As the role of distal fallopian tube as organ of serous carcinogenesis is emerging, additional literature on the role of tubal intraepithelial carcinoma (TIC) as a precursor lesion in a subset of primary peritoneal serous carcinomas (PPSC is emerging as well. TIC although fallopian tube in origin can be genetically related to ovarian/peritoneal carcinomas. The role of PAX2 in primary fallopian tube carcinomas (PFTC)/PPSC is yet to be defined. The aim of our study was to understand if the biologic properties of tumors arising in the distal fallopian tube that remain as PFTC are different when they seed on to the peritoneal surface (PPSC). A panel of 6 polymorphic microsatellite markers corresponding to p53, PAX2, and WT1 tumor suppressor genes were studied. Invasive carcinomas as well as TIC arising in the distal fallopian tube when remain as PFTC appears to exhibit different LOH patterns in comparison to PPSC. PAX 2 LOH patterns might represent a “hidden PAX 2 signature” analogous to p53 signatures. PAX 2 might be an emerging marker for detection of early serous carcinomas particularly in BRCA + women.
Ovarian cancer is divided into two (2) histopathologic groups with distinct molecular pathogenesis as well as propensity for extra ovarian spread. Group 1 includes tumors of borderline malignancy, low-grade serous carcinomas, endometrioid and mucinous carcinomas. These tumors arise from ovarian parenchyma, confined to one or more cysts, and do not typically originate from or involve the ovarian surface and often exhibit BRAF, KRAS, P-TEN, b-catenin mutations. Group 2 includes serous carcinomas that arise from the ovarian surface epithelium or mullerian inclusions, fallopian tube mucosa, and mullerian epithelium in peritoneal cavity and often exhibit p53 mutations; these tumors are rapidly evolving and lethal [
MWH pathology files were searched between the years January 2006— December 2009. All tumors that met WHO 2001 criteria for PPSC were selected [
The criteria included the following Both ovaries normal size/enlarged by benign process. Extra ovarian involvement > surface of ovaries. Ovarian involvement-nonexistent—confined to surface w/no stromal invasion (
SEE-FIM protocol, a procedure for sectioning the entire fimbriated end (SEE-FIM protocol) in prophylactic oophorectomy specimens tubes, are first fixed for 4–6 hours: the fimbriated end (a) is amputated, (b) open-sectioned longitudinally to expose the maximum surface area, (c) submitted in toto with the remainder of the tube sectioned at 2-3 mm intervals, was adopted at MWH in 2006 [
PPSC were grouped into
All tumors that met the WHO criteria for PFTC were selected as well [
Hematoxylin & Eosin (H&E) slides were reviewed and the diagnosis was confirmed.
Figures
Hematoxylin and Eosin section of primary fallopian tube carcinoma (PFTC) with high grade serous papillary morphology: “nuclear” expression for PAX 2, p53, and WT-1 immunostains.
Hematoxylin and Eosin (H&E) section of primary peritoneal serous carcinomas (PPSC) with high-grade serous papillary morphology: “nuclear” expression for PAX 2, p53, and WT-1 immunostains.
Hematoxylin and Eosin (H&E) section of tubal intraepithelial carcinoma (TIC) with stratified epithelium, marked nuclear atypia associated with a subset of PPSC as well as PFTC: “nuclear” expression for PAX 2, p53, and WT-1 immunostains.
Representative tumor blocks as well as associated TIC in the corresponding fallopian tube sections were selected.
Wilm’s tumor gene 1 (WT-1), p53, and PAX 2 gene markers were selected for loss of heterozygosity (LOH) and immunohistochemical (IHC) analysis.
One or two paraffin blocks were selected to represent tumor and normal tissue from each patient. Ten (10) serial sections were obtained from paraffin-embedded tissue blocks. The first and last sections were stained with H&E to confirm that the intervening blank slides contained the lesional material. Tissue fragments were microdissected under stereomicroscopic visualization from 6–8 blank deparaffinized slides for each of the marked targets. Areas that contained large amounts blood, necrosis, or contaminating normal tissue were avoided during microdissection. DNA was isolated using DNEasy tissue extraction kit (Qiagen, Chatsworth, Calif.; USA). PCR amplification was performed using fluorescently labeled primers flanking microsatellite repeat polymorphisms located in close proximity to the specific genes of interest. Six microsatellite loci situated adjacent to three known tumor suppressor genes on chromosome 10q (PAX2 D10S.520, D10S.1173), 11p (WT-1 D11S.2370, D11S.995), and 17p (p53 D17S.1844, D17S.516) were assessed.
Amplification products were detected using fluorescence capillary electrophoresis and Gene Mapper Software 4.0 (ABI 3730, Applied Biosystems, Foster City, CA). Allelic peak heights of each electropherogram in relative fluorescence units were analyzed. The presence or absence of allelic imbalance was accessed by comparing allelic peak heights for each tumor sample with corresponding normal tissue. The sample was considered as noninformative (NI) if the tissue sample demonstrates only a single peak. Individuals with a heterozygous allele pattern (two peaks) were designated as informative for that particular locus (Figure
This figure shows the Loss of Heterozygosity (LOH) patterns of the normal (a) in comparison to the corresponding tumor (b), (c).
Immunohistochemical stains were performed on the blank sections on the corresponding paraffin-embedded block selected for LOH analysis. The details of the antibodies are specified in Table
Antibodies, sources, and conditions.
Antibody | Vendor | Reference no. | Clone | Dilution | Pre-Treatment | Detection |
---|---|---|---|---|---|---|
WT-1 protein | Cell Marque; Rocklin, CA | CMA788 | 6F-H2 | Pre-dilute | CC1 mild plus Protease 3, 4 min; Vetana Medical | Iview DAB plus Amplification; Ventana |
PAX-2 | Zymed-Invitrogen; Carlsbad, CA | 71–6000 | Rabbit Polyclonal | 1 : 30 | CC1 standard | Iview DAB |
P53 | Ventana Medical; Tucson, AZ | 790–2912 | DO-7 | Pre-dilute | CC1 standard | Iview DAB |
A cumulative score (CS) is calculated incorporating PS as well as IS. The score is derived by the summation of PS and IS, ranged from 0 to 7 and is further divided into
All the tumors are categorized into three groups.
This group included all cases of primary fallopian tube carcinomas (PFTCs). All tumors (100%) were present in the background of TIC.
Table
Clinical and pathologic features of Group 1 [primary fallopian tube carcinoma (PFTC)] arising in the background of tubal intraepithelial carcinoma (TIC).
PFTC ( | Age | Histology Diagnosis | Tumor size(cm) | Lymph nodemetastasis | FIGO stage | |
Positive | Negative | |||||
Case 1 | 48 | High grade serous | 8.0 | 0 | 6 | T1a |
Case 2 | 81 | High grade serous | 0.8 | 0 | 6 | T1a |
Case 3 | 73 | High grade serous | 5.5 | 0 | 9 | T2a |
Case 4 | 58 | High grade serous | 3.0 | 0 | 9 | T1a |
Case 5 | 79 | High grade serous | 5.0 | 0 | 6 | T1a |
Case 6 | 71 | High grade serous | 4.5 | 1 | 4 | T3c |
Case 7 | 77 | High grade serous | 1.6 | 0 | 21 | T3a |
Case 8 | 44 | High grade serous | 6.0 | 6 | 15 | T3c |
Case 9 | 67 | High grade serous | 1.5 | 23 | 0 | T3c |
PFTC: Primary fallopian tube carcinomas.
there is heterogeneity in PAX 2 immunostain expression in both TIC as well as the adjacent invasive carcinoma. A moderate-strong staining is seen in 40% (4/10) cases of both TIC as well as the adjacent invasive carcinoma. LOH was seen in up to 30% (3/10) in the invasive carcinomas. The foci of TIC were able to be captured for LOH analysis in four (4) out of ten (10) cases. LOH was in 75% (3/4) cases.
immunohistochemical expression for WT-1 and p53 were similarly seen consistently. A strong nuclear staining is seen in 100% (10/10) of all TIC and 80% (8/10) adjacent invasive carcinoma. LOH was seen in up to 50% (2/4) of TIC and 30% (3/10) of invasive carcinomas.
This group includes all cases of PPSC without associated TIC in the fallopian tubes.
there is heterogeneity in the expression of PAX 2 within this group. A strong PAX expression is seen in 33.3% (3/9) and moderate expression in 33.3% (3/9). LOH was seen in a higher frequency in 7/9 (79%) of cases.
immunohistochemical expression for WT-1 and p53 were similarly seen consistently. A strong nuclear staining is seen in 100% (10/10) of all TIC and 80% (8/10) adjacent invasive carcinoma. LOH was seen in up to 44% (4/9) at the WT-1 locus and 44% (4/9) at the p53 locus.
This group includes all cases of PPSC with associated TIC in the fallopian tubes.
there is heterogeneity in the expression of PAX 2 immunostain expression in both TIC as well as PPSC. A moderate-strong staining is seen in 40% (2/5) cases PPSC, and 20% (1/5) of associated TIC. No LOH was seen in both TIC as well as PPSC at the PAX gene.
immunohistochemical expression for WT-1 and p53 were similarly seen consistently. A strong nuclear staining is seen in 100% (5/5) of all TIC and 80% (4/5) adjacent invasive carcinoma. No LOH was seen in both TIC as well as PPSC at the WT-1 or p53 gene loci. One (1) out of five (5) cases showed LOH at WT-1 gene locus in both TIC as well as PPSC. Tables
Immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of Group 1: primary fallopian tube carcinoma (PFTC) and associated tubal intraepithelial carcinoma (TIC).
PPSC/+ | Associated TIC | PFTC | ||||||||||
PAX 2 | WT-1 | P53 | PAX 2 | WT-1 | P53 | |||||||
*
| LOH |
*
| LOH |
*
| LOH |
*
| LOH |
*
| LOH |
*
| LOH | |
♦Case 1 | 0 | No LOH | 7 | No LOH | 7 | 0 | No LOH | 6 | No LOH | 7 | ||
Case 2 | 0 | 3 | 7 | No LOH | 3 | 7 | No LOH | 7 | No LOH | |||
Case 3 | 6 | 7 | 0 | 6 | No LOH | 7 | 7 | No LOH | ||||
Case 4 | 0 | 7 | 7 | 6 | No LOH | 7 | 7 | |||||
♦Case 5 | 2 | 7 | No LOH | 0 | 3 | No LOH | 7 | No LOH | 0 | No LOH | ||
♦Case 6 | 7 | 6 | 7 | No LOH | 7 | 7 | No LOH | 7 | No LOH | |||
Case 7 | 0 | 7 | 7 | 0 | No LOH | 7 | No LOH | 7 | No LOH | |||
Case 8 | 3 | 7 | 7 | **0 | 7 | No LOH | 7 | |||||
♦Case 9 | **0 | 7 | **0 | 0 | No LOH | 7 | 0 | No LOH | ||||
Case 10 | 6 | 7 | 7 | 6 | No LOH | 7 | No LOH | 7 | No LOH |
*
(**): PAX signature; (♦): 4 cases of TIC on which LOH was performed.
Immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of Group 2: primary peritoneal serous carcinomas (PPSC)
PPSC/− | PAX 2 | WT-1 | P53 | |||
*
| LOH | * | LOH |
*
| LOH | |
Case 1 | 7 | 7 | No LOH | 7 | No LOH | |
Case 2 | **0 | 7 | 7 | No LOH | ||
Case 3 | **0 | 7 | No LOH | 7 | No LOH | |
Case 4 | 7 | 7 | 7 | |||
Case 5 | **0 | 7 | No LOH | 7 | No LOH | |
Case 6 | 7 | No LOH | 7 | 3 | ||
Case 7 | 3 | 7 | 7 | |||
Case 8 | 3 | No LOH | 7 | No LOH | 7 | No LOH |
Case 9 | 3 | 7 | No LOH | 7 |
*
(**): PAX signature.
Immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of Group 3: primary peritoneal serous carcinomas (PPSC)
PPSC/+ | Associated TIC | PPSC | ||||||||||
PAX 2 | WT-1 | P53 | PAX 2 | WT-1 | P53 | |||||||
*
| LOH |
*
| LOH |
*
| LOH |
*
| LOH |
*
| LOH |
*
| LOH | |
Case 1 | 1 | No LOH | 7 | No LOH | 7 | No LOH | 0 | No LOH | 7 | No LOH | 7 | No LOH |
Case 2 | 0 | No LOH | 7 | No LOH | 7 | No LOH | 4 | No LOH | 7 | No LOH | 7 | No LOH |
Case 3 | 5 | No LOH | 7 | No LOH | 7 | No LOH | 6 | No LOH | 7 | No LOH | 7 | No LOH |
Case 4 | No LOH | 7 | No LOH | 7 | No LOH | 0 | No LOH | 7 | No LOH | 6 | No LOH |
*
Primary fallopian tube carcinomas (PFTCs) are rare and comprises up to 1.1% of all gynecologic cancers. In order to be considered for PTFC, the tumor must be macroscopically located within the tube or at the fimbriated end and the uterus and ovary should be free of the tumor. Presence of in situ carcinoma designated as TIC qualifies “fallopian tube” as the origin. A great majority of the tubal carcinomas are predominantly “serous” in histology. Primary peritoneal serous carcinoma (PPSC) is diagnosed in the absence of an “in situ” lesion. The spread of PPSC is similar to ovarian carcinoma and sometimes involvement of adjacent ovary makes it difficult to determine the primary tumor origin and often classified either as ovarian or primary peritoneal in origin based on the “bulk of the tumor.”
Tubal intraepithelial carcinoma (TIC), though fallopian tube in origin has been shown in recent studies to be associated with nearly half of OSC as well as PPSC [
Distal fallopian tube (Fimbria) is a unique region with large surface area exposed to biologic events that impact the ovarian surface. It acts as a junction between mesothelium and mullerian epithelium. The reported frequency of PFTC at the fimbriae ranges from 40%–100%. These tumors are diagnosed effortlessly in the presence of a distended fallopian tube with a large centrally located tumor. However most of the tumors in this region are less commonly recognized as PFTC when a microscopic invasive tumor or a focus of TIC is present, until they spread to other sites. Additionally, fallopian tubes are not thoroughly examined in all PPSC for a tubal source. Interestingly, in the tumors arising from the distal fallopian tube though they share a common “precursor lesion” TIC, some of them appear to remain as PFTC and others seed on to the peritoneal surface to become PPSC. One of the primary goals of our study was to understand the biologic properties of the tumors arising in the distal fallopian tube that remain as PFTC are similar when they seed on to the peritoneal surface to become PPSC. Two tumor suppressor gene markers p53 and WT-1 were chosen along with an emerging mullerian marker PAX2 to understand these biologic properties.
P53 is a tumor suppressor gene located on the short arm of chromosome 17. Mutations of p53 are common in many human cancers; overexpression of p53 protein is associated with poor prognosis in a variety of cancers. Mutations of p53 are seen in Type 2, rapidly evolving tumors. P53 mutations are seen in up to 75% (8/12) of p53 signatures and a much higher incidence in TIC as well [
Using the three markers, we observed a great majority of our PPSC
Fallopian tube has been shown to be an organ of serous carcinogenesis. Early serous carcinomas were diagnosed in 2%–10% of BRCA (+) women undergoing prophylactic bilateral salpingo-oopherectomies [
In conclusion, tumors arise in distal FT remaining as PFTC appears to exhibit different LOH patterns when seeds on peritoneal surface (PPSC) though there is no difference in the expression of these tumors by immunohistochemistry. TIC in association with PFTC demonstrates different LOH patterns in comparison to PPSC w/TIC (? Tubal origin). PAX 2 LOH patterns in PPSC represent “PAX signature.” Understanding precursor lesions in distal fallopian tube helps in detection of early serous carcinomas (BRCA). Our study is the first to demonstrate the LOH and IHC of PAX2 in these tumors and might be an emerging biologic marker for early detection. Studies are in progress using these molecular markers in BRCA (+) women w/prophylactic bilateral salpingo-oophorectomies.
This study was done with support of Scaife Foundation pilot grant from MWRI research Institute. This study was presented in part as Platform presentation at United States and Canadian Academy (USCAP), March 2010, Washington DC, USA.