the normal prostate luminal cells , which are the histological origin of most prostate 
                 malignancies , are physically separated from the stroma by the basal cells and 
                 basement membrane ( bm ) .
basal cells are joined by intercellular junctions and 
                 adhesion molecules , constituting a continuous sheet encircling luminal cells 1 - 2 .
the 
                 bm is composed of type iv collagen , laminins , and other molecules , forming a 
                 continuous lining surrounding and attaching to the basal cell layer 3 - 4 .
the 
                 epithelium is normally devoid of blood vessels and lymphatic ducts , and totally 
                 relies on the stroma for its metabolic and even survival needs .
due to these 
                 structural relationships , the disruption of both the basal cell layer and the bm is 
                 a pre - requisite for prostate tumor invasion .
it is a commonly held belief that human prostate tumor invasion is a multistage 
                 process , progressing sequentially from normal to hyperplasia , to prostatic 
                 intraepithelial neoplasia ( pin ) , and to invasive or metastatic stages 5 - 8 . 
                
progression from pin to invasion is believed to be triggered by cancer cells that 
                 increasingly produce proteolytic enzymes with tumor progression , which cause 
                 degradation of the bm 9 - 10 .
these theories are consistent with results of studies in 
                 tissue cultures and animal models , whereas are hard to interpret the following 
                 critical facts : ( 1 ) our 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 , ( 2 ) recent studies detected a dna phenotype 
                 that is identical to that of invasive prostate cancer in some 
                  healthy  men , and in morphologically normal prostate tissues 
                 adjacent to prostate cancer 14 - 17 , ( 3 ) a vast majority of pin express high 
                 levels of proteolytic enzymes , while only 10 - 30% of untreated pin progress to 
                 invasive lesions during patients ' lifetime 18 - 21 . unfortunately , none of the 
                 current approaches could predict which pin lesions will progress 22 - 25 . 
                
the only established approach to monitor pin progression is repeat biopsy 22 - 25 , 
                 which is costly and painful , and ( 4 ) results from all at clinical trials of prostate 
                 cancer treatment or prevention with corresponding proteolytic enzyme inhibitors have 
                 been very disappointing 26 - 28 .
together , these facts argue that alternative pathways of prostate tumor progression 
                 and invasion may exist or even play more direct roles .
since over 90% of prostate 
                 cancer related mortality result from invasion - related illness , and the incidence of 
                 pin could be up to 16.5%-25% prostate biopsies 24 - 28 , there is an urgent need to 
                 uncover the intrinsic mechanism of tumor invasion .
promoted by the fact that the 
                 basal cell layer is the sole source of tumor suppressor p63 and maspin 29 - 32 , 
                 and that degradation of basal cell layers is a pre - requisite for tumor invasion , our 
                 recent studies have attempted to identify early signs of basal cell degradation .
our 
                 initial study examined the physical integrity of 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 cases to over 1/3 of the ducts or acini with fbcld in 17 
                 cases 33 . compared to their non - disrupted counterparts ,
focally disrupted basal cell layers 
                 showed a significantly lower frequency of tumor suppressor expression and 
                 proliferation , but a significantly higher rate of degeneration and leukocyte 
                 infiltration 33 .
in contrast , epithelial 
                 cells overlying focally disrupted basal cell layers had a significantly higher rate 
                 of proliferation and expression of tumor invasion related genes 33 - 34 . 
                 based on these and other findings , we have proposed that prostate tumor invasion or 
                 progression is triggered by fbcld induced auto - immunoreactions , which facilitate 
                 formation of more aggressive cell clones or monoclonal proliferation of tumor stem 
                 cells overlying focally disrupted basal cell layers .
our hypothesis and supporting 
                 data have been recently published in multiple peer - reviewed journals 33 - 36 . 
                
as the basement membrane surrounds and attaches to the basal cell layer , our current 
                 study attempted to assess whether fbcld would impact the physical integrity of the 
                 associated basement membrane .
formalin - fixed and paraffin embedded tissue blocks from 25-human prostate tumors with 
                 both pre - invasive and invasive components were selected from our previous studies 
                     33 - 36 .
consecutive sections at 4 m thickness were prepared and placed 
                 on positively charged slides .
the first and last sections from each case were 
                 stained with hematoxylin and eosin ( h&e ) for morphological classification , 
                 based on our published criteria 11 . to identify focal basal cell layer disruptions ( fbcld ) , two sections from each case 
                
were subjected to double immunohistochemistry with basal cell phenotypic markers p63 
                 ( clone : 4a4 ; cell marque , foster city , ca ) at a 1:50 dilution and cytokeratin ( ck ) 
                 34e12 ( clone : m0630 ; dako , carpinteria , ca ) at a 1:50 dilution according 
                 to the manufacturers ' protocols .
a fbcld was defined as the focal absence of basal cells 
                 resulting in a gap larger than the combined size of at least three basal cells in at 
                 least two immediate adjacent sections . to identify the potential impact of fbcld on the physical integrity of the associated 
                 basement membrane
, two sections immediate adjacent to double immunostained ones that 
                 harbored fbcld from each case were subjected to double immunohistochemistry to 
                 simultaneously elucidate the basal cell layer and the basement membrane using a 
                 previously published protocol .
briefly , deparaffinized sections were incubated in 1x 
                 antigen retrieval solution ( cat # : rv1000 m ; biocare medical , concord , ca ) overnight 
                 ( ? ) at 70 in a regular oven .
after incubation , the sections were washed 
                 in tap water and pbs ( ph 7.4 ) , each for 5 - 10 minutes , and then , incubated with 
                 antibodies to p63 ( at a 1:50 dilution ) and ck 34e12 ( at a 1:50 dilution ) 
                 for 2 - 3 hours at room temperature .
after the incubation , the sections were washed in 
                 three changes of pbs , each for 2 - 3 minutes , and then , incubated with the 
                 corresponding secondary antibody .
the antigen and antibody complex was detected with 
                 an abc detection kit and a dab chromogen kit ( vector laboratories , burlingame , ca ) , 
                 according to the instructions provided by the manufacturer .
after chromogen 
                 reaction , the sections were washed in tap water and pbs , each for 5 - 10 minutes . 
                
then , the sections were incubated with proteinase k ready - to - use solution ( cat # : 
                 s3020 ; dako , carpinteria , ca ) at room temperature for 3 - 5 minutes . after the 
                 proteinase k digestion
, sections were incubated with mouse monoclonal antibodies to 
                 collagen iv ( clone : civ 22 ; dako ; carpinteria , ca ) at a 1:50 dilution or laminin 
                 ( clone : vp - l551 ; vector laboratories , burlingame , ca ) at a 1:25 dilution at room 
                 temperature for 2 - 3 hours . after the incubation
, the sections were washed in three 
                 changes of pbs , each for 2 - 3 minutes , and then , incubated with the corresponding 
                 secondary antibody .
the antigen and antibody complex was detected with an abc 
                 detection kit and an ap red - chromogen kit ( cat # : 00 - 2203 ; zymad , south san 
                 francisco , ca ) according to the instructions provided by the manufacturers . to assess the specificity of the immunostaining
first , different negative controls were used , which included ( 1 ) the 
                 substitution of the primary antibody with normal serum , ( 2 ) the omission of the 
                 secondary antibody from the immunostaining sequence , ( 3 ) serial dilutions of the 
                 primary antibody , and ( 4 ) the inclusion of sections from normal lymph - nodes in the 
                 normal immunostaining process .
second , the same immunostaining protocol was used on 
                 the same cases , but substituting with different detection system and substrates . 
                
third , the immunostaining procedure was repeated at least twice using the same 
                 protocol and under the same condition and immunostained sections were independently 
                 evaluated by at least two investigators . using p63 and cke12 double immunostained sections as references , the 
                 corresponding sites of fbcld in sections double immunostained for both basal cell 
                 phenotypic and basement membrane markers were photographed , and large prints were 
                 made and examined , to determine whether alterations in the basal cell layer and the 
                 basement membrane are correlated events .
correlated alterations of the basal cell 
                 layer and basement membrane were defined as a simultaneous focal loss of both 
                 structures on the same sites .
distinct immunoreactivities to collagen or laminin were preferentially seen 
                 in the basement membrane , but were also seen in the stroma and blood vessels , which 
                 contain abundant collagen and laminin as structural elements .
all negative controls 
                 completely lacked distinct immunoreactivities to any of the markers used . in sections
double immunostained for basal cell phenotypic and basement membrane 
                 markers , both p63 and cke12 could elucidate the basal cell layer , while 
                 p63 appeared to be able to better differentiate between the basal cell layer from 
                 the basement membrane ( fig 1 ) .
a vast majority 
                 of the ducts and acini with normal morphology or with hyperplastic or pin lesions 
                 contained a non - disrupted basal cell layer and a continuous basement membrane , 
                 whereas their adjacent invasive lesions laced both ( fig 1 ) .
the physical integrity of the basal cell layer and basement 
                 membrane appeared to be largely independent of the ductal or acinar lumen or tumor 
                 size ( fig 1e-1h ) .
of a total of 89 fbcld encountered , 76 ( 85 % ) showed focal disruption or 
                 fragmentations in the overlying basement membrane ( table 1 ) , whereas none showed the integrity of basement membrane is 
                 impaired while the basal cell layer is normal . over 60% of the focal disruptions in 
                 both the basal cell layer and the overlying basement membrane
were seen in pin ( fig 
                     2 ) , while about 30% of these focal 
                 disruptions were seen in ducts or acini with benign morphology ( fig 3 ) .
the size of these focal disruptions varied 
                 substantially , from a few cells ( fig 2a-2d ) to more than a half of the entire basal cell 
                 layer and the basement membrane ( fig 2e-2h ) .
the size of these focal disruptions in 
                 normal or hyperplastic lesions was generally small and varied in numbers ( fig 3 ) .
the basement membrane overlying the remaining 13 ( 15% ) fbcld showed significant 
                 attenuation or reduction of the immunostaining intensity , compared to its adjacent 
                 counterpart overlying the non - disrupted basal cell layer ( fig 4 ) .
the basement membrane in all or nearly all ducts or acini 
                 with p63 positive basal cells was substantially thicker and more uniform than that 
                 in ducts or acini without p63 positive basal cells ( fig 4a-4b ) , and also , a vast 
                 majority of the focal disruptions occurred near basal cells that lack p63 expression 
                 ( not shown ) .
the pattern and frequency of fbcld seen in our current study are in total agreement 
                 with those of our previous studies , and also those from other groups .
our findings 
                 of the loss or fragmentations of the basement membrane are also in line with 
                 previous reports . to our best knowledge ,
our finding of correlated alterations in 
                 the basal cell layer and the underlying basement membrane , and malignancy - associated 
                 morphologic alterations ( focal disruptions in both the basal cell layer and basement 
                 membrane ) in morphologically normal or hyperplastic duct or acinar clusters , 
                 however , have not been previously reported . since the epithelium is normally devoid of both blood vessels and lymphatic ducts , 
                 and
the basal cell layer is the sole source of several tumor suppressors 29 - 32 , a 
                 focal disruption in the basal cell layer and its underlying basement membrane could 
                 potentially have a number of consequences , including : ( 1 ) a loss or reduction of 
                 tumor suppressors and the paracrine inhibitory functions , which allow the luminal 
                 cells to undergo elevated proliferation 37 - 41 , ( 2 ) 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 42 - 44 , ( 3 ) the exposure of luminal cells to different cytokines , which 
                 facilitates vasculogenic mimicry and tumor angiogenesis 45 - 46 , ( 4 ) the physical 
                 contact between luminal and stromal cells , which augments the expression of stromal 
                 mmp and facilitates epithelial - mesenchymal transition and cell motility 47 - 49 , 
                 and ( 5 ) the physical contact between luminal and immunoreactive cells , which 
                 directly causes genomic or cellular damages that trigger a cascade reaction of 
                 malignant transformation 50 - 55 .
these alterations could individually or 
                 collectively trigger elevated proliferation in luminal cells near fbcld , which leads 
                 to the enlargement of fbcld and stretching - out of the residual basal cell layer and 
                 basement membrane . eventually , the entire basal cell layer and basement membrane 
                 becomes dissociated or degenerated ( as those shown in fig 2e-2h ) , which facilitates 
                 invasion or progression of the overlying tumor cells .
thus , ducts or acini with 
                 focal disruptions in both the basal cell layer and the underlying basement membrane 
                 are very likely at greater risk to develop invasive prostate lesions .
consequently , 
                 the development of more practical and quantitative methods to assess the physical 
                 and functional integrity of the basal cell layer and basement membrane may lead to 
                 the development of a more effective alternative for repeat biopsy to monitor tumor 
                 progression and invasion .
more importantly , our findings suggest that in addition to 
                 the multistage model , in which prostate carcinogenesis is believed to be 
                 sequentially progressing from normal , to hyperplasia , to high grade pin , and to 
                 invasive lesions , prostate tumor invasion could potentially take place at any stage , 
                 if a focal disruption of the basal cell layer and the basement membrane happens to 
                 occur near a tumor progenitor 34 - 36 .
the underling mechanism for the correlated alterations in the basal cell layer and 
                 the underlying basement membrane is unknown , but is likely to result from focal 
                 degeneration of aged or injured basal cells and resultant auto - immunoreactions .
the 
                 basal cell belongs to a self - renewal population that has to consistently undergo 
                 proliferation and differentiation to replace aged or injured cells .
a number of 
                 external or internal insults , such as radiation , carcinogens , localized trauma , 
                 inflammation , or other factors , could cause the inactivation of , or defects , in 
                 basal cell renewal - related genes , which impair the basal cell replenishment process 
                 to replace the aged or injured basal cells , resulting in a 
                  senesced  basal cell population .
these 
                  senesced  basal cells may have significantly reduced functions 
                 to produce the major building blocks of the basement membrane or may have 
                 significantly reduced affinity in their surface to attract the deposition of 
                 collagen , laminin , and other building elements of the basement membrane .
consistent 
                 with this possibility is the fact that the basement membrane in all or nearly all 
                 ducts or acini with p63 positive basal cells was substantially thicker and more 
                 uniform than that in ducts or acini without p63 positive basal cells ( fig 4a-4b ) , and 
                 also , a vast majority of the focal disruptions occurred near basal cells that lack 
                 p63 expression . in summary ,
our current study reveals for the first time that the basement membrane 
                 underlying all focally disrupted basal cell layers encountered showed either 
                 correlated focal disruptions ( 85% ) or substantial attenuation ( 15% ) , suggesting that 
                 the functional or physical status of the basal cells significantly impact the 
                 physical integrity of the associated basement membrane . as the degradation of both 
                 the basal cell layer and
the basement membrane is a pre - requisite for prostate tumor 
                 invasion or progression , ducts or acini with focally disrupted basal cell layer and 
                 basement membrane are likely at greater risk to develop invasive lesions .
thus , 
                 further elucidation of the specific molecules and mechanism associated with these 
                 events may lead to the development of a more effective alternative for repeat biopsy 
                 to monitor tumor progression and invasion .
 our recent studies revealed that focal basal cell layer disruption ( fbcld ) 
                     induced auto - immunoreactions represented a contributing factor for human 
                     prostate tumor progression and invasion . as the basement membrane surrounds and 
                     attaches to the basal cell layer , our current study assessed whether fbcld would 
                     impact the physical integrity of the associated basement membrane . 
 paraffin 
                     sections from 25-human prostate tumors were subjected to double 
                     immunohistochemistry to simultaneously elucidate the basal cell layer and the 
                     basement membrane with corresponding biomarkers . 
 the physical integrity of the 
                     basement membrane overlying fbcld was examined to determine the extent of 
                     correlated alterations . of a total of 89 fbcld encountered , 
 76 ( 85 % ) showed 
                     correlated alterations in the overlying basement membrane , which included 
                     distinct focal disruptions or fragmentations . in the remaining 13 ( 15% ) 
 fbcld , 
                     the overlying basement membrane showed significant attenuation or reduction of 
                     the immunostaining intensity . 
 the basement membrane in all or nearly all ducts 
                     or acini with p63 positive basal cells was substantially thicker and more 
                     uniform than that in ducts or acini without p63 positive basal cells , and also , 
                     a vast majority of the focal disruptions occurred near basal cells that lack p63 
                     expression . 
 these findings suggest that focal disruptions in the basal cell 
                     layer and alterations in the basement membrane are correlated events and that 
                     the physical and functional status of the basal cells could significantly impact 
                     the physical integrity of the overlying basement membrane . as the degradation of 
                     both the basal cell layer and 
 the basement membrane is a pre - requisite for 
                     prostate tumor invasion or progression , ducts or acini with focally disrupted 
                     basal cell layer and basement membrane are likely at greater risk to develop 
                     invasive lesions . 
 thus , further elucidation of the specific molecules and 
                     mechanism associated with these events may lead to the development of a more 
                     effective alternative for repeat biopsy to monitor tumor progression and 
                     invasion . 
Introduction
Materials and Methods
Results
Discussion