCC Lung neuroendocrine CA Hepatocellular CA Clear cell CA of the uterus Clear cell CA of the ovary Breast invasive ductal CA, G3 Pancreas neuroendocrine CA Testicular tumors/seminomas Breast invasive ductal CA, G2 Thyroid follicular CA

40 37 29 25 50 49 21 59 43 41 34 61 30 43 20 42 34 13 14 33 17 115 47 37

4 10 3 8 21 21 9 27 21 21 18 33 17 28 14 30 29 10 12 28 15 107 45 36

2s 3s 5s 1s/1w 8s 10s 0 16s 4 7s/1w 4s 11s 6s 6s 2s 3s 5s 2s 1s 4s 0 3s 1s 1s

0 0 3s/1w 3s/1w 6s/1w 15s 1s 10s/1w 7s/1w 10s 5s 9s 5s 7s 2s/1w 6s 0 0 0 1s 0 3s 1s 0

1s 1s 6s/1w 4s 8s/1w 2s 5s 2s/1w 6s/1w 2s 4s 4s 1s 2s 1s 1w 0 1s 1s 0 0 2s 0 0

33s 28s 10s 7s 5s 1s 6s 2s 3s 0 3s 4s 1s 0 0 2s 0 0 0 0 2s 0 0 0

AC, adenocarcinoma; CA, carcinoma; CRCC, clear cell renal cell carcinoma; FIGO, International Federation of Gynecologic Oncology; G2, grade 2; G3, grade 3; ICC, intrahepatic cholangiocarcinoma; RCC, renal cell carcinoma; s, strong; w, weak. a Cholangiocarcinoma cases include 11 TMA and 18 routine samples. Hepatocellular carcinoma cases include 17 TMA and 17 routine samples.

© American Society for Clinical Pathology

221

Am J Clin Pathol 2014;141:219-225 DOI: 10.1309/AJCPVJDS28KNYZLD

221 221

Luong-Player et al / CA IX Expression in Tumors and Normal Tissues

A

B

C

D

E

F

❚Image 1❚ Carbonic anhydrase IX (CA IX) staining results on representative cases with no reactivity on chromophobe renal cell carcinoma (A, ×400), diffuse membranous staining on clear cell renal cell carcinoma (B, ×400), routine H&E section of cholangiocarcinoma (C, ×200), diffuse membranous staining on cholangiocarcinoma (D, ×200), routine H&E section of clear cell carcinoma of the ovary (E, ×400), and membranous staining on clear cell carcinoma of the ovary (F, ×200). Note: Only two of the 27 cases of clear cell carcinoma of the ovary/uterus showed strong diffuse membranous staining with CA IX. 222 222

Am J Clin Pathol 2014;141:219-225 DOI: 10.1309/AJCPVJDS28KNYZLD

© American Society for Clinical Pathology

AJCP / Original Article

CA IX Expression in Normal Tissues All normal intrahepatic bile ducts, stomach (gastric mucosa), and duodenum showed strong and diffuse (4+) positive staining for CA IX (100%). No staining was observed in normal liver (hepatocytes), kidney, lung, parotid gland, prostate, and seminal vesicle. Very focal (

Immunohistochemical reevaluation of carbonic anhydrase IX (CA IX) expression in tumors and normal tissues.

To immunohistochemically evaluate the carbonic anhydrase IX (CA IX) expression on 1,551 cases of tumors and normal tissues from various organs...
493KB Sizes 3 Downloads 0 Views