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Histone H1.5, a novel prostatic cancer marker: an immunohistochemical study
Human Pathology (2014) 45, 2115–2119
www.elsevier.com/locate/humpath
Original contribution
Histone H1.5, a novel prostatic cancer marker: an immunohistochemical study☆,☆☆ Vadim Khachaturov MD ⁎, Guang-Qian Xiao MD, PhD, Yayoi Kinoshita DMD, Pamela D. Unger MD, David E. Burstein MD Department of Pathology, Mount Sinai School of Medicine and Medical Center, New York, NY 10029, USA Received 23 March 2014; revised 12 June 2014; accepted 19 June 2014
Keywords: Histone H1.5; Prostate adenocarinoma; Prostate cancer; AMACR; Prostate cancer marker
Summary Histone H1.5 (HH1.5) is a somatic subtype of the histone H1 family of linker proteins that are located in the nucleus and play a role in stabilizing higher-order chromatin structure, gene expression, DNA repair, and cell proliferation. Recently, differential immunohistochemical expression of HH1.5 has been found in various neuroendocrine neoplasms. This study aimed to investigate the immunohistochemical expression of HH1.5 in prostatic adenocarcinomas. Sixty-three prostate needle core biopsies, 9 radical prostatectomy specimens, and 3 metastatic prostate cancer cases were evaluated. HH1.5 immunohistochemistry revealed strong nuclear reactivity in 68 (93%) of 73 cases of prostate adenocarcinomas, compared to only 7 (9%) of 75 cases of benign prostatic glands (P ≤ .0001). In all positive benign prostate epithelium, HH1.5 was limited to focal and weak reactivity. Similarly, all 23 foci of high-grade prostatic intraepithelial neoplasia exhibited focal staining, with the vast majority having only weak nuclear reactivity. Increased HH1.5 reactivity was observed in Gleason patterns 4 and 5 as compared to Gleason pattern 3, 72% and 56%, respectively (P ≤ .02). All 3 metastatic prostate cancer cases showed strong nuclear reactivity. HH1.5 may be a useful diagnostic tool in evaluating prostatic biopsies, particularly with small foci of cancer. Further studies are needed to support these findings and investigate the possible prognostic significance of HH1.5 in prostatic adenocarcinomas. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Diagnosis of prostate cancer on core needle biopsy specimens can be challenging because of small foci of cancer or the presence of benign mimickers [1–4]. Immunohisto☆
Conflict of interest and funding disclosure: None of the authors have any conflict of interests or funding disclosures. ☆☆ Submission declaration: Submission of an article implies that the work described has not been published previously, except in the form of an abstract at USCAP 2013. Institutional review board approval was obtained prior to starting the research, and all procedures were performed in compliance with relevant laws and institutional guidelines. ⁎ Corresponding author. Annenberg Building, 15th Floor, 1468 Madison Avenue, New York, NY 10029. E-mail address: [email protected] (V. Khachaturov). http://dx.doi.org/10.1016/j.humpath.2014.06.015 0046-8177/© 2014 Elsevier Inc. All rights reserved.
chemical stains such as high–molecular weight keratin, p63, and α-methylacyl-CoA racemase (AMACR) have greatly assisted in identifying prostate cancer [5–7], but they are not without limitations. The occasional absence of basal cells in partial atrophy and adenosis along with outpouching of highgrade prostatic intraepithelial neoplasia (HPIN) may yield false-negative results with basal cell markers (high–molecular weight keratin and p63) [6,7]. AMACR reactivity is absent in approximately 25% of prostate cancers [7,8] and 36% of HPIN [9]. Other limitations of AMACR include cytoplasmic staining, which can be ambiguous, and the lack of increased staining intensity with increasing Gleason pattern [5,6]. Histone H1 is a family of linker histone proteins that are located in the nucleus and play a role in stabilizing higher-
2116 order chromatin structure, gene expression, DNA repair, and cell proliferation [10–15]. There are 11 known subtypes, many with specific and multiple functions that are essential for cell survival [12,14,15]. Histone H1.5 (HH1.5) is one of the 7 somatic subtypes, which is expressed in a replicationdependent manner and has been found to alter gene expression by regulating transcription through modification of chromatin structure [10,12,16]. In a recent study, Li et al [17] found that, in differentiated cells, HH1.5 binds to genes encoding membrane proteins that function in cell to cell communication. Depletion of HH1.5 in fibroblasts resulted in deregulation of genes and alteration of the cell cycle leading to decreased cell growth [17]. Recently, HH1.5 has been immunohistochemically detected in several cancers, including atypical carcinoids and large and small cell neuroendocrine carcinomas [18]. In regards to prostate cancer, histone H1.0, which is a terminally differentiated subtype, showed immunohistochemical expression that is directly associated with Gleason pattern, cell proliferation, and androgen receptor expression [19]. However, its expression in normal prostatic tissue precludes its usefulness diagnostically. To our knowledge, the expression of HH1.5 has not been previously studied in prostatic adenocarcinoma. The aim of this study was to investigate the immunohistochemical expression of HH1.5 in prostate adenocarcinoma, benign prostatic glands, and HPIN as a potential diagnostic as well as prognostic tool.
2. Materials and methods Sixty-three prostate needle core biopsies from 59 patients, 9 radical prostatectomy specimens, and 3 metastatic prostate adenocarcinoma cases were obtained from The Mount Sinai Hospital pathology archives between the years of 2008 and 2012. Sixty-two of 64 biopsies had prostatic adenocarcinoma ranging from Gleason scores 6 to 9 with a variable amount of cancer per slide (5%-95%). The radical prostatectomy specimens had a similar range of Gleason scores (6-9) with 30% to 95% of cancer per specimen. The metastatic cases consisted of 2 pelvic lymph node specimens and a T8 vertebra biopsy, each from different patients. All 3 metastatic cases had high-grade carcinoma with Gleason pattern 4 or 5. All specimens were routinely fixed in 10% neutral buffered formalin and paraffin embedded. Prior to immunostaining, epitope retrieval was performed by boiling slides with 10 mmol/L citrate buffer (pH 6) for 4 minutes in a pressure cooker at 125°C with slow cooling. Charged slides with 5-μm-thick sections of tissue were incubated with a rabbit polyclonal antibody reactive against HH1.5 (Abcam, Cambridge, MA). The HH1.5 antibody was diluted at 1:800 using 5% goat serum in antibody diluent (Invitrogen). UltraVision Quanto Detection System HRP DAB (Thermo Scientific) was used as a secondary antibody and signal detection.
V. Khachaturov et al. HH1.5 immunohistochemical expression was assessed in prostatic adenocarcinoma, HPIN, benign glands, and prostatic stroma. Only nuclear staining was considered positive and was scored based on the percentage of reactive nuclei within each individual gland or cluster of reactive glands. Staining of less than 10% was considered negative; 11% to 50% and greater than 50% were reported as 1+ and 2+, respectively. For example, in Fig. D, more than 95% of the malignant nuclei are staining for HH1.5. Therefore, it was scored as 2+ (N50% reactivity). Each Gleason pattern of prostatic adenocarcinoma was separately evaluated for HH1.5 reactivity. Positive controls consisted of tonsillar and lymph node tissue because lymphocytes consistently demonstrate nuclear expression of HH1.5. When present, lymphocytes and seminal vesicles were also evaluated for HH1.5 reactivity. All cases were reviewed by 2 genitourinary pathologists.
3. Results Prostatic adenocarcinoma showed nuclear reactivity for HH1.5 in 68 (93%) of 73 cases, in contrast to only 7 (9%) of 75 cases of benign prostatic glands. The difference in HH1.5 staining between malignant and benign prostate glands was statistically significant (P ≤ .0001, Table 1). All positive cases of prostatic adenocarcinoma demonstrated diffuse and moderate to strong nuclear reactivity for HH1.5. In contrast, only weak and focal reactivity was present in 7 cases of benign glands. Five cases of prostate adenocarcinoma were negative for HH1.5 and higher-grade Gleason patterns were more often negative as compared to lower-grade Gleason patterns (13% vs. 3%). There were no cases displaying both moderate or strong nuclear reactivity for HH1.5 and less than 11% staining of the prostatic glands. Out of the 23 foci of HPIN lesions, all showed focal reactivity, with the vast majority having only weak nuclear reactivity for HH1.5 (Table 1). Increased HH1.5 reactivity was observed in higher-grade compared to lower-grade Gleason patterns, 72% and 56%, respectively (P ≤ .02, Table 2). All 3 metastatic prostate cancer cases yielded strong nuclear reactivity for HH1.5. When present, lymphocytes consistently stained strongly positive for HH1.5. On the contrary, prostatic stroma and seminal vesicles, when present, were consistently negative for HH1.5.
4. Discussion Histone H1 is a family of linker histone proteins, consisting of 11 subtypes that directly interact with the DNA between nucleosome particles, also known as linker DNA [14]. Their function appears to be of 2 major roles: a general one in forming and stabilizing higher-order chromatin structures, which is shared by all subtypes, and a more subtype-specific function involving gene regulation [14,15]. The latter results from alteration of chromatin structure [10], DNA methylation, or direct interaction with transcription
HH1.5, a novel prostatic cancer marker
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Fig A, High-grade prostatic intraepithelial neoplasia demonstrating focal and predominately weak nuclear reactivity for HH1.5 with occasional cells showing moderate reactivity (immunohistochemistry, original magnification ×200). B, Gleason pattern 3 prostate adenocarcinoma (right) showing nuclear expression of HH1.5 with adjacent nonreactive benign prostate glands (left). Scattered lymphocytes in benign glands and stroma serve as an internal control (immunohistochemistry, original magnification ×100). C, Prostate adenocarcinoma, Gleason pattern 3 (left), exhibiting nuclear reactivity for HH1.5 and reactive lymphoid aggregate (lower right) acting as an internal control (immunohistochemistry, original magnification ×100). D, Focal low-grade prostate adenocarcinoma (top right) with nuclear expression of HH1.5 and adjacent nonreactive benign prostate glands (immunohistochemistry, original magnification ×100). E, Diffuse and strong nuclear expression of HH1.5 in Gleason pattern 4 adenocarcinoma (immunohistochemistry, original magnification ×100 and ×400 [inset]). F, Benign nonreactive prostate glands surrounded by Gleason pattern 5 prostate adenocarcinoma that is strongly and diffusely reactive for HH1.5 (immunohistochemistry, original magnification ×100). G, Metastatic prostate adenocarcinoma to T8 vertebra showing mature bone fragment with surrounding tumor cells demonstrating strong nuclear reactivity for HH1.5 (immunohistochemistry, original magnification ×400).
factors [12,14,15]. Recently, specific histone subtypes have been implicated in tumorigenesis [18–20], which will be discussed later on.
HH1.5 is one of 7 somatic subtypes, which is present in all cells albeit with varying expression levels, depending on the cell type and degree of differentiation [10–12,15,17].
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Table 1 HH1.5 expression in prostate cancer, benign glands, and HPIN Prostate cancer HH1.5 Total a b c
Benign
a
b
68 (93%) 73
7 (9%) 75
HPIN 23 c (100%) 23
Strong nuclear positivity. Weak nuclear positivity. Mostly weak nuclear positivity.
Ongoing studies reveal that HH1.5 is not immunohistochemically expressed in all cells, nor is it specific for prostate cancer. Differential immunohistochemical expression of HH1.5 has been reported in pulmonary neuroendocrine tumors, particularly those of higher grade [18]. Additionally, HH1.5 was one of several mutated genes discovered from sequencing of colorectal carcinoma [21]. This study demonstrates that immunohistochemical detection of HH1.5 is common in prostate cancer cells and rare in benign prostatic epithelium. The positive staining exhibited in rare benign prostatic glands was focal and weak. Similarly, HPIN showed mostly weak reactivity for HH1.5 with occasional cells demonstrating moderate nuclear reactivity. Importantly, the staining observed in benign prostatic glands and HPIN was distinctly different from the strong nuclear reactivity observed in prostatic adenocarcinomas. Rare false-negative results were encountered with HH1.5; however, false-positive results showing moderate-tostrong nuclear reactivity were not observed. Higher grade Gleason patterns had a higher incidence of false-negatives than compared with lower grade Gleason patterns. The explanation for this is currently unclear. There were no cases with moderate-to-strong HH1.5 expression and less than 11% involvement of prostatic glands. However, if such cases are encountered, it may be best to regard them as atypical or suspicious for prostatic adenocarcinoma and further work them up. Lymphocytes, when present, serve as an excellent internal control. Cases with significant chronic inflammation should be interpreted cautiously, to prevent erroneous interpretation of lymphocytes for individual tumor cells. Histomorphologic correlation is paramount.
Table 2 HH1.5 intensity: Gleason pattern 3 vs. 4/5 vs. metastatic prostate cancer Intensity
Gleason 3
Gleason 4/5
Metastatic
Neg 1+ 2+ Total
2 (3%) 24 (39%) 35 (57%) 61
6 (13%) 7 (15%) 34 (72%) 47
0 0 3 a (100%) 3
NOTE. Intensity of HH1.5 is based on percentage of nuclear reactivity of prostatic glands: negative, 0% to 10%; 1+, 11% to 50%; 2+, N50%. a All cases were Gleason pattern 4 or 5.
Interestingly, HH1.5 exhibited an overall increased immunoreactivity in high-grade Gleason patterns 4 and 5 prostate adenocarcinoma and metastatic cases as compared to low-grade Gleason pattern 3 prostate cancers. These findings indicate not only a diagnostic but also a potential prognostic role of HH1.5. Pluripotent cells show higher HH1.5 expression than differentiated cells [15]. Therefore, increased HH1.5 expression in prostate cancer may contribute to its self-renewal mechanism. Aside from HH1.5, histone H1, a terminally differentiated histone protein variant, has also been implicated in prostate and breast cancer [12,19]. Histone H1 immunohistohemical expression positively correlated with the Gleason grade, Ki67 positivity, and androgen receptor expression in prostate cancer [19]. However, its expression in normal luminal cells, basal cells, and stromal cells precludes its diagnostic utility for prostate cancer [19]. Routine examination of prostate core needle biopsies for cancer consists of evaluating 3 main histological parameters: glandular architecture, cytologic features, and absence of a basal cell layer. The presence of the basal cell layer may be difficult to appreciate on routine hematoxylin and eosin sections. Immunohistochemical staining with high-molecular-weight cytokeratins (34βE12) and p63 can highlight the presence or absence of the basal cell layer [7]. Prostate cancer by definition lacks a basal cell layer. However, atypical adenomatous hyperplasia, atrophy, HPIN, and some morphologically benign prostatic glands may show partial or complete absence of basal cell staining [3,5,22,23]. Therefore, the absence of basal cell staining in a subset of cases may lead to an erroneous diagnosis of malignancy. AMACR is involved in β-oxidation of branched fatty acids and is overexpressed in prostate cancer [5,24]. It can be used as an immunohistochemical marker of prostate cancer, but it shows variable sensitivity and specificity [6,8,9]. Additionally, noncancerous lesions such as HPIN, atrophic glands, and AAH have been shown to be positive with AMACR in 64%, 36%, and 18%, respectively [6]. HPIN may show moderate to even strong staining with AMACR [9]. In our experience, AMACR has approximately 75% sensitivity and specificity. AMACR immunohistochemical staining limitations may hinder a correct diagnosis in a subset of small foci of prostate cancer or in one of its benign mimickers. In summary, this is the first study using HH1.5 immunohistochemistry in distinguishing prostate cancer from benign prostatic glands, with sensitivity and specificity superior to those of AMACR. Other advantages of HH1.5 over AMACR include clear nuclear staining and lymphocytes serving as an internal control. For small foci of prostate cancer, HH1.5 may be a useful tool in combination with hematoxylin and eosin and other immunological markers, particularly cytoplasmic staining with high–molecular weight cytokeratins. Further studies are needed to confirm these findings and investigate the histoprognostic significance of HH1.5 in prostate cancer.
HH1.5, a novel prostatic cancer marker
References [1] Epstein JI. Mimickers of prostatic intraepithelial neoplasia. Int J Surg Pathol 2010;18:142S-8S. [2] Epstein JI. Diagnostic criteria of limited adenocarcinoma of the prostate on needle biopsy. HUM PATHOL 1995;26:223-9. [3] Gaudin PB, Epstein JI. Adenosis of the prostate: histologic features in needle biopsy specimens. Am J Surg Pathol 1995;19:737-47. [4] Thorson P, Humphrey PA. Minimal adenocarcinoma in prostate needle biopsy tissue. Am J Surg Pathol 2000;114:896-909. [5] Beach R, Gown AM, De Peralta-Venturina MN, et al. P504S immunohistochemical detection in 405 prostatic specimens including 376 18-gauge needle biopsies. Am J Surg Pathol 2002;26:1588-96. [6] Evans AJ. α-Methylacyl CoA racemase (P504S): overview and potential uses in diagnostic pathology as applied to prostate needle biopsies. J Clin Pathol 2003;56:892-7. [7] Hameed O, Sublett J, Humphrey PA. Immunohistochemical stains for p63 and alpha-methylacyl-CoA racemase, versus a cocktail comprising both, in the diagnosis of prostatic carcinoma a comparison of the immunohistochemical staining of 430 foci in radical prostatectomy and needle biopsy tissues. Am J Surg Pathol 2005;29:579-87. [8] Sung MT, Jiang Z, Montironi R, MacLennan GT, Mazzucchelli R, Cheng L. alpha-Methylacyl-CoA racemase (P504S)/34ßE12/p63 triple cocktail stain in prostatic adenocarcinoma after hormonal therapy. HUM PATHOL 2007;38:332-41. [9] Zhou M, Chinnaiyan AM, Kleer CG, Lucas PC, Rubin MA. α-MethylacylCoA racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions. Am J Surg Pathol 2002;26:926-31. [10] Happel N, Doenecke D. Histone H1 and its isoforms: contribution to chromatin structure and function. Gene 2008;431:1-12. [11] Albig W, Meergans T, Doeneck D. Characterization of the H1.5 gene completes the set of human H1 subtype genes. Gene 1997;184:141-8. [12] Sancho M, Diani E, Beato M, Jordan A. Depletion of human H1 variants uncovers specific roles in gene expression and cell growth. PLoS Genet 2008;4:1-17.
2119 [13] Hizume K, Yoshimura SH, Takeyasu K. Linker histone H1 per se can induce three-dimensional folding of chromatin fiber. Biochemistry 2005;44:12978-89. [14] Izzo A, Kamieniarz K, Schneider R. The histone H1 family: specific members, specific functions? Biol Chem 2008;389:333-43. [15] Terme JM, Sesé B, Millán-Ariño L, et al. Histone H1 variants are differentially expressed and incorporated into chromatin during differentiation and reprogramming to pluripotency. J Biol Chem 2011;286:35347-57. [16] Lee H, Habas R, Abate-Shen C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis. Science 2004;304:1675-8. [17] Li JY, Patterson M, Mikkola HK, Lowry WE, Kurdistani SK. Dynamic distribution of liner histone H1.5 in cellular differentiation. PLoS Genet 2012;8:1-13. [18] Hechtman JF, Beasley MB, Kinoshita Y, Ko HM, Hao K, Burstein DE. Promyelocytic leukemia zinc finger and histone H1.5 differentially stain low- and high-grade pulmonary neuroendocrine tumors: a pilot immunohistochemical study. H UM P ATHOL 2013;44:1400-5. [19] Sato S, Takahashi S, Asamoto M, et al. Histone H1 expression in human prostate cancer tissues and cell lines. Pathol Int 2012;62:84-92. [20] Kostova NN, Srebreva LN, Milev AD, et al. Immunohistochemical demonstration of histone H1(0) in human breast carcinoma. Histochem Cell Biol 2005;124:435-43. [21] Sjöblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006;314:268-74. [22] Jiang Z, Woda BA, Rock KL, et al. P504S: a new molecular marker for the detection of prostate carcinoma. Am J Surg Pathol 2001;25:1397-404. [23] Amin MB, Schultz DS, Zarbo RJ. Analysis of cribriform morphology in prostatic neoplasia using antibody to high–molecular weight cytokeratins. Arch Pathol Lab Med 1994;118:260-4. [24] Rubin MA, Zhou M, Dhanasekaran SM, et al. α-Methylacyl coenzyme A racemase as a tissue biomarker for prostate cancer. JAMA 2002;287:1662-70.
www.elsevier.com/locate/humpath
Original contribution
Histone H1.5, a novel prostatic cancer marker: an immunohistochemical study☆,☆☆ Vadim Khachaturov MD ⁎, Guang-Qian Xiao MD, PhD, Yayoi Kinoshita DMD, Pamela D. Unger MD, David E. Burstein MD Department of Pathology, Mount Sinai School of Medicine and Medical Center, New York, NY 10029, USA Received 23 March 2014; revised 12 June 2014; accepted 19 June 2014
Keywords: Histone H1.5; Prostate adenocarinoma; Prostate cancer; AMACR; Prostate cancer marker
Summary Histone H1.5 (HH1.5) is a somatic subtype of the histone H1 family of linker proteins that are located in the nucleus and play a role in stabilizing higher-order chromatin structure, gene expression, DNA repair, and cell proliferation. Recently, differential immunohistochemical expression of HH1.5 has been found in various neuroendocrine neoplasms. This study aimed to investigate the immunohistochemical expression of HH1.5 in prostatic adenocarcinomas. Sixty-three prostate needle core biopsies, 9 radical prostatectomy specimens, and 3 metastatic prostate cancer cases were evaluated. HH1.5 immunohistochemistry revealed strong nuclear reactivity in 68 (93%) of 73 cases of prostate adenocarcinomas, compared to only 7 (9%) of 75 cases of benign prostatic glands (P ≤ .0001). In all positive benign prostate epithelium, HH1.5 was limited to focal and weak reactivity. Similarly, all 23 foci of high-grade prostatic intraepithelial neoplasia exhibited focal staining, with the vast majority having only weak nuclear reactivity. Increased HH1.5 reactivity was observed in Gleason patterns 4 and 5 as compared to Gleason pattern 3, 72% and 56%, respectively (P ≤ .02). All 3 metastatic prostate cancer cases showed strong nuclear reactivity. HH1.5 may be a useful diagnostic tool in evaluating prostatic biopsies, particularly with small foci of cancer. Further studies are needed to support these findings and investigate the possible prognostic significance of HH1.5 in prostatic adenocarcinomas. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Diagnosis of prostate cancer on core needle biopsy specimens can be challenging because of small foci of cancer or the presence of benign mimickers [1–4]. Immunohisto☆
Conflict of interest and funding disclosure: None of the authors have any conflict of interests or funding disclosures. ☆☆ Submission declaration: Submission of an article implies that the work described has not been published previously, except in the form of an abstract at USCAP 2013. Institutional review board approval was obtained prior to starting the research, and all procedures were performed in compliance with relevant laws and institutional guidelines. ⁎ Corresponding author. Annenberg Building, 15th Floor, 1468 Madison Avenue, New York, NY 10029. E-mail address: [email protected] (V. Khachaturov). http://dx.doi.org/10.1016/j.humpath.2014.06.015 0046-8177/© 2014 Elsevier Inc. All rights reserved.
chemical stains such as high–molecular weight keratin, p63, and α-methylacyl-CoA racemase (AMACR) have greatly assisted in identifying prostate cancer [5–7], but they are not without limitations. The occasional absence of basal cells in partial atrophy and adenosis along with outpouching of highgrade prostatic intraepithelial neoplasia (HPIN) may yield false-negative results with basal cell markers (high–molecular weight keratin and p63) [6,7]. AMACR reactivity is absent in approximately 25% of prostate cancers [7,8] and 36% of HPIN [9]. Other limitations of AMACR include cytoplasmic staining, which can be ambiguous, and the lack of increased staining intensity with increasing Gleason pattern [5,6]. Histone H1 is a family of linker histone proteins that are located in the nucleus and play a role in stabilizing higher-
2116 order chromatin structure, gene expression, DNA repair, and cell proliferation [10–15]. There are 11 known subtypes, many with specific and multiple functions that are essential for cell survival [12,14,15]. Histone H1.5 (HH1.5) is one of the 7 somatic subtypes, which is expressed in a replicationdependent manner and has been found to alter gene expression by regulating transcription through modification of chromatin structure [10,12,16]. In a recent study, Li et al [17] found that, in differentiated cells, HH1.5 binds to genes encoding membrane proteins that function in cell to cell communication. Depletion of HH1.5 in fibroblasts resulted in deregulation of genes and alteration of the cell cycle leading to decreased cell growth [17]. Recently, HH1.5 has been immunohistochemically detected in several cancers, including atypical carcinoids and large and small cell neuroendocrine carcinomas [18]. In regards to prostate cancer, histone H1.0, which is a terminally differentiated subtype, showed immunohistochemical expression that is directly associated with Gleason pattern, cell proliferation, and androgen receptor expression [19]. However, its expression in normal prostatic tissue precludes its usefulness diagnostically. To our knowledge, the expression of HH1.5 has not been previously studied in prostatic adenocarcinoma. The aim of this study was to investigate the immunohistochemical expression of HH1.5 in prostate adenocarcinoma, benign prostatic glands, and HPIN as a potential diagnostic as well as prognostic tool.
2. Materials and methods Sixty-three prostate needle core biopsies from 59 patients, 9 radical prostatectomy specimens, and 3 metastatic prostate adenocarcinoma cases were obtained from The Mount Sinai Hospital pathology archives between the years of 2008 and 2012. Sixty-two of 64 biopsies had prostatic adenocarcinoma ranging from Gleason scores 6 to 9 with a variable amount of cancer per slide (5%-95%). The radical prostatectomy specimens had a similar range of Gleason scores (6-9) with 30% to 95% of cancer per specimen. The metastatic cases consisted of 2 pelvic lymph node specimens and a T8 vertebra biopsy, each from different patients. All 3 metastatic cases had high-grade carcinoma with Gleason pattern 4 or 5. All specimens were routinely fixed in 10% neutral buffered formalin and paraffin embedded. Prior to immunostaining, epitope retrieval was performed by boiling slides with 10 mmol/L citrate buffer (pH 6) for 4 minutes in a pressure cooker at 125°C with slow cooling. Charged slides with 5-μm-thick sections of tissue were incubated with a rabbit polyclonal antibody reactive against HH1.5 (Abcam, Cambridge, MA). The HH1.5 antibody was diluted at 1:800 using 5% goat serum in antibody diluent (Invitrogen). UltraVision Quanto Detection System HRP DAB (Thermo Scientific) was used as a secondary antibody and signal detection.
V. Khachaturov et al. HH1.5 immunohistochemical expression was assessed in prostatic adenocarcinoma, HPIN, benign glands, and prostatic stroma. Only nuclear staining was considered positive and was scored based on the percentage of reactive nuclei within each individual gland or cluster of reactive glands. Staining of less than 10% was considered negative; 11% to 50% and greater than 50% were reported as 1+ and 2+, respectively. For example, in Fig. D, more than 95% of the malignant nuclei are staining for HH1.5. Therefore, it was scored as 2+ (N50% reactivity). Each Gleason pattern of prostatic adenocarcinoma was separately evaluated for HH1.5 reactivity. Positive controls consisted of tonsillar and lymph node tissue because lymphocytes consistently demonstrate nuclear expression of HH1.5. When present, lymphocytes and seminal vesicles were also evaluated for HH1.5 reactivity. All cases were reviewed by 2 genitourinary pathologists.
3. Results Prostatic adenocarcinoma showed nuclear reactivity for HH1.5 in 68 (93%) of 73 cases, in contrast to only 7 (9%) of 75 cases of benign prostatic glands. The difference in HH1.5 staining between malignant and benign prostate glands was statistically significant (P ≤ .0001, Table 1). All positive cases of prostatic adenocarcinoma demonstrated diffuse and moderate to strong nuclear reactivity for HH1.5. In contrast, only weak and focal reactivity was present in 7 cases of benign glands. Five cases of prostate adenocarcinoma were negative for HH1.5 and higher-grade Gleason patterns were more often negative as compared to lower-grade Gleason patterns (13% vs. 3%). There were no cases displaying both moderate or strong nuclear reactivity for HH1.5 and less than 11% staining of the prostatic glands. Out of the 23 foci of HPIN lesions, all showed focal reactivity, with the vast majority having only weak nuclear reactivity for HH1.5 (Table 1). Increased HH1.5 reactivity was observed in higher-grade compared to lower-grade Gleason patterns, 72% and 56%, respectively (P ≤ .02, Table 2). All 3 metastatic prostate cancer cases yielded strong nuclear reactivity for HH1.5. When present, lymphocytes consistently stained strongly positive for HH1.5. On the contrary, prostatic stroma and seminal vesicles, when present, were consistently negative for HH1.5.
4. Discussion Histone H1 is a family of linker histone proteins, consisting of 11 subtypes that directly interact with the DNA between nucleosome particles, also known as linker DNA [14]. Their function appears to be of 2 major roles: a general one in forming and stabilizing higher-order chromatin structures, which is shared by all subtypes, and a more subtype-specific function involving gene regulation [14,15]. The latter results from alteration of chromatin structure [10], DNA methylation, or direct interaction with transcription
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2117
Fig A, High-grade prostatic intraepithelial neoplasia demonstrating focal and predominately weak nuclear reactivity for HH1.5 with occasional cells showing moderate reactivity (immunohistochemistry, original magnification ×200). B, Gleason pattern 3 prostate adenocarcinoma (right) showing nuclear expression of HH1.5 with adjacent nonreactive benign prostate glands (left). Scattered lymphocytes in benign glands and stroma serve as an internal control (immunohistochemistry, original magnification ×100). C, Prostate adenocarcinoma, Gleason pattern 3 (left), exhibiting nuclear reactivity for HH1.5 and reactive lymphoid aggregate (lower right) acting as an internal control (immunohistochemistry, original magnification ×100). D, Focal low-grade prostate adenocarcinoma (top right) with nuclear expression of HH1.5 and adjacent nonreactive benign prostate glands (immunohistochemistry, original magnification ×100). E, Diffuse and strong nuclear expression of HH1.5 in Gleason pattern 4 adenocarcinoma (immunohistochemistry, original magnification ×100 and ×400 [inset]). F, Benign nonreactive prostate glands surrounded by Gleason pattern 5 prostate adenocarcinoma that is strongly and diffusely reactive for HH1.5 (immunohistochemistry, original magnification ×100). G, Metastatic prostate adenocarcinoma to T8 vertebra showing mature bone fragment with surrounding tumor cells demonstrating strong nuclear reactivity for HH1.5 (immunohistochemistry, original magnification ×400).
factors [12,14,15]. Recently, specific histone subtypes have been implicated in tumorigenesis [18–20], which will be discussed later on.
HH1.5 is one of 7 somatic subtypes, which is present in all cells albeit with varying expression levels, depending on the cell type and degree of differentiation [10–12,15,17].
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V. Khachaturov et al.
Table 1 HH1.5 expression in prostate cancer, benign glands, and HPIN Prostate cancer HH1.5 Total a b c
Benign
a
b
68 (93%) 73
7 (9%) 75
HPIN 23 c (100%) 23
Strong nuclear positivity. Weak nuclear positivity. Mostly weak nuclear positivity.
Ongoing studies reveal that HH1.5 is not immunohistochemically expressed in all cells, nor is it specific for prostate cancer. Differential immunohistochemical expression of HH1.5 has been reported in pulmonary neuroendocrine tumors, particularly those of higher grade [18]. Additionally, HH1.5 was one of several mutated genes discovered from sequencing of colorectal carcinoma [21]. This study demonstrates that immunohistochemical detection of HH1.5 is common in prostate cancer cells and rare in benign prostatic epithelium. The positive staining exhibited in rare benign prostatic glands was focal and weak. Similarly, HPIN showed mostly weak reactivity for HH1.5 with occasional cells demonstrating moderate nuclear reactivity. Importantly, the staining observed in benign prostatic glands and HPIN was distinctly different from the strong nuclear reactivity observed in prostatic adenocarcinomas. Rare false-negative results were encountered with HH1.5; however, false-positive results showing moderate-tostrong nuclear reactivity were not observed. Higher grade Gleason patterns had a higher incidence of false-negatives than compared with lower grade Gleason patterns. The explanation for this is currently unclear. There were no cases with moderate-to-strong HH1.5 expression and less than 11% involvement of prostatic glands. However, if such cases are encountered, it may be best to regard them as atypical or suspicious for prostatic adenocarcinoma and further work them up. Lymphocytes, when present, serve as an excellent internal control. Cases with significant chronic inflammation should be interpreted cautiously, to prevent erroneous interpretation of lymphocytes for individual tumor cells. Histomorphologic correlation is paramount.
Table 2 HH1.5 intensity: Gleason pattern 3 vs. 4/5 vs. metastatic prostate cancer Intensity
Gleason 3
Gleason 4/5
Metastatic
Neg 1+ 2+ Total
2 (3%) 24 (39%) 35 (57%) 61
6 (13%) 7 (15%) 34 (72%) 47
0 0 3 a (100%) 3
NOTE. Intensity of HH1.5 is based on percentage of nuclear reactivity of prostatic glands: negative, 0% to 10%; 1+, 11% to 50%; 2+, N50%. a All cases were Gleason pattern 4 or 5.
Interestingly, HH1.5 exhibited an overall increased immunoreactivity in high-grade Gleason patterns 4 and 5 prostate adenocarcinoma and metastatic cases as compared to low-grade Gleason pattern 3 prostate cancers. These findings indicate not only a diagnostic but also a potential prognostic role of HH1.5. Pluripotent cells show higher HH1.5 expression than differentiated cells [15]. Therefore, increased HH1.5 expression in prostate cancer may contribute to its self-renewal mechanism. Aside from HH1.5, histone H1, a terminally differentiated histone protein variant, has also been implicated in prostate and breast cancer [12,19]. Histone H1 immunohistohemical expression positively correlated with the Gleason grade, Ki67 positivity, and androgen receptor expression in prostate cancer [19]. However, its expression in normal luminal cells, basal cells, and stromal cells precludes its diagnostic utility for prostate cancer [19]. Routine examination of prostate core needle biopsies for cancer consists of evaluating 3 main histological parameters: glandular architecture, cytologic features, and absence of a basal cell layer. The presence of the basal cell layer may be difficult to appreciate on routine hematoxylin and eosin sections. Immunohistochemical staining with high-molecular-weight cytokeratins (34βE12) and p63 can highlight the presence or absence of the basal cell layer [7]. Prostate cancer by definition lacks a basal cell layer. However, atypical adenomatous hyperplasia, atrophy, HPIN, and some morphologically benign prostatic glands may show partial or complete absence of basal cell staining [3,5,22,23]. Therefore, the absence of basal cell staining in a subset of cases may lead to an erroneous diagnosis of malignancy. AMACR is involved in β-oxidation of branched fatty acids and is overexpressed in prostate cancer [5,24]. It can be used as an immunohistochemical marker of prostate cancer, but it shows variable sensitivity and specificity [6,8,9]. Additionally, noncancerous lesions such as HPIN, atrophic glands, and AAH have been shown to be positive with AMACR in 64%, 36%, and 18%, respectively [6]. HPIN may show moderate to even strong staining with AMACR [9]. In our experience, AMACR has approximately 75% sensitivity and specificity. AMACR immunohistochemical staining limitations may hinder a correct diagnosis in a subset of small foci of prostate cancer or in one of its benign mimickers. In summary, this is the first study using HH1.5 immunohistochemistry in distinguishing prostate cancer from benign prostatic glands, with sensitivity and specificity superior to those of AMACR. Other advantages of HH1.5 over AMACR include clear nuclear staining and lymphocytes serving as an internal control. For small foci of prostate cancer, HH1.5 may be a useful tool in combination with hematoxylin and eosin and other immunological markers, particularly cytoplasmic staining with high–molecular weight cytokeratins. Further studies are needed to confirm these findings and investigate the histoprognostic significance of HH1.5 in prostate cancer.
HH1.5, a novel prostatic cancer marker
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