1. Describe the diagnostic criteria and clinical significance of PIN and ASAP
2. Explain the strengths and limitations of Gleason grading in contemporary needle biopsies.
3. Understand the factors that the pathologist relies upon in predicting cancer aggressiveness in biopsy specimens.
4. Address the value of extent of prostate cancer in needle biopsies and the potential limitations.

INTRODUCTION
In prostate needle biopsies, two histologic findings-- high-grade prostatic intraepithelial neoplasia (PIN) and atypical small acinar proliferation (ASAP)-- are each highly predictive of subsequent prostatic adenocarcinoma, and the identification of either without concurrent cancer warrants follow-up with repeat biopsy. [1, 2, 3, 4, 5, 6, 7, 8] PIN is present in 4-16% of contemporary needle biopsies, whereas ASAP is observed in a bout 2% of biopsies. [9, 10, 11, 12, 13, 14, 15, 16] PIN and ASAP can occur together in the same biopsy set without concomitant cancer. We refer to the coexistence of the two lesions in the same high-power microscopic field as "PIN/ASAP".

HIGH-GRADE PROSTATIC INTRAEPITHELIAL NEOPLASIA (PIN)
High-grade PIN is the earliest accepted stage in carcinogenesis, possessing most of the phenotypic, biochemical, and genetic changes of cancer without invasion of through the basement membrane and into the fibromuscular stroma. [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33] PIN is the defined as an abnormal epithelial proliferation within the pre-existing ducts and ductules with nucleomegaly and nucleolomegaly involving at least 10% of the cells . [34, 35, 36] The diagnostic term "prostatic intraepithelial neoplasia" has been endorsed at every multidisciplinary and pathology consensus meeting on the subject around the world since into introduction in 1986 [22, 30, 32, 37, 38, 39, 40, 41, 42] ,and the interobserver agreement between pathologists has been determined to be "good to excellent" [43, 44] for high-grade PIN. Terms such as dysplasia, malignant transformation, carcinoma in situ, and intraductal carcinoma are formally discouraged. [30, 45, 46]

Prostatic intraepithelial neoplasia was originally graded from 1–3, but current recommendations recognize two grades of PIN (low grade and high grade). Grade 1 is synonymous with low-grade prostatic intraepithelial neoplasia (low grade PIN), whereas grades 2 and 3 are considered together as high-grade PIN; currently, conventional use of the term "PIN" without qualification refers to only high-grade PIN. High-grade PIN is a standard diagnosis that must be included as part of the reported pathologic evaluation of prostate biopsies, transurethrally resected prostate chips, and radical prostatectomy specimens. [32, 39, 41] The high level of interobserver variability and apparent lack of predictive value for cancer with low grade PIN limits its clinical utility, and most pathologists do not report this finding except in research studies, including us.

Epidemiology of PIN
In the United States, an estimated 1,300,000 prostate biopsies are performed annually to detect about 230,900new cases of prostate cancer. [47, 48] The incidence of isolated high-grade PIN averages 9% (range 4–16%) of prostate biopsies, representing 115,000 new cases of high-grade PIN without cancer diagnosed each year (Table 1). [49]

Table 1: Estimated frequency of men harboring high-grade PIN in the United States

Age	High-grade % PIN	US population (1990 U.S. census)	Number of PIN
40-49	15.2	20,550,000	3,123,600
50-59	24.0	14,187,000	3,404,880
60-69	47.3	9,312,000	4,404,576
70-79	58.4	6,926,000	4,044,784
80-89	70.0	2,664,000	1,864,800
	Total	53,639,000	16,842,640

The incidence and extent of PIN appear to increase with patient age (Table 1). An autopsy study of step-sectioned whole mount prostates from older men showed that the prevalence of PIN in prostates with cancer increased with age, predating the onset of carcinoma by more than five years. [50] A similar study of young men revealed that PIN is first seen in men in their twenties and thirties (9% and 22% frequency, respectively), and precedes the onset of carcinoma by more than ten years. } The volume of [50,] high grade PIN also increases with patient age. [52]

Race and geographical location may also influence the incidence of high-grade PIN. [21] When age groups are compared between races, there are significant differences in the frequency of high-grade PIN. For example, African-American men have a greater prevalence of high-grade PIN than Caucasians in the 50–60 year age group, the decade preceding the manifestation of most clinically detected prostate cancers. [37, 53, 54, 55] African-American men also have the highest incidence of prostate cancer (about 50% more than Caucasians). [37, 53, 54, 56, 57] In contrast, Japanese men living in Osaka, Japan have a significantly lower incidence of high-grade PIN compared to men residing in the United States, and Asians have the lowest clinically detected rate of prostate cancer [58, 59] Interestingly, Japanese men diagnosed with high-grade PIN also have an increased likelihood of developing prostate cancer, indicating that high-grade PIN is also a precursor of clinical prostate cancer in Asian men. [60] Thus, the differences in the frequency of high-grade PIN in the 50–60 year age group across races essentially mirror the rates of clinical prostate cancer observed in the 60–70 year age group. [53, 58]

The causal association of high-grade PIN with prostatic adenocarcinoma is based on the fact that the prevalence of both high-grade PIN and prostate cancer increases with patient age and that high-grade PIN precedes the onset of prostate cancer by less than one decade (Table 1). [50, 53, 54, 61] The severity and frequency of high-grade PIN in prostates with cancer is greatly increased (73% of 731 specimens) when compared to prostates without cancer (32% of 876 specimens). [52, 62, 63, 64] When high-grade PIN is found on sextant needle biopsy, there is a 50% risk of finding carcinoma on subsequent biopsies over 3 years [41] , although this risk is cut in half when twice as many cores are obtained. There is also evidence to suggest that high-grade PIN may represent a precursor to a more aggressive form of prostate cancer phenotype than to those that are more likely to remain indolent. [26, 60, 65]

Incidence of PIN
The incidence of PIN varies according to the population of men under study (Table 2). [66, 67, 68, 69, 70, 71, 72] The lowest likelihood is in men participating in PSA screening and early detection studies, with an incidence of PIN in biopsies ranging from 0.7– 20%. [66, 67, 68, 69, 70, 71, 72] Men seen by urologists in practice have PIN in 4.4– 25% of needle biopsies. Those undergoing transurethral resection also have a high likelihood of PIN, varying from 2.8 – 33%. [71, 73, 74] In such cases, all tissue should be examined, but serial sections of suspicious foci are usually not necessary. Unfortunately, needle biopsy specimens fail to show the suspicious focus on deeper levels in about half of cases, precluding assessment by immunohistochemistry and compounding the diagnostic dilemma.

Diagnostic Criteria of PIN
There are four main patterns of high grade PIN: tufting, micropapillary, cribiform, and flat. [75] The tufting pattern is the most common, present in 97% of cases, although most cases have multiple patterns. There are no known clinically important differences between the architectural patterns of high grade PIN, and their recognition appears to be only of diagnostic utility. Nonetheless, one report suggested that the cribriform pattern may indicate higher risk of coexistent cancer, but this has been refuted. Other unusual patterns of PIN include the signet ring-cell pattern, small cell neuroendocrine pattern, and foamy-gland pattern. [76] The presence of extensive PIN appears to be more predictive of cancer than the more common isolated single acinus with PIN.

There is inversion of the normal orientation of epithelial proliferation with PIN; proliferation in the benign epithelium normally occurs in the basal cell compartment, whereas, in PIN, the greatest proliferation occurs on the luminal surface, similar to pre-invasive lesions in the colon (tubular adenoma) and other sites.

Table 2. Incidence of Isolated High Grade PIN in Prostatic Needle Biopsies

Reference	Patient Population	No. men	Incidence of PIN (%)
Screening Programs
Mettlin et al., 1991 [156]	American Cancer Society National Prostate Cancer Detection Project	330	5.2
Richie et al., 1994 [66]	Screening population	163	8.6
Feneley et al., 1997 [157]	Screening population in Gwent, England, 1991-1993	212	20
Hoedemaeker et al., 1999 [68]	PSA screening study in Rotterdam (Netherlands)	1824	0.7
Urology Practice
Lee et al., 1989 [100]	Consecutive biopsies of hypoechoic lesions at St. Joseph Mercy Hospital	256	11
Bostwick et al., 1995 [158]	Consecutive biopsies at Mayo Clinic	200	16.5
Bostwick et al., 1995 [158]	Consecutive biopsies at Glendale Hospital (CA.)	200	10.5
Langer et al., 1996 [69]	Consecutive biopsies at University of Pennsylvania Med. Ctr.	1275	4.4
Wills et al., 1997 [70]	Consecutive biopsies at Johns Hopkins Hospital	439	5.5
Skjorten et al., 1997 [71]	Consecutive biopsies from 1974-1975 at Ullevaal and Lovisenberg Hospitals, Oslo, Norway	79	7.6
Perachino et al., 1997 [72]	Consecutive biopsies	148	14.1
Feneley et al., 1997 [67]	Consecutive biopsies at University College London Hospitals 1988-1994	1205	11
Feneley et al., 1997 [67]	Consecutive biopsies of symptomatic men at St. Bartholomew's Hospital, London, 1993-1994	118	25

PIN spreads through prostatic ducts in multiple different patterns, similar to prostatic carcinoma. In the first pattern, neoplastic cells replace the normal luminal secretory epithelium, with preservation of the basal cell layer and basement membrane. This pattern often has a cribriform or near-solid appearance. Foci of high grade PIN are usually indistinguishable from intraductal/intra-acinar spread of carcinoma by routine light microscopy, [77] In the second pattern, there is direct invasion through the ductal or acinar wall, with disruption of the basement membrane and basal cell layer. In the third pattern, neoplastic cells invaginate between the basal cell layer and columnar secretory cell layer ("pagetoid spread"), a very rare finding.

Early stromal invasion, the earliest evidence of carcinoma, occurs at sites of acinar outpouching and basal cell disruption in acini with high grade PIN. Such microinvasion is present in about 2% of high power microscopic fields of PIN, and is seen with equal frequency with all architectural patterns. [75] In equivocal cases, we prefer the term PIN/ASAP to avoid over-diagnosis of tangential cutting of PIN as cancer.

The mean volume of PIN in prostates with cancer is 1.2–1.32 cc, and the volume increases with increasing pathologic stage, Gleason grade, positive surgical margins, and perineural invasion. } PIN is multicentric in 72% of radical prostatectomies with cancer, including 63% [52,] of those involving the non-transition zone and 7% of those involving the transition zone; 2% of cases have concomitant single foci in all zones [52] . The peripheral zone of the prostate, the area in which the majority of prostatic carcinomas occur (70% or more), is also the most common location for PIN. [52, 75] Cancer and PIN are frequently multicentric in the peripheral zone, indicating a "field" effect similar to the multicentricity of urothelial carcinoma of the bladder. [59]

High-grade PIN and prostate cancer are morphometrically and phenotypically similar. High-grade PIN occurs primarily in the peripheral zone and is seen in areas that are in continuity with prostate cancer. [22, 52, 55, 64, 79, 80] High-grade PIN and prostate cancer are multifocal and heterogeneous. [52, 81, 82] Increasing rates of aneuploidy and angiogenesis as the grade of PIN progresses are further evidence that high-grade PIN is a precancer. [17, 80, 83, 84, 85, 86] Prostate cancer and high-grade PIN also have similar proliferative and apoptotic indices. [17, 19, 58, 87, 88, 89, 90, 91, 92]

It is often difficult with small foci in needle biopsies to separate cancer from suspicious foci (atypical small acinar proliferation suspicious for but not diagnostic of malignancy) when there is coexistent high-grade PIN; the difficulty is based on the inability to separate tangential cutting of the larger pre-existing acini of PIN (that may appear as small separate adjacent acini) from the smaller discrete acini of cancer.

Recent renewed efforts to introduce the term "intraductal carcinoma" rely on the abandoned concept that dysplasia (defined here as malignancy arising at that specific site within the epithelium) can be separated reliably from intraductal/intracinar spread of cancer (defined here as extension of malignant cells through the pre-existing lumens of the prostate); however, this concept was rejected by consensus on multiple occasions owing to lack of reproducible criteria for making this distinction, and the non-committal term intraepithelial neoplasia was internationally adopted and repeatedly reconfirmed as it begs the question of site of origin of the process. Those who persist with the belief that "intraductal carcinoma" can be diagnosed rely on proximity of the epithelial abnormality to invasive cancer, but this criterion is arbitrary and not based on valid objective confirmatory data. More importantly, there is no clinical utility at present that requires separation of dysplasia and intraductal/intra-acinar spread of cancer—the clinical response is the same. It is conceivable that future studies may allow diagnostic separation of dysplasia and intraductal/ intra-acinar spread of cancer; if so, then these steps in the biologic progression of prostate cancer may be shown to have differential predictive value for prostate cancer. We agree that identification of subsets of high-grade PIN that indicate greater risk of cancer is a clinically important area of investigation.

Useful Immunohistochemical Markers for the diagnosis of PIN (Table 3)
The secretory luminal cells of high-grade PIN invariably stain with PSA and PAP. Select antibodies such as anti-keratin 34ß-E12 (high molecular weight keratin) or p63 may be used to stain tissue sections for the presence of basal cells, recognizing that PIN retains an intact or fragmented basal cell layer whereas cancer does not. Increasing grades of PIN are associated with progressive disruption of the basal cell layer, according to studies utilizing anti-keratin 34ß-E12. Basal cell layer disruption is present in 56% of cases of high grade PIN, and is more frequent in acini adjacent to invasive carcinoma than in distant acini. The amount of disruption increases with increasing grades of PIN. Early invasive carcinoma occurs at sites of glandular out-pouching and basal cell discontinuity in association with PIN. [36] The cribriform pattern of PIN may be mistaken for cribriform adenocarcinoma or ductal carcinoma, and the use of anti-keratin staining

Table 3. Markers of Basal Cell Differentiation in the Prostate

Biomarker	Function	Findings
PCNA	Cell proliferation marker	Up to 79% of labeled cells are basal cells
MIB 1	Cell proliferation marker	Up to 77% of labeled cells are basal cells
Ki-67	Cell proliferation marker	Up to 81% of labeled cells are basal cells
Androgen Receptors	Nuclear receptors which are necessary for prostatic epithelial growth	Strong immunoreactivity; also present in cancer cells
Prostate-specific antigen	Enzyme which liquifies the seminal coagulum	Present in rare basal cells; mainly in secretory luminal cells
Keratin 8.12	Keratins 13, 16	Strong immunoreactivity
Keratin 4.62	Keratin 19	Moderate immunoreactivity
Keratin PKK1	Keratins 7,8,17,18	Moderate immunoreactivity
Keratin 312C8-1	Keratin 14	Strong immunoreactivity
Keratin 34B-E12	Keratins 5,10,11	Strong immunoreactivity; most commonly used for diagnostic purposes
P63	A member of the p53 family.	Stains basal cells nuclei; most commonly used for diagnostic purposes
Epidermal Growth Factor Receptor	Membrane bound 170-kd glycoprotein which mediates the activity of EGF	Strong immunoreactivity; rare in cancer
CuZn-Superoxide Dismutase	Enzyme which catalyzes superoxide anion radicals	Strong immunoreactivity
Type IV Collagenase	Enzyme involved in extracellular matrix degradation	Strong immunoreactivity; decreased in cancer
Type VII Collagen	Part of the hemidesmosomal complex	Strong immunoreactivity; lost in cancer
Integrins alpha1,2,4,6,and v; Beta 1 and 4	Extracellular matrix adhesion molecules	Strong immunoreactivity; decrease in most with cancer, although alpha 6 and Beta 1 are retained
Estrogen Receptors	Hormone receptor	Moderate immunoreactivity
bcl-2	Oncoprotein which suppresses apoptosis	Strong immunoreactivity; also found in most cancers
c-erbB2	Oncogene protein in the EGF family	Strong immunoreactivity; also found in most cancer
Glutathione S- transferase gene (GSTP1)	Enzyme which inactivates electrophilic carcinogens	Strong immunoreactivity; rare in cancer
C-CAM	Epithelial cell adhesion molecule	Strong immunoreactivity; absent in cancer
TGF-B	Growth factor which regulates cell proliferation and differentiation	Strong immunoreactivity; absent in cancer
Cathepsin B	Enzyme which degrades basement membranes; may be involved in tumor invasion and metastases	Present in many basal cells, and rarely in luminal secretory cells; also found in cancer cells
Progesterone Receptors	Hormone receptor	Moderate immunoreactivity

is invaluable in making this distinction. [93] There are a number of other markers of basal cell markers, including nuclear stain for p63 that is gaining in popularity in recent years. [94, 95]

A new molecular marker, racemase (alpha-methylacyl-CoA racemase, P504S) was introduced for separating benign and neoplastic acini. This marker has proven useful for evaluation of PIN and ASAP (atypical small acinar proliferation suspicious for but diagnostic of cancer) and separation of cancer from hormonally-treated benign acini. It's advantage over anti-keratin 34ß-E12 is positive granular cytoplasmic staining in cancer cells, with little or no staining in benign acini. The gene for alpha-methylacyl-CoA racemase (AMACR) is greatly overexpressed in prostate cancer cells.

Differential Diagnosis of PIN
The histologic differential diagnosis of PIN includes lobular atrophy, post-atrophic hyperplasia, atypical basal cell hyperplasia, cribriform hyperplasia, and metaplastic changes associated with radiation, infarction, and prostatitis. Many of these display architectural and cytologic atypia, including enlarged nucleoli, and small specimens, and cauterized or distorted specimen. Cribriform adenocarcinoma, ductal (endometrioid) carcinoma, and urothelial carcinoma involving prostatic ducts and acini may also be confused with PIN. Biopsies submitted with incomplete patient history should be interpreted with caution.

PIN may be overdiagnosed as adenocarcinoma. Our retrospective review of transurethral resections from the Mayo Clinic files between 1960–1970 revealed that PIN was often diagnosed as adenocarcinoma. [96] Similarly, fine needle aspiration of the prostate may yield cell clusters of PIN that are over-diagnosed as cancer; this issue is critically important to consider in evaluating studies from Sweden and other countries that have, perhaps erroneously, relied on fine needle aspiration diagnoses for patients treated with watchful waiting (expectant management).

CLINICAL SIGNIFICANCE OF PIN

PIN Does Not Elevate PSA
Biopsy remains the definitive method for detecting PIN and early invasive cancer. Serum PSA concentration may be elevated in patients with PIN [97] , although these results have been refuted. [98, 99] There is a poor correlation of PIN and PSA density according to studies of radical prostatectomy specimens and preoperative serum. [99] Mean PSA increased from 8.4 to 11.6 ng/mL in patients with PIN who developed cancer within two years; those with PIN who did not develop cancer during this interval had an increase in PSA from 4.8 to 5.9 ng/mL or decrease from 5.1 to 4.6 ng/mL; however, these findings have not been confirmed. The ratio of free to total PSA is the same for patients with high grade PIN and cancer, unlike low grade PIN and hyperplasia, although this has also been refuted. Many patients in these studies were later found to have cancer, so the elevation in serum PSA concentration and its derivatives may have resulted from the undetected cancer.

The PSA antigen is excreted to the outside via the prostatic ducts. PIN does not significantly elevate serum PSA concentration is due to the architectural integrity of the gland lumina involved by PIN. Conversely, the cells of the neoplastic acinar structures found in prostate cancer release prostate specific antigen into the surrounding stroma where it is absorbed into the bloodstream, raising serum PSA.

Transrectal Ultrasound Cannot Detect PIN
By transrectal ultrasound, PIN may be hypoechoic like carcinoma, although these findings have not been confirmed. [100] Today, most urologists and radiologists do not believe that PIN is detectable by transrectal ultrasound because PIN is a microscopic finding which is below the detection threshold for this form of imaging.

Men with PIN Develop Prostate Cancer
As a risk factor, the presence of isolated PIN in a set of sextant needle biopsies connotes a risk ratioof 14.9. PIN is a far stronger predictor for subsequent cancer than the independent predictors of patient age (>65 years old vs. ≤65 years old) and serum prostate specific antigen (PSA) (>4 ng/ml vs. ≤4 ng/ml); for these, the respective risk ratios are 3.5 and 3.64. [2] PIN coexists with cancer in more than 85% of cases, according to studies employing whole-mounted totally embedded prostates. [101] In one report, the likelihood of finding cancer increased with the biopsy time interval. The investigators reported a 32% incidenceof cancer if repeat biopsy was performed within 1 year, compared with a 38% incidence in biopsies obtained after 1 year. [2] Other series have also found a high predictive value of PIN for cancer, although recent reports based on obtaining a greater number of cores shows a lower predictive value (Table 4). [3, 4, 5, 6, 7, 8, 69, 72, 73, 74, 102, 103, 104] Results in recent cohorts of patients with isolated PIN biopsied between 1999 and 2001 are listed in Table 1B. The reported frequencies of cancer at follow-up were 23%, 25%, 27%, and 28%. [105, 106, 107, 108, 109, 110, 111, 112] High grade PIN in transurethral resection specimens is also an important predictive factor for prostate cancer (Table 5). [73, 113, 114] These data underscore the strong association of PIN and adenocarcinoma and indicate that vigorous diagnostic follow up is needed.

Table 4. Cancer Detection in Patients with High Grade Pin

Reference	Pt. population	No. men	% patients with cancer on repeat biopsy
Brawer et al. [74]	Urology practice	10	100
Ellis and Brawer [3]	Urology practice	5	100
Aboseif et al. [1]	Urology practice	24	79.1
Weinstein and Epstein [4]	Urology practice	19	53
Keetch et al. [5]	PSA screening	37	51
Davidson et al. [2]	Two urology practices	100	35
Markham [102]	Urology practice	32	41
Raviv et al. [159, 103]	Urology practice	48	47.9
Langer et al. [69]	Urology practice	53	27
Berner et al. [160]	Oncology practice	37	38
Shepherd et al. [8]	PSA screening	66	58
Perachino et al. [72]	Urology practice	21	71.1
Krishnamurthi et al. [161]	Urology practice	74	31
Rovner et al. [162]	Urology practice	19	31.6
Park et al. [15]	Urology practice	43	51
Kronz et al. [163]	Urology practice	245	32
Igel et al. [164]	Urology practice	88	43
Park et al. [104]	Urology practice	104	22
Gokden et al. [106]	Urology practice	221	28%
Sakr et al [105]	Urology Practice	540	27%
Siever et al. [107]	Urology Practice	145	25%
Schlesinger & Bostwick [108]	Urology Practice	335	23%
Bishara et al. [109]	Urology Practice	132	28.8%
Mendrinos et al. [110]	Urology Practice	80	67% for PIN in multiple cores 38.6% for isolated PIN
Pierson et al. [111]	Urology Practice	249	21%
Varma et al. [112]	Urology Practice	37	5.4%

We believe that the following factors account for the decline in the predictive accuracy of HGPIN for cancer.The major role is played by use of extended biopsy techniques that result in more thorough prostate sampling and in higher cancer detection rates. Conversely, by these actions there remains a smaller pool of patients who receive isolated diagnoses of PIN. Another contributor is the lower detection rate for, and difficulty in the detection of, the remaining small cancers; larger

Table 5. Incidence of Isolated High Grade Pin in Prostatic Transurethral Resections

Reference		Patient Population	Number of men	Incidence of PIN (%)
Gaudin et al., 1997 [73]	TURP	Consecutive TURPs without cancer at Johns Hopkins Hospital	158	3.2
Pacelli and Bostwick, 1997 [113]	TURP	Consecutive TURPs without cancer at Mayo Clinic	570	2.8
Skjorten et al., 1997 [71]	TURP	Consecutive TURPs from 1974-1975 at Ullevaal and Lovisenberg Hospitals, Oslo, Norway	731	33

significant tumors may also escape detection. These factors lead to a higher frequency of negative repeat biopsies. These results may reflect a new steady state and a newly reached low plateau in the predictive accuracy of these markers.

Androgen Deprivation Therapy Eliminates PIN
There is a marked decrease in the prevalence and extent of high grade PIN in cases after androgen deprivation therapy when compared with untreated cases. [115, 116, 117] This decrease is accompanied by epithelial hyperplasia, cytoplasmic clearing, and prominent glandular atrophy, with decreased ratio of glands to stroma. These findings indicate that the dysplastic prostatic epithelium is hormone dependent. In the normal prostatic epithelium, luminal secretory cells are more sensitive to the absence of androgen than basal cells, and these results indicate that the cells of high grade PIN share this androgen sensitivity. The loss of some normal, hyperplastic, and dysplastic epithelial cells with androgen deprivation is probably due to acceleration of programmed single cell death. A recent report suggested that PIN is not substantially decreased after hormonal therapy, but those authors failed to use current criteria for PIN, so the results are not comparable. [118]

Neoadjuvant hormone deprivation with monthly leuprolide and flutamide 250mg PO TID for three months resulted in a 50% reduction in high-grade PIN [39] . Longer therapy with 6 months of neoadjuvant androgen deprivation therapy prior to radical prostatectomy in the European Randomized Study of Screening for Prostate Cancer (ERSPC) study reduced high-grade PIN even more [41] . Flutamide decreased the prevalence and extent of high-grade PIN and induced epithelial atrophy [119] . There is also evidence that cessation of flutamide resulted in return of high-grade PIN [32, 120] .

The results of 5-alpha-reductase (finasteride) treatment in high-grade PIN are controversial and the cumulative number of cases studied is probably too small to draw firm conclusions. Two reports found no apparent effect on the histologic appearance or extent of high-grade PIN [121, 122] , whereas a third study of three cases described atrophy and involution with decreased prevalence. [123]

Radiation Therapy Eliminates PIN
The prevalence and extent of PIN is decreased after radiation therapy. [124, 125, 126] However, one study paradoxically noted a higher incidence (70%) of PIN after radiation therapy than expected [125] , but they failed to employ accepted diagnostic criteria for PIN, so their results are not comparable with others. A recent report from Memorial Sloan-Kettering found PIN in 8.8% of biopsies following a course of 3-dimensional external beamconformal radiation therapy. [126] Following radiation therapy, PIN retains the features characteristic of untreated PIN, and is readily recognized in tissue specimens. The key pathologic features include nuclear crowding, nuclear overlapping and stratification, nuclear hyperchromasia, and prominent nucleoli. The basal cell layer is present, but often fragmented. The most common patterns of PIN are tufting and micropapillary, similar to those reported in untreated PIN.

The long-term efficacy of radiation treatment may depend on eradication of cancer as well as pre-cancerous lesions that may otherwise lead to evolution of secondary metachronous invasive cancers. Identification of residual or recurrent cancer portends a worse prognosis. The questions remain whether recurrent cancer after irradiation is due to regrowth of incompletely eradicated tumor or progression from incompletely eradicated PIN. Further studies of salvage prostatectomy specimens and post-RT needle biopsies are justified in an attempt to establish the significance of high-grade PIN as a source of long-term treatment failure among these patients. If PIN is associated with treatment failure, adjuvant chemoprevention strategies that ablate this lesion may reduce the risk of late cancer recurrence.

Should Men with High Grade PIN Be Treated?
The clinical importance of recognizing PIN is based on its strong association with prostatic carcinoma. PIN has a high predictive value as a marker for adenocarcinoma, so its identification in biopsy specimens warrants further search for concurrent invasive carcinoma. If all procedures fail to identify coexistent carcinoma, close surveillance and follow-up are indicated. As high-grade PIN progresses, the likelihood of basal cell layer disruption increases, very much like what is observed for carcinoma in situ (CIS) of the urinary bladder. CIS of the urinary bladder, like PIN, may become invasive and is treated aggressively. The standard of care for management of CIS of the bladder is intravesical instillation of chemotherapy or BCG, and, in some cases, radical cystectomy.

Follow-up biopsy is suggested at three to six month intervals for two years, and thereafter at twelve-month intervals for life. [2, 127] Some urologists have performed "saturation" biopsies, consisting of more than 12–15 biopsies in one session, often with brief general anesthesia in the operating theatre, in an effort to definitively exclude cancer. Most authors agree that the identification of PIN in the prostate should not influence or dictate therapeutic decisions. [127] We are aware of 21 radical prostatectomies that were purposely (3 cases) or inadvertently performed (18 cases) in patients whose biopsies contained only high grade PIN; all but two of the cases contained adenocarcinoma in the surgical specimen (DG Bostwick, personal communication, 2003).

Currently, routine treatment is not available for patients who have high-grade PIN. Prophylactic radical prostatectomy or radiation is not an acceptable treatment for patients who have high-grade PIN only [128] . The development and identification of acceptable agents to treat high-grade PIN would fill a therapeutic void [40] .As noted above, androgen deprivation therapy and radiation therapy induce acinar atrophy and apoptosis that result in regression of high-grade PIN. [88, 115, 116, 117, 128, 129, 130, 131]

Chronic therapy, however, would most likely be required to prevent new high-grade PIN lesions from invading and becoming clinical prostate cancer. Although more toxicity is likely to be tolerated for the treatment agents targeted to regress or inhibit high-grade PIN [39] ,as compared to treating healthy patients to reduce prostate cancer incidence, androgen deprivation therapy has too many adverse effects in men to be clinically useful. Newer agents with better safety and lower side effect profile are greatly needed since patients may be taking the agent at least until they attain 70 years of age [128] . Acapodene, an anti-estrogen is currently in a Phase IIb multicenter, randomized, prospective placebo-controlled human clinical trial to determine if it can treat high-grade PIN and reduce prostate cancer incidence; preliminary results are encouraging. [49]

PIN offers promise as an intermediate endpoint in studies of chemoprevention of prostatic carcinoma. Recognizing the slow growth rate of prostate cancer and the considerable amount of time needed in animal and human studies for adequate follow-up, the non-invasive precursor lesion PIN is a suitable intermediate histologic marker to indicate subsequent likelihood of cancer.

PIN Does Not Predict Cancer Recurrence
PIN was not predictive of PSA (biochemical) failure at 32 months in patients undergoing radical prostatectomy and androgen deprivation therapy. [115]

ATYPICAL SMALL ACINAR PROLIFERATION
SUSPICIOUS FOR BUT NOT DIAGNOSTIC OF MALIGNANCY (ASAP)

ASAP Is a Valid Diagnostic Category
The diagnostic quandary in cases in which the diagnosis of ASAP is rendered usually results from one or a combination of the reasons listed in Table 6. All of these may hinder a definitive

Table 6. Reasons for ASAP Diagnosis

Size Small number of acini in the focus of concern Small focus size, average 0.4 mm in diameter [165] Lesion breakage at core tip, indicating that the focus is incompletely sampled Histology Lack of clear histologic detail (e.g., thick section, overstained nuclei, etc.) Distortion of acini Lack of convincing malignant features, e.g, small number of cells with nucleomegaly or nucleolomegaly Clustered growth pattern mimicking a benign process such as atypical adenomatous hyperplasia or atrophy Loss of the focus of concern in deeper levels obtained for high molecular weight cytokeratin or racemase immunostains. Focally positive high molecular weight cytokeratin or negative racemase immunostain in focus. Presence of associated high-grade PIN raising the possibility of tangential cutting of PIN. Inflammation Prominent inflammation in which the adjacent benign acini show distortion, raising concern for overdiagnosis of reactive changes or atrophy. Inflammatory cellular reactive atypia with nuclear and nucleolar enlargement

diagnosis of carcinoma, but, in such cases, the possibility cannot be definitively excluded. The need for this category is based on our "absolute uncertainty" regarding the diagnosis. That this need exists is manifested by the variety of terms or synonyms currently in use that include the word "atypical" to describe this diagnosis, although ASAP is now the preferred term that is most widely used. The diagnosis of ASAP indicates to the clinician that the biopsy in question exhibits histologic features that are neither clearly malignant nor clearly benign and that follow-up of the patient is warranted.

For pathologists, two questions need to be answered prior to the diagnosis of ASAP or cancer in a small lesion: "Would you be absolutely confident of this biopsy diagnosis if it were followed by a negative radical prostatectomy?"; and similarly "Can you confidently support a diagnosis of malignancy based solely on this biopsy?" If the answer to either question is "No," then we recommend use of the more conservative diagnosis of ASAP. In this setting, we believe that "ASAP" is a valid diagnostic category as long as it is employed judiciously and that maximum information has been obtained from the available tissue (see Methods). Other evidence useful in supporting a cancer diagnosis, including patient age, serum PSA concentration, and high-molecular weight cytokeratin and racemase expression cannot substitute for convincing hematoxylin and eosin (H&E) microscopic findings. To avoid bias, the above information should be considered only after microscopic examination is performed.

ASAP Predicts Cancer
In studies published between 1997 and 2001, the reported incidence of prostate cancer in repeat biopsies following a diagnosis of ASAP ranged from 34% to 60%. [Table 7]. [10, 11, 12, 13, 14, 15, 16] These studies involved patient cohorts that underwent biopsy between 1989 and 1996, with the exception of one investigator who reported a cohort in which biopsies were performed between 1995 and 2000 [16] .

In a recent study, the investigators reported the results of follow-up prostate needle biopsies in a patient population with long term close clinical follow-up, in whom earlier, smaller lesions are detected. Only patients biopsied after January 1, 2000 were included in order to accurately reflect the current state of clinical practice. The follow-up cancer detection rates were compared to rates in earlier cohorts of patients, in whom longitudinal repetitive PSA screening had been more recently introduced. They found that the contemporary predictive value of ASAP for subsequent cancer was 37%. [108, 132] This is compared with historic rates of 34-60% for ASAP listed in Table 6. [105] These findings indicate that the predictive accuracies of ASAP for cancer have decreased since the original studies on this topic were published. A similar lower cancer detection rate of 34% for ASAP was also noted in a 1998 report. [13]

Table 7. Cancer Detection in Patients with Asap

Date of publication	Study dates	Authors	# Subjects with repeat biopsy(ies)	Frequency of diagnosis	ASAP?CA
1997	1993-1995	Cheville et al. [10]	n=25	ASAP- 4.8%	60%
1997	1993-1996	Iczkowski et al. [11]	n= 33		45%
1998	1991-1995	Iczkowski et al. [148]	n= 295		42%
1998	1989-1996	Renshaw et al. [13]	n= 59		34%
1999	1992-1993	Chan et al. [14]	n= 144		49%
2001	1991-1998	Park et al. [15]	n= 45		51%
2001	1995-2000	Borboroglu et al. [16]	n= 48	ASAP- 3.8%	48%
2002	1990-2002	Iczkowski et al. [9]	n= 129	ASAP- 3.2%	45%
2003	2001-2003	Schlesinger & Bostwick [108]	n= 78		37%
2004	?-2004	Fadare et al. [132]	n=55	ASAP-2.8%	37.5%

During the past two decades, there has been a significant downward stage migration and decrease in positive surgical margins. [133]The following factors may account for the decline in predictive accuracies of PIN and ASAP for prostate cancer. A major contribution is the use of extended biopsy techniques that increase the diagnostic yield for cancer. Chen et al. demonstrated via a computer generated composite image the frequent origin of tumors from the lateral base and medial apex, sites not sampled by sextant [134] . Cancer detection rates of 16- 35% or higher in extended vs. sextant biopsies have been shown by other investigators. [135, 136, 137, 138, 139] Extended biopsy is now recognized as a common method of prostate sampling [139, 140, 141, 142] . Conversely, more thorough prostate sampling reduces the number of undetected cancers, particularly significant cancers, leaving a smaller pool of patients to receive isolated diagnoses of PIN, ASAP, or PIN/ASAP. Smaller tumor volumes in the remaining cancers make detection more difficult, and thus higher likelihood of a negative repeat biopsy.

Following the introduction of PSA screening [143, 144] , an unprecedented and continuous increase in localized and regional prostate cancers continued through 1992 and then decreased. [145] Beginning in 1992, there were also declines in distant stage disease and in 2-year cancer mortality rates [SEER data]. [146] These observations probably reflect lead time bias, as well as what one author referred to as a "harvesting" effect, with reference to advanced tumors. [147] In 2003, the relatively dramatic decreases in prostate cancer volumes and stage have already occurred. Thus, any subsequent evolution in cancer characteristics will, of necessity, be of lesser magnitude. We believe that our findings reflect some of these less dramatic changes, and that a steady state may have been reached.

Clinical Significance of ASAP
Like high-grade prostatic intraepithelial neoplasia, ASAP holds a significant [10, 11, 148] predictive value for cancer on repeat biopsy. When repeat biopsy is undertaken, use of at least sextant (or more) sampling method is best. Sampling only the side or sextant site initially diagnosed as ASAP missed cancer in 39% of patients whose cancer was detected exclusively at other sites. [149] The repeat biopsy rates by urologists in 1997 reports were 46% [148] and 47% [11] but, in more recent studies, ranged up to 60%, [150] similar to our 56% rate.

In one report, the investigators found that ASAP represented undersampled cancer in at least 40% of cases. They observed that some men with ASAP in first set of biopsy and benign or high-grade prostatic intraepithelial neoplasia findings on the second biopsy may still have had cancer that was not detected. [148] False-negative results on repeat sextant biopsy in untreated men with documented adenocarcinoma occurred in 23% of repeat sextant biopsies. [151] These results also suggest that the current practice of performing 6 to 12 biopsies per prostate is not lowering the frequency of ASAP diagnosis. A declining volume of residual cancer at prostatectomy was noted 5 years ago, [152] and is probably reflective of increased screening and multiple sampling. Thus, as smaller volume cancers are detected through increased sampling, many will be undersampled and not resolvable by immunostains, leading to an irreducible rate of ASAP diagnosis.

PIN/ASAP
The combination of high grade prostatic intraepithelial neoplasia and atypical small acinar proliferation lesions, found in up to 16% of all biopsies, has an intermediate predictive value of 33% for cancer. [108] Thus, it is slightly lower than isolated ASAP but higher than isolated PIN. In previous reports, associated PIN occured in 23% [153] and 31% of ASAP cases [11] , respectively. In a recent study, the frequency of associated PIN was higher, occurring in 41% of total cases containing "ASAP" in the diagnosis,, but most foci were not adjacent or contiguous.

In a similar investigation, a lesion containing both PIN and ASAP was reported to have a 46% follow-up cancer detection rate. This lesion was strictly defined, and corresponded to our definition of contiguous PIN/ASAP lesions. [154] Three reasons might account for the difference in predictive values, 33% vs. 46%, seen in these 2 studies. First, the latter study was restricted to contiguous cases; this type of lesion might have an intrinsically higher predictive value for cancer than in our series, in which the frequency of contiguous lesions was about half. The selection bias present in cases referred for consultation also may have influenced the study results as compared with unselected primary cases in another study cohort. Also, the number of patients reported in each study was few enough that skewing of data in either direction might occur. In another small study of 12 patients with PIN and adjacent atypical glands (ASAP), 75% had cancer on repeat biopsy. [155] Additional studies involving larger numbers of patients with a combination of PIN and ASAP lesions would be of interest.

CONCLUSION
High grade PIN is the most likely precursor of prostatic adenocarcinoma, according to virtually all available evidence. The clinical importance of recognizing PIN is based on its strong association with prostatic carcinoma. PIN has a high predictive value as a marker for adenocarcinoma, and its identification in biopsy specimens of the prostate warrants further search for concurrent invasive carcinoma. Studies to date have not determined whether PIN remains stable, regresses, or progresses, although the implication is that it can progress.

The predictive accuracy for cancer is lower for both PIN and for ASAP in a highly screened patient population compared with previously reported populations. However, the presence of either or both histologic markers in a biopsy set is still a significant predictor for concurrent/subsequent Cancer compared to the cohorts of patients lacking these lesions. Thus, even though the predictive value of these lesions has decreased, they remain as significant risk factors for prostate cancer.

PROSTATIC ADENOCARCINOMA

Histologic grade is a powerful predictive factor in prostatic adenocarcinoma, and is valuable even in contemporary 18-gauge needle biopsies. More than 40 grading systems have been proposed since the pioneering work of Broders more than 70 years ago (Broders, 1926). All systems successfully identify well differentiated adenocarcinoma which progresses slowly and poorly differentiated adenocarcinoma which progresses rapidly (Bostwick, 1994b). However, grading systems are less successful in subdividing the majority of moderately differentiated adenocarcinomas which have intermediate clinical behavior (Gleason 1966; Mostofi, 1975; Böcking et al., 1982; Gleason, 1990; Gleason, 1992). Problems with grading include interobserver and intraobserver variability, imprecise predictive value, and lack of a single universal system. In biopsies, these problems are compounded by small sample size, tumor heterogeneity, and undergrading of biopsy samples. Also, significant histologic changes in adenocarcinoma occur as a result of radiation and androgen deprivation therapy which make grading difficult and of questionable value. This chapter describes the current role of grading in prostatic adenocarcinoma, correlation of biopsy grade with prostatectomy grade, and clinical significance of grade. Emphasis is placed on the Gleason grading system, the most commonly used system (Gardner et al., 1988) .

Gleason Grading System (Veterans Administration Cooperative Urological Research Group Grading System; VACURG System)

The Gleason grading system is based on prospective study of more than 4000 patients between 1960 and 1975, and is the de facto grading standard in the United States and other parts of the world (Gleason, 1966; Gleason, 1990; Gleason, 1992). Other systems in use internationally are the World Health Organization (Mostofi, 1975) and Böcking (Böcking et al., 1982) systems. These systems are clinically useful, showing a positive correlation with tumor volume, preoperative serum PSA concentration, the likelihood of pelvic lymph node metastases, and tumor recurrence after surgery and radiation therapy. The Gleason system is based on the degree of architectural differentiation. Tumor heterogeneity is accounted for by assigning a primary pattern for the dominant grade and a secondary pattern for the non-dominant grade; the histologic score is derived by adding these two values together. Early studies described the addition of the clinical stage (1-4 scale) to create the Gleason "sum," but this did not achieve widespread use (Gleason et al., 1974). Some contemporary reports use the term sum for Gleason score (Greene et al., 1994). The success of Gleason grading is due to four factors. First, histologic patterns are identified by the degree of acinar differentiation without relying on morphogenetic or histogenetic models. Second, a simplified and standardized drawing is available which has been popular among pathologists throughout the world. Third, Gleason and coworkers provided abundant prospective information that allowed objective development of this self-defining grading system which combined nine separate patterns into five grades. Finally, unlike any other grading system in the body, the Gleason system provided for tumor heterogeneity by identifying primary and secondary patterns. Gleason noted that more than 50% of adenocarcinomas in his series contained two or more patterns (Gleason, 1992). Similarly, Aihara et al. recently found an average of 2.7 different Gleason grades per case (range, 1-5) in a series of 101 totally embedded prostatectomies, and more than 50% of adenocarcinomas contained at least 3 different grades (Aihara et al., 1994). The number of grades increases with increasing cancer volume, and the most common finding is high grade adenocarcinoma within a larger well or moderately differentiated adenocarcinoma (53% of cases).

Grading needle biopsies

Each pattern represents a blend of the growth pattern of the tumor and the amount of acinar differentiation. Recognition of these features in biopsies is frequently difficult due to small size and incomplete sampling. The primary grade is the most common or predominant grade. The secondary grade is the next most common, but should comprise at least 5% of the tumor. It is often hard to apply this rule when the amount of cancer in the specimen is small; in such cases, there may be no secondary pattern, and the primary grade is simply doubled. Most cancers are moderately differentiated, so biopsies usually contain Gleason pattern 3. We report cancer as "Adenocarcinoma (Gleason #+#=#)" to avoid possible confusion of what constitutes grade and score.

Gleason Pattern 1 (Grade 1)

Gleason pattern 1 adenocarcinoma is uncommon and difficult to diagnose, particularly in biopsies. It consists of a circumscribed mass of simple round acini which are uniform in size, shape, and spacing. Circumscription is the single most important criterion to separate pattern 1 and pattern 2, but is usually difficult to identify in biopsies because most foci of cancer extend beyond the edge of the needle core. In such cases, it is best to consider the focus as pattern 2, recognizing that focal loss of the rounded circumscribed border disqualifies pattern 1. The acini in pattern 1 are monotonously replicated, with round contours, evenly spaced acini, uniform round lumens, and distinct cell membranes. Even spacing of the acini is an important but little-appreciated criterion for pattern 1 and pattern 2 cancer. The acini are usually separated from each other by a distance of less than one acinar diameter, and may appear closely packed in some areas. Irregular spacing and separation of more than a few glands at the periphery suggests a higher grade. The cells in Gleason pattern 1 tend to be rectangular, with pale or clear cytoplasm. Nuclear and nucleolar enlargement are moderate, but allow and often define separation from one of its closest mimicks, atypical adenomatous hyperplasia (AAH). In small foci on needle biopsies, this distinction may be difficult, particularly when confounding factors such as crush artifact or drying artifact is present; however, AAH is rare in needle biopsies. Acidic luminal mucin is usually scant and wispy in patterns 1 and 2 carcinoma. Crystalloids are observed in more than half of cases, more than in other patterns.

Gleason Pattern 2 (Grade 2)

Gleason pattern 2 is very similar to pattern 1 except for the lack of circumscription of the focus, indicating the ability of the cancer to spread through the stroma. Slightly greater variation in acinar size and shape is observed, but the acinar contours are chiefly round and smoothly sculpted. Acinar packing is somewhat more variable than pattern 1, and separation is usually less than one acinar diameter. The cytologic features of pattern 2 are indistinguishable from pattern 1.

Gleason Pattern 3 (Grade 3)

Pattern 3 is the most common pattern of prostatic adenocarcinoma, and encompasses a wide and diverse group of lesions. This diversity is reflected in the three main variants: A,B, and C. Pattern 3A, also referred to descriptively as the large gland variant of pattern 3, is easily distinguished from pattern 3B, the small gland variant, and 3C, the cribriform variant. Mixtures of these patterns are frequently observed. The hallmark of pattern 3 adenocarcinoma is prominent variation in size, shape, and spacing of acini. We often use an arbitrary cut point of greater than two-fold variation in acinar size to separate Gleason pattern 3 from pattern 2. Despite this variation, the acini remain discrete and separate, unlike the fused acini of pattern 4 (see below). Some acini of pattern 3 display apparent rigidity, with irregular sharply angulated contours and twisted elongated forms which stand in contrast with the rounded contours of lower grades. Acinar spacing is often variable, usually more than one diameter apart; however, close packing may also be prominent without acinar fusion. Acini are haphazardly arranged in the stroma, sometimes with prominent stromal fibrosis. The irregular size and spacing at the edges imparts a ragged appearance. The large acinar pattern, 3A, differs from the small acinar pattern, 3B, by the average size of the acini. The cribriform pattern, 3C, is distinctive, consisting of solid aggregates of cells punctuated by fenestrations imparting a sieve-like appearance. Papillae may also be present. Ductal adenocarcinoma (endometrioid carcinoma) is included in this pattern. Patterns 3B and 3C are progressively slightly more aggressive than 3A, but they often coexist and have similar cancer-specific death rates. Thus, separation of these subgrades is not necessary and could create additional problems with consistency of grading (Gleason, 1990).

Gleason Pattern 4 (Grade 4)

The characteristic finding of pattern 4 is fusion of acini, with ragged infiltrating cords and nests at the edges. Unlike the simple entwined acinar tubules of pattern 3, this pattern consists of an anastomosing network or spongework of epithelium. The most common subtype, 4A, consists of cells with basophilic cytoplasm, unlike the clear or pale cytoplasm of 4B, the "hypernephroid" pattern. Pattern 4 adenocarcinoma is considered poorly differentiated, and is much more malignant than pattern 3.

Gleason Pattern 5 (Grade 5)

Pattern 5 adenocarcinoma is characterized by fused sheets and masses of haphazardly arranged acini in the stroma, often displacing or overrunning adjacent tissues. In biopsy specimens, these cases raise the serious concern for anaplastic carcinoma or sarcoma. Cases with scattered acinar lumins indicative of glandular differentiation are included within this pattern. Comedocarcinoma is an important subtype of this pattern, consisting of luminal necrosis within an otherwise cribriform pattern; only a single acinus has to contain necrosis to apply this designation. Pattern 5 also includes rare histologic variants such as signet ring-cell carcinoma and small cell undifferentiated carcinoma.

THIRTEEN CLUES FOR GRADING NEEDLE BIOPSIES OF THE PROSTATE
(From Bostwick DG, Dundore PA. Biopsy Pathology of the Prostate. Chapman & Hall, 1997)

CLUE #1: Small foci of cancer do not necessarily mean low grade cancer.

With the advent of serum PSA and sextant biopsy, small foci of carcinoma are frequently found in needle biopsies. However, these small foci are not low grade simply because they are small. High grade adenocarcinoma (Gleason pattern 4 and 5) often infiltrates as irregular ribbons or ragged masses immediately beneath the edge of the prostate, and a small portion may be sampled by biopsy, resulting in very few malignant acini in the specimen. In rare instances, the only evidence of carcinoma may be a few acini surrounding one or more small nerve twigs; this small focus is usually Gleason pattern 3 or 4 in our experience.

CLUE #2: It probably isn't Gleason pattern 1.

Gleason patterns 1 and 5 are the least common patterns in any prostate specimen, including radical prostatectomies and biopsies. Pattern 1 is usually present in the transition zone, an area infrequently sampled by needle biopsy. Further, these tumors are usually small. In a Mayo Clini study of over 300 needle biopsies, only one had Gleason pattern 1 (secondary pattern 1; primary pattern 2).

CLUE #3: To identify Gleason pattern 1, the cancer must be circumscribed.

The most important difference between Gleason pattern 1 and 2 is the presence or absence of circumscription, respectively. Contemporary needle biopsy rarely provides the entire focus of cancer for evaluation, precluding evaluation of the periphery for completeness of circumscription. Consequently, the default grade for partially sampled low grade cancers with uniform spacing is pattern 2.

CLUE #4: Gleason pattern 2 should satisfy the three "R's": Round, Regularly spaced, and Relatively uniform in size.

Gleason pattern 2 cancer consists of predominately round acini without sharp angulation or distorted shape. Nearly as important as acinar roundness is spacing-- pattern 2 acini have relatively uniform spacing throughout the focus, unlike pattern 3 with variable spacing.

CLUE #5: Gleason pattern 2 acini may be close to one another, but must have intervening stroma and no significant distortion of shape.

If significant acinar croding is present with some loss of intervening stroma between acini, it may be more accurately considered as pattern 3. Any significant distortion of adjacent malignant acini constitutes Gleason pattern 3.

CLUE #6: It's probably Gleason pattern 3.

The "default" grade for prostatic adenocarcinoma is pattern 3, recognizing that the great majority of cancers fall in this pattern which encompasses the center of the normal distribution curve. More that 80% of Gleason's original series was pattern 3. Don't be hesitant about assigning pattern 3+3 =6 to a needle biopsy simply because the previous five cases with small foci of cancer were the same grade.

CLUE #7: If there is a two-fold or greater variation in acinar size, it's probably Gleason pattern 3 rather than pattern 2.

When malignant acini are uniformly separated from one another, a two-fold variation in acinar size distinguishes Gleason pattern 3 from pattern 2. Any variation in acinar size less than this may represent Gleason pattern 2 (exceptions to this exist; see CLUE #8).

CLUE #8: Despite relative uniformity of acinar size, significant acinar angulation or distortion indicates Gleason pattern 3 rather than pattern 2.

Significant acinar angulation violates CLUE #4 above, precluding pattern 2. Some areas of Gleason pattern 3 may have relatively uniform acinar size with or without crowding. This pattern often has acini that are smaller than pattern 2. The lack of acinar roundness in such cases separates pattern 3 from pattern 2.

CLUE #9: Fusion is fusion is fusion (Gleason pattern 4).

Acinar fusion separates most cases of Gleason pattern 4 and 3. This is a critical cut point in grading prostate cancer, as pattern 4 indicates poorly differentiated cancer. Fortunately, this is one of the most reproducible cut points due to the requirement for acinar fusion in pattern 4. If a line can be drawn around individual acini, no mattern how tightly packed, then the acini are not fused and it is pattern 3 (See CLUE #10).

CLUE #10: If a line can be drawn between acini that have no intervening stroma (fusion) for a length of at least 4 times the width of the acinus, this constitutes Gleason pattern 4.

Tangentially cut tubular and tortuous acini of Gleason 3 may mimick pattern 4, and such "grade inflation" should be avoided. In difficult cases, if the length of "fusion" of the acinus of concern is less than 4 times its width, we consider it pattern 3.

CLUE #11: If its cribriform and nearly solid, its probably Gleason pattern 4.

Cribriform acini are usually pattern 3 (with comedo necrosis, pattern 5). However, when the sieve-like opernings lose their round rigid punched-out appearance and become collapsed and nearly solid, it is best considered pattern 4. Similarly, when the sieve-like masses lose their round contours, it often indicates transition to pattern 4.

CLUE #12: The loss of most acinar lumens within fused acini indicates Gleason pattern 5.

Most acinar lumens must be absent in order to separated Gleason pattern 5 from pattern 4. Tangential cutting and crush artifact may obscure or hide lumens. However, if most acini lack lumens, it constitutes pattern 5.

CLUE #13: If in doubt, double the pattern to create the score.

With small foci of cancer, it is often best to simply double the Gleason pattern. We invariably do this when there is less than 5% of the needle biopsy involved with cancer unless there is an obvious secondary pattern (this almost never occurs).

Four Common Misinterpretations in Prostate Cancer Grading

Misinterpretation	Comment
1. If a biopsied focus of cancer is small, it is Gleason grade 1 or 2, or "well-differentiated."	Unlikely! Most cancers (over 80% in Gleason's original series) are primary grade 3. When the size is too small to call cancer, suspicious is the prudent default. Size of the focus of cancer has no bearing on Gleason grade at prostatectomy.
2. If a biopsied focus is suspicious for cancer, it is best called Gleason grade 1 or 2, or "well-differentiated."	The prudent diagnosis in the absence of sufficient features for cancer is atypical small acinar proliferation (ASAP). Optimism seems naturally to lead one to consider low Gleason grade; but if there is cancer, it is usually moderately differentiated, since most peripheral zone cancers are moderately differentiated.
3. Confusing the large gland variant of Gleason grade 3 cancer with benign acini.	Cancer acini occasionally are rounded, and medium to large, like benign acini. Look for microvacuolated cytoplasm, nuclear enlargement, and macronucleoli to diagnose cancer.
4. All cribriform acinar formations are Gleason grade 3.	Some cribriform acinar formations are grade 4. These sieve-like spaces lose their round, rigid, punched-out contours, and elongate; the acini collapse into solid areas.

Grading Errors

Needle core biopsy underestimates tumor grade in 33-45% of cases and overestimates grade in 4-32% (Kastendieck, 1980; Catalona et al., 1982; Lange et al., 1983; Garnett et al., 1984; Mills and Fowler, 1986; Epstein and Steinberg, 1990; Bostwick, 1994c; Spires et al., 1994). Grading errors are common in biopsies with small amounts of tumor and low grade tumor, and are probably due to tissue sampling error, tumor heterogeneity, and undergrading of needle biopsies. The accuracy of biopsy is highest for the primary Gleason pattern, but the secondary pattern also provides useful predictive information, particularly when combined with primary pattern to create the Gleason score. Gleason grading should be used for all needle biopsies (Bostwick, 1994b), even those with small amounts of tumor, according to the recommendations of Gleason (Gleason, 1992). Kramer et al. compared Gleason score in 14 gauge needle biopsies with matched lymph node metastases, and found exact correlation in 17 of 42 cases (40%), + 1 in 32 of 42 cases (76%), and + 2 in 40 of 42 cases (95%) (Kramer et al., 1980; Kramer et al., 1981). The lack of a more anaplastic pattern in the metastatic deposits implies that factors other than loss of differentiation are responsible for the ability of cancer cells to metastasize (Brawn et al., 1990; Cumming et al., 1990).

Reproducibility of Gleason Grading and Comparison with Other Grading Systems

Interobserver and intraobserver variability limit the reproducibility of grading in the prostate as in other organs with their grading systems (Bain et al., 1982; Schroder et al., 1985a, 1985b, 1985c; Ten Kate et al., 1986; De Las Morenas et al., 1988; Gallee et al., 1990; Contra and Billis, 1991; Di Loreto et al., 1991; Gleason, 1992). The subjective nature of grading precludes absolute precision, no matter how carefully the system is defined, yet the significant correlation of prostatic adenocarcinoma grade with virtually every outcome measure attests to the predictive strength and utility of grading in the hands of most investigators. Gleason noted exact reproducibility of score in 50% of needle biopsies and + 1 score in 85%, similar to others (Gleason, 1992; Bain et al., 1982). Some investigators question the utility of grading because of the significant incidence of interobserver variability (Bain et al., 1982; De Las Morenas et al., 1988; Di Loreto et al., 1991). One study found a high level of disagreement in grading among 3 pathologists evaluating 41 cases of well to moderately differentiated adenocarcinoma (Di Loreto et al., 1991). Another study compared the level of interoberver agreement with four grading systems in a consecutive series of 100 prostatic adenocarcinomas, and found the Gleason grading system to be the least reproducible, with complete agreement of score in only 66% of cases (De Las Morenas et al., 1988). To perform the analysis, the authors compressed the Gleason scores into three grade groups: 2-5, 6-7, and 8-10. Gallee et al. compared the prognostic accuracy of five grading systems (Broders, Gleason, M.D. Anderson, Mostofi, and Mostofi-Schroder), and found that the Gleason system had the lowest predictive ability for recurrence and death, whereas the Broders and Mostofi-Schroder systems had reasonable accuracy (Gallee et al., 1990). Conversely, another report compared the level of intraobserver agreement with the Gleason, Mostofi, and Bocking systems, and found no significant differences; further, the level of variability was unaffected by type of specimen or amount of tissue examined (Cintra and Billis, 1991). Despite questions of reproducibility, the collective experience supports the clinical utility of grading prostatic adenocarcinoma. Using a variety of architectural and nuclear features, Bibbo et al. developed a Bayesian belief network for grading prostatic adenocarcinoma, and attained agreement with Gleason grade in 241 of 256 microscopic fields (Bibbo et al., 1990; Bibbo et al., 1993). Features used by the belief network included acinus formation, lumen area, acinar fusion, type of acinar fusion, acinar packing, acinar size, acinar uniformity, thickness of acinar epithelial layer, nuclear size, nuclear variability, nuclear shape, chromatin pattern, and nucleolar size. These authors noted that four diagnostic clues allowed unique mapping of Gleason primary patterns, and additional clues offered redundancy and robustness to the network.

Proposed Modifications to Gleason Grading

Morphometric nuclear grading Grouping of grades Amount of high grade cancer (Gleason patterns 4 and 5) Cribriform pattern as 4 rather than 3 Tertiary pattern Gleason grading after therapy Application of Gleason grade to Bladder cancer

Nuclear and Nucleolar Grading

Nuclear and nucleolar enlargement are important diagnostic clues for the diagnosis of malignancy. Morphometric methods allow objective evaluation of nuclear size, roundness, shape, chromatin texture, and other features. In an effort to create an objective method of grading prostatic adenocarcinoma, one study found that morphometric estimates of variation in nuclear size separated patients undergoing prostatectomy into two groups with differing survival rates (Blom et al., 1990). Other investigators have utilized morphometry to improve the predictive value of Gleason grading, but these methods are not used routinely (Diamond et al., 1982; Aragona et al., 1989; Robutti et al.,1989; Schultz et al., 1990;Irinopoulou et al., 1993). Nuclear roundness has been the subject of considerable interest since the first report by Diamond et al. in 1982 (Diamond et al., 1982; Epstein et al., 1984; Mohler et al., 1987; Mohler et al., 1988a; Mohler et al., 1988b; Partin et al., 1989; Armas et al., 1991; Partin et al., 1992; Schaeffer et al., 1992). Average nuclear roundness accurately predicted prognosis in patients with untreated stage T1b (A2) prostatic adenocarcinoma and other clinical stage adenocarcinomas. However, many of these reports were limited by small sample size (less than 30 patients), use of the same patient cohort in multiple publications, failure to describe the morphologic variations and nuclear roundness extremes, and patient selection bias. Nuclear roundness failed to identify patients with tumor recurrence following radiation therapy except in those with well-differentiated adenocarcinoma (Schaeffer et al., 1992). Nucleolar grading of prostatic adenocarcinoma has also been proposed, but has not been adopted (Grade 1: Large and prominent nucleoli in virtually every cell; Grade 2: Intermediate; Grade 3: Very small nucleoli which are difficult to find) (Myers et al., 1982).

Grade Compression

Many authors have simplified the Gleason grading system by compressing the scores into groups, usually creating three groups: 2-3-4, 5-6-7, and 8-9-10 (De Las Morenas et al., 1988) Unfortunately, grade compression diminishes the statistical strength of grading (Gleason, 1992). Further, the choice of grouping is often problematic; the most important "cut point" is between Gleason score 6 and 7 due to the emergence of poorly differentiated adenocarcinoma (pattern 4) in score 7, yet many studies combine these scores. Gleason argued against grade compression except in studies with a small number of patients in which grouping is unavoidable; in such cases, a cutpoint between scores 6 and 7 is preferred (Gleason, 1992; Oesterling et al., 1987). The probability of lymph node metastases is significantly greater in patients with score 7 adenocarcinoma than in those with score 6 (Thomas et al., 1982; McNeal et al., 1990).

Volume of High Grade Adenocarcinoma

The volume of high grade adenocarcinoma appears to be an important prognostic factor; as tumor volume increases, the frequency and volume of high-grade tumor increases (McNeal et al., 1986; McNeal et al., 1990; Bostwick et al., 1993; Bostwick, 1994a). Gleason grade stratifies adenocarcinoma into three subgroups with different levels of aggressiveness; Gleason pattern 1 and 2 adenocarcinoma is almost always small, usually less than 1 cc, indolent, localized, and frequently located in the transition zone whereas pattern 3 adenocarcinoma is variable in size and very common. Pattern 4 and 5 adenocarcinomas are usually larger and more aggressive than lower grade tumors, and likely to extend beyond the prostate or metastasize (McNeal et al., 1990). One study of 209 radical prostatectomies from patients with clinical stage T1 and T2 adenocarcinomas reported that the volume of high grade adenocarcinoma (Gleason grades 4 and 5) had the highest predictive value for lymph node metastases, greater even than tumor volume (McNeal et al., 1990). Twenty-two of 38 patients (58%) with more than 3.2 cc of high-grade adenocarcinoma had pelvic lymph node metastases, compared with only 1 of 171 patients (0.6%) with smaller volumes of high-grade adenocarcinoma. The extent of solid undifferentiated carcinoma shows a strong correlation with tumor progression according to one report of 24 cases (Gaffney et al., 1992). Gleason score and percent of pattern 4 and 5 adenocarcinoma show a positive correlation with tumor volume (Bostwick, 1994a). In addition, poorly differentiated adenocarcinoma was the strongest predictor of tumor progression and cancer-specific survival in a series of 107 patients with clinically localized prostatic adenocarcinoma (Egawa et al., 1993). The cumulative data suggest that the volume of high-grade adenocarcinoma is of prognostic significance, refuting Gleason's contention that prostatic carcinoma behaves according to the average of histologic grades. However, many of these studies grouped the Gleason scores, raising questions of grade compression. Histologic dedifferentiation of prostatic adenocarcinoma has been reported by numerous investigators, but these studies included only cases with more than one resection, probably selecting for adenocarcinomas which are more aggressive and thus more likely to require repeat operation (Brawn, 1983; Cumming et al., 1990; McNeal et al., 1990) Dedifferentiation occurs in 65% of repeat transurethral resctions (Brawn, 1983; Cumming et al., 1990). Dedifferentiation to high grade adenocarcinoma appears to be unusual in low-grade (Gleason patterns 1-3) and small volume (1 cc) adenocarcinoma, occurring in only 2.4% of patients in 7 years according to one study (Whittemore et al., 1991).

Recommendations of WHO, 1999

The Committee recommends that pathologists report Gleason score for all prostate specimens. Further, the relative percentage or proportion of high-grade cancer (Gleason primary pattern 4 and 5) be included. A global Gleason score should be given for the entirity of multiple biopsies containing cancer (in addition to individual specimen grading). Pathologists should pursue additional education, as appropriate, in understanding and using the Gleason system.

EXTENT OF CANCER ON NEEDLE BIOPSY: RP DATA

Cancer Volume
Biopsy cancer volume depends on multiple factors, including prostate volume, cancer volume, cancer distribution, number of biopsy cores obtained, the cohort of patients being evaluated and the technical expertise of the person undertaking the biopsy procedure. The combined results from multiple studies indicate that the biopsy extent of cancer is somewhat predictive of cancer extent in radical prostatectomy specimens and should be reported, although its predictive value for an individual patient is limited. Reliance upon this measure alone may often be misleading. There is a fair to good correlation between amount of cancer reported in biopsies and that subsequently found in radical prostatectomy specimens. This correlation is greatest for large cancers. High cancer burden on needle biopsy is strongly suggestive of large volume high stage cancerr.

Low cancer volume on needle biopsy does not necessarily indicate low-volume low-stage cancer in final prostatectomy specimens. [1, 2, 3, 4, 5, 6, 7, 8] Cupp et al. found that patients with less than 30% of needle cores replaced by cancer had a mean volume in the radical prostatectomy of 6.1 cc (range, 0.19-16.8 cc), indicating that the amount of tumor on transrectal needle biopsy was not a good predictor of tumor volume. In another report, patients with less than 10% cancer in the biopsy had a 30% risk of positive surgical margins, 27% risk of extraprostatic extension, and 22% risk of PSA biochemical progression; these risks were higher in patients with more than 10% cancer. Patients with less than 3 mm cancer and Gleason score 6 or less on needle biopsy had a 59% risk of cancer volume exceeding 0.5 cc. Those with less than 2 mm of cancer had 26% risk of extraprostatic cancer, and those with less than 3 mm had 52% risk. In another study, 66 patients diagnosed with a single microscopic focus of carcinoma on biopsy were found to have grossly visible disease on prostatectomy specimens in 92% of cases. Rubin reported that cancer volume on needle biopsy was predictive of extraprostatic extension, with the odds of pT3 cancer increasing by a factor of 1.7 times with each 20% increase in cancer volume above 40% volume. The CAP recommends that the volume of cancer in needle biopsy should be reported as the percentage of tissue involved by cancer.

Cancer volume in biopsies was a strong predictor of biochemical failure. Similarly, cancer volume in radical prostatectomy specimens was usually but not always predictive of cancer recurrence. Accordingly, the CAP recommends that cancer volume be recorded in prostatectomy specimens, although there is no accepted universal approach. Methods include computer-assisted morphometric determination, simple measurement of length X height X section thickness of the cancer (some measure the largest "index" focus, whereas others report the cumulative volumes), greatest cancer dimension , grid method, and visual estimate of the percentage of cancer. Measurements performed on fixed tissue sections may include a formalin shrinkage correction factor which varies from about 1.25 to 1.5, representing tissue shrinkage of 18 to 33%; conversely, Schned and colleagues demonstrated that shrinkage correction is unnecessary. Cancer volume is a critical element in definitions of clinically significant and insignificant prostate cancer.

Vanishing Cancer Phenomenon
In some radical prostatectomy specimens, there is little or no residual cancer within the specimen ("vanishing cancer phenomenon"). We recently reported that about 6 in 1,000 partially or totally sampled radical prostatectomies (0.5%) had no residual cancer (stage pT0) among a series of 6,843 cases (Bostwick & Bostwick, in press). The incidence of this "vanishing cancer phenomenon" declined more than ten-fold from 1966 to 1995, probably as a result of the substantial decline in the use of transurethral resection of the prostate. We now encounter cases of no residual cancer in about 2 cases per 1000 radical prostatectomies. This decline may be offset in recent years by two factors: (1) an upswing in the number of patient receiving preoperative androgen deprivation therapy (or radiation therapy) that causes apparent cancer volume reduction in some patients, although this trend was not apparent in our study because such therapy was given only rarely to our patients; and (2) increasing vigilance in screening demonstrates that prostate cancer is now being detected at smaller volume and lower stage than ever before. None of our "vanishing cancer" patients developed clinical evidence of cancer recurrence with a mean follow-up of almost 10 years (Bostwick & Bostwick). Further, those that died of intercurrent disease had no evidence of cancer at the time of death. These cumulative data strongly suggest that these patients are cured of cancer and no residual therapy is indicated.

The inability to identify residual cancer in radical prostatectomy specimens raises the question of accuracy of the original diagnosis of cancer. In reports from Johns Hopkins Medical Center, biopsies were overdiagnosed as cancer in 2 of 4 cases with no residual cancer after radical prostatectomy1 and major changes in diagnosis occurred in 1.4% of referred cases following second opinion review; however, these results have not been confirmed by other centers. Over-diagnosis of prostate cancer is also a concern, with an incidence of 21% misdiagnosis in small foci in transurethral resection specimens between 1960 and 1970. Genotypic analysis to verify patient identity in cases of "vanishing" cancer is becoming increasingly popular and appears prudent to reassure patients (DG Bostwick, unpublished observations) and exclude the possibility of improper patient identification. DNA "fingerprinting" can now be used to compare formalin-fixed paraffin-embedded biopsies and prostatectomy tissues. Substantial laboratory resources may be needed to identify minimal residual cancer, and even exhaustive sectioning may fail. How many sections are reasonable to obtain in such cases? When can one stop sectioning if no cancer is found? We believe that it is appropriate for the pathologist to submit routine sections of the entire prostatectomy for histologic evaluation in such cases; however, after submission and examination of the entire prostate, obtaining additional levels from paraffin blocks and/or re-embedding all blocks (block-flipping) are probably not necessary, as any residual cancer at that point is likely to be extremely small and of no clinical significance.

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