Vanilla Cake Batter Chocolate Truffles…only 5 Ingredients And No Baking For These Yummy Treats! | Recipe Hut
Vanilla Cake Batter Chocolate Truffles…only 5 Ingredients And No Baking For These Yummy Treats!
Categories: Uncategorized Tags: Baking, Batter, Cake, Chocolate, Ingredients, Treats, Truffles...only, Vanilla, Yummy
« Bubble Wrap Era/-/ Hazel Nut Tiramisu Cake | The Moonblush Baker Diagnostic Pathology | Full text | Immunohistochemical profiles of claudin-3 in primary and metastatic prostatic adenocarcinoma
Corresponding author:	Anil V Parwani parwaniav@upmc.edu  Equal contributors View PDF (499KB) Author affiliations
Diagnostic Pathology 2011, 6:12 doi:10.1186/1746-1596-6-12
The electronic version of this article is the complete one and can be found online at: http://www.diagnosticpathology.org/content/6/1/12
Received:2 January 2011 Accepted:21 January 2011 Published:21 January 2011 © 2011 Bartholow et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Claudins are integral membrane proteins that are involved in forming cellular tight
junctions. One member of the claudin family, claudin-3, has been shown to be overexpressed
in breast, ovarian, and pancreatic cancer. Here we use immunohistochemistry to evaluate
its expression in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia
(PIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic
Tissue microarrays were immunohistochemically stained for claudin-3, with the staining
intensities subsequently quantified and statistically analyzed using a one-way ANOVA
with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent.
Fifty-three cases of NAC, 17 cases of BPH, 35 cases of PIN, 107 cases of PCa, and
55 cases of Mets were analyzed in the microarrays.
PCa and Mets had the highest absolute staining for claudin-3. Both had significantly
higher staining than BPH (p < 0.05 in both cases) and NAC (p < 0.05 in both cases).
PIN had a lower, but non-significant, staining score than PCa and Mets, but a statistically
higher score than both BPH and NAC (p < 0.05 for both cases). No significant differences
were observed between PCa, Mets, and PIN.
To our knowledge, this represents one of the first studies comparing the immunohistochemical
profiles of claudin-3 in PCa and NAC to specimens of PIN, BPH, and Mets. These findings
provide further evidence that claudin-3 may serve as an important biomarker for prostate
cancer, both primary and metastatic, but does not provide evidence that claudin-3
can be used to predict risk of metastasis.
During 2010 alone, 32,050 deaths attributable to prostate cancer are expected to occur
in the United States, making it the second leading cause of cancer death in males. Despite this, studies conducted before the era of prostate specific antigen (PSA)
screening have shown that latent prostate cancer was often diagnosed only after autopsy
in older males[2-4]. One autopsy study of males over the age of 50, also conducted before PSA screening
was implemented, indicated that the overall age-adjusted prevalence of latent prostate
carcinoma was as high as 34.6% for whites and 36.9% for blacks in the United States, providing evidence that not all cases of prostate cancer have the same clinical
aggressiveness. Based upon cases of prostate cancer diagnosed by PSA, researchers
have estimated that clinically insignificant prostate cancer is now actually overdiagnosed
and 37% of tumors in whites and blacks, respectively.
There is limited information in the literature on predictive biomarkers to discern
which cases of prostate cancer are likely to remain latent, versus those that are
likely to metastasize and warrant more aggressive management. Understanding the protein expression patterns associated with prostate adenocarcinoma
may provide a new toolset to aid in the diagnosis of prostate cancer, provide prognostic
information about the risk of metastasis, and indicate unique treatment targets.
Cellular tight junctions have been shown to play roles in maintaining cell polarity,
regulating ion flow, and signalling[8-13]. Given that the disruption of tight junctions is believed to be one facet of tumorigenesis, claudins, one of the transmembrane protein families hypothesized to be the backbone
of tight junctions, have become a target of focus in the recent cancer literature.
One specific member of this family, claudin-3, has been shown to be overexpressed
in multiple forms of cancer, having been most thoroughly studied in ovarian cancer,
where it has been shown to be up regulated more than 80-fold in comparison to normal
ovarian cells. In ovarian serous adenocarcinoma, its increased expression has also been associated
with shorter survival times. Additional studies have shown that it is also up regulated in breast, pancreatic,
bladder, thyroid, fallopian tube, ovary, stomach, colon, and uterine cancer. Moreover, it has been observed in 18% of mesotheliomas and 90% of cases of metastatic
lung adenocarcinoma. A significant inverse relationship between claudin-3 expression and overall survival
in renal clear cell carcinoma has been documented.
In prostate cancer specifically, claudin-3 has been shown to correlate with advanced
tumor stage and recurrence, although the immunohistochemical expression profiles varied
in an examination of 141 cases of prostate cancer, where matched adjacent normal epithelium
was compared to prostatic adenocarcinoma (PCa). In 31% of cases, the expression was
actually lower than in the matched PCa .
Here, we compare the immunohistochemical profiles in a series of 53 cases of normal
tissue adjacent to prostatic adenocarcinoma (NAC), 17 cases of benign prostatic hyperplasia
(BPH), 35 cases of prostatic intraepithelial neoplasia (PIN), 107 cases of PCa, and
55 cases of metastatic prostatic adenocarcinoma (Mets) in order to obtain a more complete
understanding of the immunostaining profile of claudin-3 in primary and metastatic
prostate cancer and to further examine its potential as a biomarker for prostate cancer
and/or prostate cancer metastasis.
Cores were taken from the appropriate case specific paraffin-embedded tissue blocks
and assembled into TMAs according to a previously described protocol. The final TMAs consisted of 53 cases of NAC, 17 cases of BPH, 35 cases of PIN, 107
cases of PCa, and 55 cases of Mets. Each case was represented at least in triplicate,
however, in some cases, due to processing, only two cores for each case were represented.
In these instances, the cases were still included in the analysis.
TBS buffer rinse. A Dako Autostainer was then used to stain the TMAs with anti-claudin-3
(working dilution 1:150), a rabbit polyclonal antibody (Catalogue # RB-9251-PO) from
Thermo Scientific (Waltham, MA, USA). Immunolabeling was conducted using Rabbit Envision
+ HRP Polymer (Catalogue # K4003) from Dako (Carpinteria, CA). Slides were counterstained
Slides were scored on a scale of staining intensity, with 0 representing no staining,
1 representing weak staining, 2 representing moderate staining, and 3 representing
strong staining. Each staining value was multiplied by the percentage of the core
that stained for that intensity, expressed as a whole number. For cores where more
than one intensity was represented, the sum of the staining values was calculated.
An average value was obtained for each case, and subsequently, each tissue type. This
scoring procedure was modified from a scoring protocol previously used by Parwani,
Means by Gleason score and tumor stage, where available, were also obtained. All means
were reported with standard error. A one-way ANOVA with subsequent Tukey tests for
multiple comparisons or nonparametric Kruskal-Wallis test with post-hoc Dunn's tests
(α = 0.05 in both cases) were used to compare the tissue types, PCa carcinoma stages,
and PCa Gleason scores.
The mean staining scores for NAC, BPH, PIN, PCa, and Mets were 71.42 ± 8.78, 60.23
± 9.42, 108.76 ± 7.67, 117.03 ± 5.38, and 116 ± 7.66 (Figure 1).
Figure 1. Mean claudin-3 staining score by prostatic tissue type. Mean claudin-3 staining score by prostatic tissue type. Significant differences
were seen between NAC and PIN, NAC and PCa, NAC and Mets, BPH and PIN, BPH and PCa,
and BPH and Mets (p < 0.05 for each). No differences were seen between PCa, Mets,
A Kruskal-Wallis test (p = < 0.001), with subsequent Dunn's tests showed significant
differences between NAC and PCa, BPH and PCa, NAC and Mets, BPH and Mets, NAC and
PIN, and BPH and PIN (p < 0.05 in all cases). No significant differences were seen
between PCa and Mets or PCa and PIN (p > 0.05 in both cases). Twelve of 53 cases (23%)
of NAC, 2 of 17 cases (12%) of BPH, 20 of 35 cases (57%) of PIN, 64 of 107 cases (60%)
of PCa, and 33 of 55 cases (60%) of Mets had staining scores higher than 100.
When classified by Gleason score, the average staining score was 113.63 ± 12.08 (n
= 17) for those with a score of 6 or less, 116.81 ± 8.42 (n = 51) for those with a
score of 7, and 118.80 ± 8.53 (n = 39) for those with a score of 8 or more (Figure
2). A resultant one-way ANOVA showed no significant differences (p = 0.9504). Twelve
of 17 cases (71%) with a Gleason score of 6 or less, 29 of 51 cases (57%) with a Gleason
score of 7, and 24 of 39 cases (62%) with a Gleason score of 8 or higher had staining
scores higher than 100.
Figure 2. Mean claudin-3 staining score by Gleason score. Mean claudin-3 staining score by PCa Gleason score. No significant differences were
seen by this classification (P = 0.950).
When classified by tumor stage, the mean scores were stage 2, 134.34 ± 10.25 (n =
39), stage 3, 98.25 ± 6.91 (n = 38), and stage 4, 113.95 ± 8.88 (n = 29) (Figure 3). A resultant one-way ANOVA (p = 0.013) with subsequent Tukey tests for multiple
comparisons showed significant differences between stage 2 and stage 3 (p = 0.010).
Twenty-eight of 39 cases (72%) of stage 2 carcinoma, 18 of 38 cases (47%) of stage
3 carcinoma, and 18 of 29 cases (62%) of stage 4 carcinoma had staining scores higher
Figure 3. Mean claudin-3 staining score by carcinoma stage. Mean claudin-3 staining score by PCa carcinoma stage. Significant differences were
seen between stage 2 and stage 3 (p = 0.010).
Representative photomicrographs of the TMA cores are shown in Figure 4. Claudin-3 staining was a predominantly membranous in positive cases, although in
some cores, depicted most prominently in the primary and metastatic photomicrographs,
additional cytoplasmic staining was also noted.
Figure 4. Photomicrographs of TMA cores. Photomicrographs of TMA cores (20x). A) NAC, B) BPH, C) PIN, D) PCA, and E) Mets.
Average staining was higher in PCa and Mets than in NAC and BPH. The immunostain was
predominantly membranous in positive cases, however cytoplasmic staining was additionally
noted in select cores as depicted most prominently here in the photomicrographs of
PCa and Mets.
In the claudin-3 stained specimens, the average staining scores were highest in PCa
and Mets. PIN had a lower absolute staining score than PCa and Mets, although the
differences were not significant. Both BPH and NAC had significantly less staining
than PCa and Mets (Figure 1). These differences support the notion that claudin-3 is upregulated in many cases
of prostatic adenocarcinoma. It does not appear that stronger expression exists in
all cases, however, as there were 42 cases of PCa and 22 cases of Mets that had staining
scores below 100.
Previous studies have shown that Gleason scores of 7 or higher were more frequently
associated with lower expression of claudin-1. Additionally, Landers, et al., demonstrated that claudin-4 was upregulated in primary
and metastatic prostate cancer, although they state that it tended to be expressed
more in primary tumors with a Gleason score of 6 than those with a score of 7 or higher. In this current study, no significant differences were seen when the cases of PCa
were compared by Gleason score (Figure 2).
Sheehan, et al., have also reported that claudin-3 expression correlates with advanced
stage and tumor recurrence in prostate cancer , which coincides with another study of urothelial carcinoma, where claudin-3 has
also been shown to correlate with advanced stage and poor survival. In this current study, however, the highest average staining appeared to be in stage
2 PCa, which was significantly higher than stage 3 PCa staining, but not significantly
higher than that noted in stage 4 PCa (Figure 3). Although statistically different, it is worth noting that these absolute staining
scores are similar, reflecting that the difference in stage staining may not be of
enough magnitude to use clinically. In a study of gastric cancer, claudin-3 has also
been shown to be less expressed in advanced stage cases, which may suggest that claudin family expression patterns vary by cancer type.
As claudin-3 is a tight junction protein, it is interesting to note that in addition
to membranous staining, cytoplasmic staining was also seen in select cores, most prominently
in cases of PCa and Mets (Figure 4). Rangel, et al., have reported positive cytoplasmic immunostaining for claudin-3
in ovarian tumors and have suggested that this mislocation may be the result of abnormal
pathway activation in cancer. This finding may indicate that a similar occurrence takes place in prostate cancer
and may warrant further investigation.
Based on this study, it appears that claudin-3 is moderately to strongly expressed
in the majority of cases of prostate cancer and may serve as an important biomarker
for prostate cancer diagnosis, both primary and metastatic, however the discrimination
between primary and metastatic cases in this study is not great enough to merit its
usage as a marker to predict the risk of metastasis.
Some groups have speculated on the specific mechanism whereby claudin-3 may be involved
in carcinogenesis. D'Souza, et al., have shown that tight junction strength decreased
when claudin-3, when designed to contain a T192D mutation mimicking a phosphorylated
state, was overexpressed in ovarian cancer cell line OVCA433, a mechanism possibly
enabling invasion. Additionally, Agarwal, et al, have noted that ovarian epithelial cells specifically
designed to constitutively express claudin-3 were found to have higher activity of
matrix metalloproteinase-2, which may also contribute to invasion, although this level
did not decrease following siRNA knockdown of claudin-3, possibly indicating that
other pathways also promote its expression. This could be one explanation for the fact that strong claudin-3 staining is not
noted in all cases of PCa.
Finally, in addition to its potential role as a predictive biomarker for cancer, claudin-3
has also been shown to bind clostridium perfringens enterotoxin, subsequently leading
to toxin-mediated cytolysis, prompting some to suggest that this may indicate a future
therapy in select cases of prostate cancer[31,32]. Should such a therapy be developed, the immunohistochemical profiles of patients
for this marker may become even more important, as it may serve to guide therapy selection.
These results provide a basis for the characterization of claudin-3 staining in both
primary prostatic adenocarcinoma and metastatic prostatic adenocarcinoma. In both
types of tissue, increased expression of claudin-3 was seen in the majority of cases
examined, and was seen more frequently than in non-neoplastic tissue, indicating that
it may serve as an important biomarker for prostate cancer. Despite this, the differences
in primary and metastatic cancers were not enough to indicate that a claudin-3 immunostain
could provide prognostic information about the risk of metastasis. Furthermore, no
significant differences were seen when the cases of PCa were compared by Gleason score.
While stage 2 mean staining was significantly greater than stage 3, the numerical
differences were similar in absolute. Given its high expression in the majority of
cases of prostate cancer, and the fact that it has been implicated as a possible therapeutic
target, claudin-3 warrants additional studies to evaluate its potential as a clinically
useful biomarker in the diagnosis of prostate cancer.
and the Clinical Scientist Training Program, funded by the Office of the Dean at the
Jemal A, Siegel R, Xu J, Ward E: Cancer statistics. CA Cancer J Clin 2010, 60(5):277-300. PubMed Abstract | Publisher Full Text Konety BR, Bird VY, Deorah S, Dahmoush L: Comparison of the incidence of latent prostate cancer detected at autopsy before and
after the prostate specific antigen era. J Urol 2005, 174(5):1785-1788. discussion 1788 PubMed Abstract | Publisher Full Text Breslow N, Chan CW, Dhom G, Drury RA, Franks LM, Gellei B, Lee YS, Lundberg S, Sparke B, Sternby NH, et al.: Latent carcinoma of prostate at autopsy in seven areas. The International Agency for
Research on Cancer, Lyons, France. Int J Cancer 1977, 20(5):680-688. PubMed Abstract | Publisher Full Text Holund B: Latent prostatic cancer in a consecutive autopsy series. Scand J Urol Nephrol 1980, 14(1):29-35. PubMed Abstract | Publisher Full Text Yatani R, Chigusa I, Akazaki K, Stemmermann GN, Welsh RA, Correa P: Geographic pathology of latent prostatic carcinoma. Int J Cancer 1982, 29(6):611-616. PubMed Abstract | Publisher Full Text Etzioni R, Penson DF, Legler JM, di Tommaso D, Boer R, Gann PH, Feuer EJ: Overdiagnosis due to prostate-specific antigen screening: lessons from U.S. prostate
adjacent to cancer and prostate tissue from cancer free organ donors. BMC Cancer 2005, 5:45. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text Tsukita S, Furuse M: The structure and function of claudins, cell adhesion molecules at tight junctions. Ann N Y Acad Sci 2000, 915:129-135. PubMed Abstract | Publisher Full Text Van Itallie CM, Anderson JM: Claudins and epithelial paracellular transport. Annu Rev Physiol 2006, 68:403-429. PubMed Abstract | Publisher Full Text Tsukita S, Furuse M, Itoh M: Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2001, 2(4):285-293. PubMed Abstract | Publisher Full Text Cereijido M, Shoshani L, Contreras RG: Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight
junctions and epithelial polarity. Am J Physiol Gastrointest Liver Physiol 2000, 279(3):G477-482. PubMed Abstract | Publisher Full Text Cereijido M, Valdes J, Shoshani L, Contreras RG: Role of tight junctions in establishing and maintaining cell polarity. Annu Rev Physiol 1998, 60:161-177. PubMed Abstract | Publisher Full Text D'Atri F, Citi S: Molecular complexity of vertebrate tight junctions (Review). Mol Membr Biol 2002, 19(2):103-112. PubMed Abstract | Publisher Full Text Morin PJ: Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res 2005, 65(21):9603-9606. PubMed Abstract | Publisher Full Text Ouban A, Ahmed AA: Claudins in human cancer: a review. Histol Histopathol 25(1):83-90. PubMed Abstract | Publisher Full Text Hough CD, Sherman-Baust CA, Pizer ES, Montz FJ, Im DD, Rosenshein NB, Cho KR, Riggins GJ, Morin PJ: Large-scale serial analysis of gene expression reveals genes differentially expressed
in ovarian cancer. Cancer Res 2000, 60(22):6281-6287. PubMed Abstract | Publisher Full Text Choi YL, Kim J, Kwon MJ, Choi JS, Kim TJ, Bae DS, Koh SS, In YH, Park YW, Kim SH, et al.: Expression profile of tight junction protein claudin 3 and claudin 4 in ovarian serous
adenocarcinoma with prognostic correlation. Histol Histopathol 2007, 22(11):1185-1195. PubMed Abstract | Publisher Full Text Hewitt KJ, Agarwal R, Morin PJ: The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer 2006, 6:186. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text Soini Y, Kinnula V, Kahlos K, Paakko P: Claudins in differential diagnosis between mesothelioma and metastatic adenocarcinoma
of the pleura. J Clin Pathol 2006, 59(3):250-254. PubMed Abstract | Publisher Full Text | PubMed Central Full Text Lechpammer M, Resnick MB, Sabo E, Yakirevich E, Greaves WO, Sciandra KT, Tavares R, Noble LC, DeLellis RA, Wang LJ: The diagnostic and prognostic utility of claudin expression in renal cell neoplasms. Mod Pathol 2008, 21(11):1320-1329. PubMed Abstract | Publisher Full Text Sheehan GM, Kallakury BV, Sheehan CE, Fisher HA, Kaufman RP Jr, Ross JS: Loss of claudins-1 and -7 and expression of claudins-3 and -4 correlate with prognostic
variables in prostatic adenocarcinomas. Hum Pathol 2007, 38(4):564-569. PubMed Abstract | Publisher Full Text Kajdacsy-Balla A, Geynisman JM, Macias V, Setty S, Nanaji NM, Berman JJ, Dobbin K, Melamed J, Kong X, Bosland M, et al.: Practical aspects of planning, building, and interpreting tissue microarrays: the
Cooperative Prostate Cancer Tissue Resource experience. J Mol Histol 2007, 38(2):113-121. PubMed Abstract | Publisher Full Text Yin M, Dhir R, Parwani AV: Diagnostic utility of p501s (prostein) in comparison to prostate specific antigen
(PSA) for the detection of metastatic prostatic adenocarcinoma. Diagn Pathol 2007, 2:41. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text Seo KW, Kwon YK, Kim BH, Kim CI, Chang HS, Choe MS, Park CH: Correlation between Claudins Expression and Prognostic Factors in Prostate Cancer. Korean J Urol 51(4):239-244. PubMed Abstract | Publisher Full Text | PubMed Central Full Text Landers KA, Samaratunga H, Teng L, Buck M, Burger MJ, Scells B, Lavin MF, Gardiner RA: Identification of claudin-4 as a marker highly overexpressed in both primary and metastatic
prostate cancer. Br J Cancer 2008, 99(3):491-501. PubMed Abstract | Publisher Full Text | PubMed Central Full Text Nakanishi K, Ogata S, Hiroi S, Tominaga S, Aida S, Kawai T: Expression of occludin and claudins 1, 3, 4, and 7 in urothelial carcinoma of the
upper urinary tract. Am J Clin Pathol 2008, 130(1):43-49. PubMed Abstract | Publisher Full Text Jung H, Jun KH, Jung JH, Chin HM, Park WB: The Expression of Claudin-1, Claudin-2, Claudin-3, and Claudin-4 in Gastric Cancer
Tissue. J Surg Res, in press. PubMed Abstract | Publisher Full Text Rangel LB, Agarwal R, D'Souza T, Pizer ES, Alo PL, Lancaster WD, Gregoire L, Schwartz DR, Cho KR, Morin PJ: Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian
cancer but not in ovarian cystadenomas. Clin Cancer Res 2003, 9(7):2567-2575. PubMed Abstract | Publisher Full Text D'Souza T, Agarwal R, Morin PJ: Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates
tight junction barrier function in ovarian cancer cells. J Biol Chem 2005, 280(28):26233-26240. PubMed Abstract | Publisher Full Text Agarwal R, D'Souza T, Morin PJ: Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and
is associated with increased matrix metalloproteinase-2 activity. Cancer Res 2005, 65(16):7378-7385. PubMed Abstract | Publisher Full Text Long H, Crean CD, Lee WH, Cummings OW, Gabig TG: Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4
in prostate cancer epithelium. Cancer Res 2001, 61(21):7878-7881. PubMed Abstract | Publisher Full Text Santin AD, Bellone S, Marizzoni M, Palmieri M, Siegel ER, McKenney JK, Hennings L, Comper F, Bandiera E, Pecorelli S: Overexpression of claudin-3 and claudin-4 receptors in uterine serous papillary carcinoma:
novel targets for a type-specific therapy using Clostridium perfringens enterotoxin
(CPE). Cancer 2007, 109(7):1312-1322. PubMed Abstract | Publisher Full Text close	Sign up to receive new article alerts from Diagnostic Pathology