Malignant Transformation and Stromal Invasion from Normal Appearing Prostate Tissues: True or False?

The human prostate epithelium, which is the histological origin of most prostate malignancies, is physically separated from the stroma by a layer of basal cells and the basement membrane. Basal cells are inter-connected by intercellular junctions and adhesion molecules, constituting a continuous sheet encircling luminal cells [1-2]. The basement membrane is composed of type IV collagen, laminins, and other molecules, forming a continuous lining surrounding and attaching to the basal cell layer [3-4] (Fig 1). The basement membrane and the basal cell layer are intermixed to form a dense fibrous capsule surrounding all epithelial cells. Due to these relationships, disruption of the basal cell layer and the basement membrane is a pre-requisite for tumor invasion or metastasis. It is a commonly held belief that prostate carcinogenesis progresses sequentially from normal to hyperplasia, to prostatic intraepithelial neoplasia (PIN), and to invasive or metastatic lesions [5-8]. Progression from PIN to invasion is believed to be triggered by overproduction of proteolytic enzymes primarily by cancer cells, which cause degradation of the tumor capsule [9-10]. These theories are consistent with laboratory findings from cell cultures and animal models, whereas are hard to reconcile with a number of critical facts. First, previous studies revealed that some healthy men between 19 and 29 years old had a spectrum of proliferative lesions, including hyperplasia, PIN, and incipient adenocarcinoma [11-13]. Second, recent studies have detected a DNA phenotype identical to that of invasive prostate cancer in some “healthy” men, and also in normal prostate tissues adjacent to prostate cancer [14-17]. Third, a majority of PIN express high levels of proteolytic enzymes, but only 10-30% of untreated PIN progress to invasive lesions during patients’ lifetime [1821]. Fourth, cancer of unknown primary site is one of the 10 most frequent cancers worldwide and the 4th most common cause of cancer deaths [22-24]. Together, these facts argue that the linear model of carcinogenesis [5-8] and enzyme theory of tumor invasion [9-10] are not universally applicable to all prostate cancer cases. These facts also suggest that the past efforts to classify tumor progression and invasion purely based on the profiles of epithelial cells may have overlooked some essential factors. As over 90% of prostate cancer related mortality result from invasion related diseases, and the incidence of PIN could be up to 16.5%-25% in prostate biopsies [25-27], there is an urgent


Introduction
The human prostate epithelium, which is the histological origin of most prostate malignancies, is physically separated from the stroma by a layer of basal cells and the basement membrane. Basal cells are inter-connected by intercellular junctions and adhesion molecules, constituting a continuous sheet encircling luminal cells [1][2]. The basement membrane is composed of type IV collagen, laminins, and other molecules, forming a continuous lining surrounding and attaching to the basal cell layer [3][4] (Fig 1). The basement membrane and the basal cell layer are intermixed to form a dense fibrous capsule surrounding all epithelial cells. Due to these relationships, disruption of the basal cell layer and the basement membrane is a pre-requisite for tumor invasion or metastasis. It is a commonly held belief that prostate carcinogenesis progresses sequentially from normal to hyperplasia, to prostatic intraepithelial neoplasia (PIN), and to invasive or metastatic lesions [5][6][7][8]. Progression from PIN to invasion is believed to be triggered by overproduction of proteolytic enzymes primarily by cancer cells, which cause degradation of the tumor capsule [9][10]. These theories are consistent with laboratory findings from cell cultures and animal models, whereas are hard to reconcile with a number of critical facts. First, previous studies revealed that some healthy men between 19 and 29 years old had a spectrum of proliferative lesions, including hyperplasia, PIN, and incipient adenocarcinoma [11][12][13]. Second, recent studies have detected a DNA phenotype identical to that of invasive prostate cancer in some "healthy" men, and also in normal prostate tissues adjacent to prostate cancer [14][15][16][17]. Third, a majority of PIN express high levels of proteolytic enzymes, but only 10-30% of untreated PIN progress to invasive lesions during patients' lifetime [18][19][20][21]. Fourth, cancer of unknown primary site is one of the 10 most frequent cancers worldwide and the 4 th most common cause of cancer deaths [22][23][24]. Together, these facts argue that the linear model of carcinogenesis [5][6][7][8] and enzyme theory of tumor invasion [9][10] are not universally applicable to all prostate cancer cases. These facts also suggest that the past efforts to classify tumor progression and invasion purely based on the profiles of epithelial cells may have overlooked some essential factors. As over 90% of prostate cancer related mortality result from invasion related diseases, and the incidence of PIN could be up to 16.5%-25% in prostate biopsies [25][26][27], there is an urgent www.intechopen.com need to uncover the intrinsic mechanism of tumor invasion and to distinguish aggressive and indolent PIN for optimal or personalized treatment. Unfortunately, none of the current approaches could predict which PIN lesions will progress [28][29][30][31]. The only established approach to monitor PIN progression is repeat biopsy [28][29][30][31], which is costly and painful. Fig. 1. Structural relationships among the epithelium (circles), basal cell layer (thick arrows), basement membrane (thin arrows), and stroma (asterisks). Human prostate tissue sections were double immunostained for collagen IV (brown) and cytokeratin 34βE12 (red). A and C: 150X. B and D: a higher (500X) magnification of A and C.
Promoted by the fact that the basal cell layer is the sole source of tumor suppressor p63 and maspin [32][33][34][35], and that degradation of the basal cell layers is a pre-requisite for tumor invasion, our resent studies have attempted to identify early signs of basal cell degradation. Our initial study examined the physical integrity of the basal cell layers in 50 patients with co-existing pre-invasive and invasive prostate tumors. Of 2,047 ducts and acini examined, 197 were found to harbor focal disruptions (the absence of basal cells resulting in a gap greater than the combined size of at least 3 basal cells) in their basal cell layers. The frequency of focal basal cell layer disruptions (FBCLD) varied from none in 22 (44%) cases to over 1/3 of the ducts or acini with FBCLD in 17 (34%) cases (Table 1) [36]. Of the 17 cases with a high frequency of FBCLD, 5 harbored large acinar or duct clusters that are morphologically normal in H& E stained sections, but all harbored focal disruptions in the surrounding capsule in immunostained sections. As shown in Fig 2a,

D. significantly elevated leukocyte infiltration:
In sections double immunostained for CK 34βE12 and leukocyte common antigen (LCA), most structures with FBCLD showed leukocyte infiltration, but most structures with non-disrupted layers had no leukocyte infiltration (Table 4). Most leukocytes were located near FBCLD (Fig 6).   Table 4. Leukocyte infiltration in epithelial structures with and without focal basal cell layer disruption

E. a total loss of the expression of all basal cell phenotypic markers:
In addition to focal alterations, the entire basal cell layer in some epithelial structures of some cases showed degenerative changes. These basal cell layers were morphologically distinct, surrounding PIN or normal-appearing duct or acinar clusters (Fig 7). All the basal cells, however, lacked the expression of basal cell specific markers (Fig 7). Epithelial associated with these basal cell layers often showed malignant cytology, including enlarged nuclei and nucleoli. In addition, these basal cell layers were devoid of expression of PCNA, in contrast to normal basal cells and associated tumor cells, which were strongly positive for PCNA (Fig. 8).
Epithelial structures with altered basal cell layers often had mast cell infiltration, which was not seen in structures with intact basal cell layers (Fig 8). Together, these findings suggest that focally disrupted basal cell layers are likely to be under degeneration. As the basal cell layer is the sole source of several tumor suppressors [29][30][31][32], degenerated basal cells are very likely to have impaired or reduced paracrine inhibitory functions on tumor cell growth and invasion. In contrast to degenerative alterations in basal cells, luminal cells overlying FBCLD showed several signs of proliferative alterations that were not seen in their adjacent counterparts distant from the disruptions: A. significantly elevated proliferation: In section double immunostained for basal cell and proliferation markers, epithelial structures with FBCLD had a significantly higher proliferation index than their morphologically similar counterparts without FBCLD, and most proliferating cells were located at or near FBCLD ( Fig.9; Table 5).  Table 5. Cell proliferation in epithelial structures with and without focal basal cell layer disruption

Duct or acinar type
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B. significantly higher expression of malignancy-and tumor invasion-related molecules:
Elevated expression of prostate specific antigen (PSA) and alpha-methylacyl-CoA racemase (AMACR), are seen in cells overlying FBCLD ( Fig.10a & b), and also in normal ducts lacked the expression of basal cell markers ( Fig. 10c & d). In contrast, cells within the same duct, and adjacent ducts with intact basal cell layers were negative (Fig 10).

C. physical continuity with, and morphological resemblance to, invasive prostate cancer:
A vast majority of these normal appearing acinar or duct clusters were immediately adjacent to, or blended with, invasive cancers. In some cases, cells overlying FBCLD had significantly enlarged nuclei and nucleoli, and were often in physical continuity with, or morphologically similar to, their adjacent invasive counterparts (Fig 11).
In some cases, multiple epithelial cell nests appeared to be "budding" from the same acinus or duct (Fig.12). These "budding" cells had a higher proliferation and were similar to adjacent invasive cancer cells. The only difference was that "budding" cells are often associated with residual basal cells (Fig 12, thin arrows).

D. Significantly higher expression of chromogranin A:
In sections double immunostained for CK 34βE12 and chromogranin A, a neuroendocrine differentiation-related marker correlating with tumor progression and the status of hormone refractoriness [45][46][47], chromogranin A positive cells were exclusively or preferentially seen in epithelial structures with FBCLD (Fig 13). Compared to morphologically similar counterparts, microdissected epithelial structures with chromogranin A-positive cell clusters had a more than 5-and 7fold lower expression of Micro-RNAs 146a and 146b-5p (miR-146a and miR-146b-5p; Fig 14), which have been documented to correlate with prostate tumor invasion and hormone refractoriness [38].

E. significantly elevated expression of tumor invasion-related genes:
Compared to their adjacent counterpart associated with the residual basal cell layer within the same duct, microdissected cell clusters overlying FBCLD consistently had significantly higher expression of cell proliferation, apoptosis, angiogenesis, immuno-response, and stem cell related genes [38] (Fig 15; Table 6).   Table 6. Differentially expressed genes between cells overlying FBCLD and their adjacent cells The above alterations were consistently seen in all 17 cases with a high frequency of FBCLD, while were only seen in 1 (9.1%) of the 11 cases with a low frequency of FBCLD, and in none of the 22 cases with non-disrupted basal cell layers. Together, these findings suggest that the physical and functional status of the basal cell layer significantly impact the biological presentation of associated epithelial cells. These findings also suggest that malignant transformation and stromal invasion could occur in morphologically normal prostate tissues, and that FBCLD may represent a trigger factor for prostate tumor progression and invasion. To our best knowledge, our findings have not been previously reported by others. The most likely reasons are: [1] the enzyme theory has dominated the direction of researches in the field, and the roles of basal cells have been ignored, and [2] these alterations can be seen only by double immunohistochemistry to simultaneously elucidate the basal and epithelial cells. Double immunostaining, however, has not been commonly used in the clinical studies.

Our hypothesis of tumor invasion
Based on the above findings, we strongly believe that these normal appearing epithelial structures represent a population of maturation arrested tumor progenitors derived from monoclonal proliferation of genetically altered primitive stem cells at early stages of the prostate morphogenesis probably by trauma, radiation, inflammation, or other factors. These clusters retain the potential for unlimited cell proliferation or multi-lineage differentiation, and could progress directly to invasive lesions through two pathways: (1) In situ malignant transformation, and (2) Progenitor-mediated cell budding. These pathways are likely to contribute to early onset of prostate cancer at young ages, to biologically and clinically more aggressive prostate tumors, and also to highly heterogeneous genetic and biochemical profiles among prostate tumors.

The hypothesized main steps of tumor invasion
Our hypothesized main steps of invasion for these normal appearing epithelial structures are the followings: 1. At the early stage of prostate morphogenesis, the prostate of these patients exposed to external or internal insults, such as radiation, carcinogens, localized trauma, inflammation, or other factors, which caused permanent damages in the DNA structures of some primitive stem cells. 2. Localized DNA structural damages caused the inactivation of, or defects, in basal cell renewal-related genes, which impaired the basal cell replenishment process to replace the aged or injured basal cells, resulting in a "senesced" basal cell population with significantly reduced functions. 3. Localized DNA structural damages also caused the inactivation of, or defects in, apoptosis-, or cell cycle control related genes in the luminal cell population, which allow these cells to escape from programmed death, to continuously proliferate, and to generate their own vascular structures. 4. Deregulated proliferation in epithelial cells and impaired self-renewal in basal cells resulted in the overstretch and disassociation of the basal cell layer and the basement membrane, which lead to focal breakdown and degeneration of these two structures. 5. The degradation products of degenerated basal cells and the diffusible molecules of the overlying luminal cells function as self-epitopes to attract migration and infiltration of immunoreactive cells or auto-antibodies into the affected sites. 6. The direct physical contact between IRC and degenerated basal cells results in the discharge of the digestive enzymes from IRC, leading to the physical destruction of altered basal cell layers and the local basement membrane, resulting in a focal disruption in these structures. 7. As the epithelium is normally devoid of blood vessels and lymphatic ducts, and the basal cell layer is the sole source of several tumor suppressors, a FBCLD could lead to several focal alterations, including: a. A loss or reduction of tumor suppressors and the paracrine inhibitory functions, which allow the luminal cells to undergo elevated proliferation [48][49][50][51][52]. b. Alterations in the permeability for oxygen or growth factors, which selectively triggers the exit of stem or progenitor cells from quiescence, and favor proliferation of cells overlying FBCLD [53][54][55]. c. The exposure of luminal cells to different cytokines, which facilitates vasculogenic mimicry and tumor angiogenesis [56][57].
d. The physical contact between luminal and stromal cells augments the expression of stromal MMP, facilitating epithelial-mesenchymal transition (EMT) and cell motility [58][59][60]. e. The physical contact between luminal and immunoreactive cells directly cause genomic or cellular damages that trigger a cascade reaction of malignant transformation [61][62][63][64]. 8. These alterations could individually or collectively trigger malignant transformation and stromal invasion through two different, but correlated pathways: a. In situ malignant transformation, in which the entire basal cell layer undergoes extensive degeneration and multiple focal disruptions (Fig 16) that expose the entire luminal cell population directly to the stroma when all surrounding myoepithelial cells become degenerated. Although these cells might not possess all properties of invasive cancer cells, the changed microenvironment may act as a second "hit" to trigger a cascade reaction of malignant transformation that rapidly alters the genetic and biochemical profiles of these cells. b. Progenitor-mediated cell budding, in which focal basal cell degeneration-induced lymphocyte infiltration causes focal disruptions in the tumor capsules, which selectively favor proliferation and invasion of the overlying tumor stem cells or a biologically more aggressive cell clone. Cells overlying FBCLD gradually increases in volume and form finger-like projections invading into the surrounding stroma (Fig 17). 9. The above events may occur and progress immediately after the external or internal insults, leading to the onset of prostate cancer at young ages. On the other hand, cells of these normal appearing epithelial structures may become maturation-arrested after a few cycles of cells divisions, and remain idle until a new insult [65], representing "bad seeds for bad crops" at later ages. www.intechopen.com

The main differences between our hypothesis and other theories for tumor invasion
Based on our own and other findings, we had proposed that prostate tumor invasion is triggered by focal basal cell degeneration-induced aberrant infiltration of lymphocytes that causes focal disruptions in the tumor capsules, which selectively favor monoclonal proliferation and invasion of the overlying tumor stem or progenitor cells [38][39]. Our hypothesis differs from the enzyme theory of tumor invasion in five aspects: (1) the stage of tumor invasion, (2) the cellular origin of the invasive lesions, (3) the significance of the immunoreactive cells, (4) the significance of stromal cells, and (5) the potential approaches for interventions and prevention of tumor invasion. Our studies of breast tumors have obtained similar results and conclusions [66][67][68][69][70][71][72][73][74]. The new hypothesis presented in our current study is the expansion of our previous hypothesis with the following new points of views: A. the preservation of large clusters of genetically damaged stem or progenitor cells: Our previous hypothesis proposes that cell clusters overlying FBCLD represent tumor stem or progenitor cells, which undergo a series of immunohistochemical and morphologic changes, and finally transform into invasive lesions [38]. Our current hypothesis suggests that it is also possible that multiple genetically damaged primitive stem or progenitor cells within the same site may generate large duct or acinar clusters that harbor the same genetic defects. These clusters may be formed immediately followed the external or internal insults during the early stage of morphogenesis, and progress rapidly, leading to the early onset of prostate cancer at young ages. These clusters could also become maturation arrested at patients' early ages, while they retain the potential for unlimited proliferation and multi-lineage differentiation, representing "bad seeds for bad crops" at later ages. Our speculation is supported by the results of our gene expression profiling studies, which have detected a over 5-fold higher expression of two stem cell lineage markers, KIT and NCOR2, in cell clusters overlying FBCLD compared to their adjacent counterpart associated with the residual basal cell layers [38] (Fig 15; Table 6).

B. direct transformation of the entire duct or acinar cluster into invasive lesions:
Our previous hypothesis believes that invasive cells are derived exclusively or preferentially from monoclonal proliferation of stem or progenitor cells overlying FBCLD [38]. Our current hypothesis suggests that, in addition to monoclonal proliferation, it is possible that the entire duct or acinar cluster may directly transform into invasive lesions after the disappearance of all surrounding basal cells and the basement membrane. C. angiogenesis by genetically altered tumor stem cells: Our previous hypothesis proposes that a subset of luminal cell clusters overlying FBCLD are in direct physical continuity with vascular-or lymphatic duct-like structures, which allows them to progress directly to metastasis [38]. Our current hypothesis further suggests that some normal appearing duct or acinar clusters may retain genetically damaged primitive stem cells that could manufacture their own blood vessels or lymphatic ducts, which directly lead to metastasis. Our speculation is consistently supported by our immunohistochemical findings, which show that some of the cell clusters overlying FBCLD appear to directly invade the vascular structures (Fig 11). Our speculation is also supported by the results of our gene expression profiling studies, which have detected a over 6-and 12-fold higher expression of two endothelial cell markers, END and ICAM2, respectively, in cell clusters overlying FBCLD compared to their adjacent counterpart associated with the residual basal cell layers [38] (Fig  15; Table 6). D. Potential histological origin for cancer of unknown primary site: Cancer of unknown primary site (CUP) is one of the 10 most frequent cancers worldwide and ranks as the 4 th most common cause of cancer-related death [22][23][24]. The development of early, uncommon, systemic metastasis, and resistance to therapy are hallmarks of this clinical entity. Currently, no consensus exists on whether CUP is a group of metastatic tumors with unidentified primaries or a distinct entity with unique genetic/phenotypic aberrations that define it as "primary metastatic disease [22][23][24]. The normal appearing epithelial structures seen in our current study may represent a potential histological origin for CUP for the following reasons: (1) they are morphologically indistinguishable from clear-cut normal prostate tissues under low magnification on H & E stained sections, which allow them to escape from early detection, (2) they retain the property of stem cells and appear to be able to directly invade the stroma and vascular structures, based on our previous [38] and current studies, and (3) they share the same DNA phenotype with invasive prostate cancer based on previous reports [14][15][16][17].

The significance of our hypothesis
During the past 30-years, the cancer research community has been predominantly, if not exclusively, focused on the roles of epithelial cells in prostate tumor progression and invasion. Hundreds and thousands of epithelium-derived molecules have been implicated in the development and progression of prostate cancers. However, only prostate-specific antigen (PSA) has been approved and validated as a clinical diagnostic marker and only growth factor-and androgen receptor-based therapeutic agents have been approved and validated for the clinical trials for prostate cancers. In addition, none of epithelium-derived markers has significant value in predicating the tumor biology or invasiveness, or in identifying the specific individuals with pending prostate cancer, or at higher risk to develop prostate cancer. These findings strongly suggest that the epithelium alone is very unlikely to be sufficient to trigger tumor progression and invasion.
Our hypothesis, if confirmed, could have several significant implications. Scientifically, it could lead to a new direction to explore novel approaches for early detection, intervention, and prevention of prostate tumor invasion. For example, as non-disrupted basal cell layers have significant inhibitory functions on epithelial cell growth, the development of therapeutic agents to stimulate basal cell growth or regeneration may provide a more effective approach for treatment and prevention of prostate cancer invasion. Clinically, it could potentially bring the following benefits: 1. Better recognition of these clusters may avoid misdiagnosis and facilitate early interventions, which may significantly improve prognosis. 2. Double immunohistochemical staining to assess the physical integrity of the basal cell layer, or an quantitative measurement of basal cell degeneration-related molecules in the blood or biopsies, may facilitate early identification of individuals at greater risk to develop invasive lesions. 3. As genetic alterations not only define the scope and extent of, but also precede, both biochemical and morphological alterations, elucidation of the genetic profile of these normal appearing duct or acinar clusters may lead to the identification of the specific molecules that trigger the initiation of prostate carcinogenesis, progression, and invasion. 4. Currently, the only established approach to monitor prostate tumor progression is repeat biopsy [28][29][30][31], which is costly and painful. Our technical approaches of assessing the physical and functional status of the basal cells may be used as a more reliable alternative for repeat biopsy to monitor tumor progression and invasion. More importantly, our hypothesis may be also applicable to progression and invasion of other epithelium derived tumors.

Acknowledgment
This study was supported in part by grants DAMD17-01-1-0129, DAMD17-01-1-0130, and PC051308 from the US Congressionally Directed Medical Research Programs, grant BCTR0706983 from The Susan G. Komen Breast Cancer Foundation, grant 05AA from the AFIP/ARP joint research initiative project, grant 2008-02 from the US Military Cancer Institute and Henry Jackson Foundation, and grant 2006CB910505 from the Ministry of Chinese Science and Technology Department. The tissue blocks or unstained tissue sections used in this study were obtained from our antibody testing service or collaborative research projects with the National Cancer Institute, Toms Jefferson University Medical School, Fairfax Hospital, Howard University Hospital, and Beijing 301 Hospital. The study was conducted in the Armed Forces Institute of Pathology with IRB approved protocols (05-AA and 07-DJ).