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Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

With the completion of the Human Genome Project and high-throughput screening methods using cDNA array and tissue microarray (TMA) technology, there is a pressing need to manage the voluminous data sets generated from these types of investigations. Herein is described a database model to handle 1) clinical and pathology data, 2) TMA location information, and 3) web-based histology results. The model is useful for managing clinical, pathology, and molecular data on >1300 prostate cancer patients dating back to 1995 from the University of Michigan Specialized Program of Research Excellence for prostate cancer. The key components in this multidatabase model are 1) the TMA database, 2) the TMA-image database (TMA-I DB), and 3) the prostate pathology and clinical information databases. All databases were created in Microsoft Access (Microsoft, Redmond, WA). Desired patient, tissue, block, diagnosis, array location, and respective clinical and pathology information is obtained by linking the unique identifier fields among database tables. The TMA database is comprised of interrelated data from 336 prostate cancer patients transferred into 19 TMA blocks with 5451 TMA biopsy cores. Tissue samples include 1695 normal prostate, 3171 prostate cancer, 464 prostatic intraepithelial neoplasia, and 121 atrophy. All 19 TMA blocks have been analyzed over the Internet for several immunohistochemical biomarkers including E-cadherin, prostate-specific antigen, p27 Kip1 , and Ki-67 labeling index. This system facilitates the statistical analysis of high-density TMA data with clinical and pathology information in an efficient and cost-effective manner. Because the review is performed over the Internet, this system is ideal for collaborative multi-institutional studies.

E-cadherin is a calcium 2+-dependent cell-adhesion molecule that determines epithelial development in the embryo and maintains adult differentiated epithelium and homeostasis. Aberrant or decreased expression has been reported to be associated with prostate carcinoma progression. The degree of E-cadherin expression in prostate cancer remains controversial. Some studies have reported decreased expression of E-cadherin as tumors advance and metastasize. Other studies have not demonstrated this relationship. To address these variations, we undertook a study to systematically evaluate E-cadherin expression in a broad range of prostate tissue. Benign prostate, clinically localized prostate cancer, and hormone-refractory metastatic prostate cancer were analyzed under uniform conditions using high-density tissue microarrays (TMA). Formalin-fixed, paraffin-embedded prostate carcinoma from men with clinically localized prostate carcinoma and autopsy material from men who died of widely metastatic, hormone-refractory prostate carcinoma were arrayed into 6 high-density TMA blocks. Benign and atrophic prostate tissue and high-grade prostatic intraepithelial neoplasia (PIN) were also included from the clinically localized cases. Immunohistochemistry was performed using the immunoglobulin G1 mouse monoclonal antibody (HECD-1; Zymed, San Francisco, CA). Membranous staining was recorded as low (aberrant) or high (normal). E-cadherin expression was considered aberrant if less than 70% of the cells had strong membranous staining. A total of 1,220 prostate TMA samples were analyzed. High (normal) E-cadherin expression was seen in 87% of 757 benign, 80% of 41 high-grade PIN, 82% of 325 prostate carcinoma and 90% of 97 hormone-refractory prostate carcinoma TMA samples. Mean E-cadherin expression was determined for each of the 128 clinically localized prostate cancer cases. Aberrant E-cadherin expression showed a statistical trend toward an association with positive surgical margins (P =.012), higher Gleason score (P =.18), and prostate-specific antigen (PSA) failure (Kaplan-Meier analysis, log-rank P =.09). There was a statistically significant association between aberrant E-cadherin expression and larger tumor size (P =.01). No significant associations were seen with extraprostatic extension and seminal vesicle invasion. The current study shows a broad-spectrum approach to evaluating E-cadherin protein expression in prostate carcinoma. Clinically localized prostate tumors, treated with surgery alone, show a high level of E-cadherin expression. Aberrant expression was identified in tumors with positive surgical margins, higher Gleason score, and a higher rate of PSA failure. However, these trends were not statistically significant. A statically significant association between aberrant E-cadherin expression and larger tumor size was identified. In the metastatic hormone-refractory prostate tumors, E-cadherin expression was vastly expressed, and only rare cases had aberrant expression. Therefore, the findings of this study are most consistent with a transient down-regulation of E-cadherin in localized prostate cancer. Metastatic prostate cancer shows strong E-cadherin expression as determined by anti-E-cadherin antibody HECD-1.

Postatrophic hyperplasia (PAH) of the prostate gland often demonstrates overlapping histological features with prostatic adenocarcinoma (PCA). These features include small acinar growth and enlarged nuclei with prominent nucleoli. Recent work has demonstrated that PAH is a proliferative, noninvoluting lesion. PAH is also histologically distinct from simple atrophy (SA), which has intermediate- to large-sized glands, minimal cytoplasm, and inconspicuous nuclei. However, despite overlapping features between PAH and PCA, high-grade prostatic intraepithelial neoplasm (HGPIN) is still considered the only direct neoplastic precursor to PCA. HGPIN resembles PCA in its topographic distribution, cytological appearance, and molecular alterations including chromosome 8p loss and chromosome 8 centromeric gain. To examine the hypothesis that PAH is the earliest histologically distinct precursor to HGPIN or PCA, the frequency, distribution, proliferative state, and chromosome 8 gain of benign prostate, SA, PAH, HGPIN, and PCA were analyzed. Forty radical prostatectomy specimens from men with clinically localized PCA were systematically analyzed. Proliferation was determined by Ki-67 immunohistochemistry (MIB-1) on formalin-fixed, paraffin-embedded tissue and quantified by digital image analysis from a total of 5,510 sample areas with benign, SA, PAH, HGPIN, and PCA. A tissue microarray was constructed to evaluate 8c gain using interphase fluorescence in situ hybridization. SA foci (n = 129) and PAH foci (n = 114) were identified in the 40 cases of which 74% (95 of 129) and 88% (100 of 114) were seen in the peripheral zone, respectively (P = 0.006). PAH and SA were identified adjacent to PCA in 28% (32 of 114) and 14% (18 of 129) of foci examined, respectively (P = 0.007). The median number of proliferating nuclei increased significantly from benign (1.20%), SA (2.67%), PAH (3.62%), HGPIN (6.14%), to PCA (12.00%) (P < 0.001). The median percentage of nuclei with more than three centromeric probe signals (chromosome 8c gain) for SA, HGPIN, PAH, and PCA were 2.1, 2.8, 4.0, and 6.0%, respectively, as compared to benign prostate with 1.3% (P = 0.006). In conclusion, the present study identified a strong topographic association between PAH and PCA. PAH is also seen often to be closely associated with chronic inflammation. Proliferation of PAH is significantly greater than benign prostatic epithelium and SA but less than HGPIN or PCA. Gain of 8c is significantly greater in PAH than benign prostate, SA, and even HGPIN. These findings demonstrate a strong association between PAH and PCA, supporting its role as a neoplastic precursor.

Radical prostatectomy provides excellent cancer control in men with clinically localized prostate carcinoma. However, to our knowledge preoperative parameters for distinguishing indolent from clinically significant cancer are not well characterized. In fact, recent evidence suggests that the percent of Gleason pattern 4/5 carcinoma in the complete radical prostatectomy specimen is one of the strongest predictors of prostate cancer progression and a valid measure of cancer severity. However, it is unclear whether preoperative parameters, including biopsy Gleason pattern 4/5 carcinoma, may predict radical prostatectomy Gleason pattern 4/5 disease and, thereby, distinguish indolent from clinically significant cancer.We prospectively obtained 101 consecutive radical prostatectomy specimens and processed them in whole mount fashion. In addition to total tumor volume, we determined tumor volume for each Gleason pattern. Biopsy tumor area was measured in a similar fashion. Univariate and multivariate analyses were performed to identify preoperative clinical and pathology parameters for predicting Gleason pattern 4/5 carcinoma on prostatectomy specimens.Biopsy Gleason score 7 or greater, Gleason pattern 4/5 carcinoma, perineural invasion and biopsy tumor area had statistically significant associations for identifying Gleason pattern 4/5 carcinoma on prostatectomy specimens. Logistic regression models for predicting any or greater than 10% Gleason pattern 4/5 carcinoma on prostatectomy specimens revealed that an area of pattern 4/5 disease of greater than 0.01 cm.2 on biopsy was the best single predictor with odds ratios of 15.0 (95% confidence interval 3.3 to 69.0, p = 0.0005) and 3.9 (95% confidence interval 1. 4 to 10.9, p = 0.009), respectively. For predicting any pattern 4/5 carcinoma on prostatectomy specimens a biopsy area of pattern 4/5 disease of greater than 0.01 cm.2 had only 38% sensitivity but 96% specificity. Similarly for predicting significant pattern 4/5 disease on prostatectomy specimens, defined as 10% or greater pattern 4/5, sensitivity and specificity for a biopsy area of greater than 0.01 cm.2 were 34% and 88%, respectively. Therefore, due to high false-negative rates these models had limited predictive value on an individual basis.Biopsy parameters such as Gleason pattern 4/5 carcinoma may provide adequate specificity for predicting clinically significant cancer, as defined by high grade Gleason patterns in the corresponding radical prostatectomy specimen. However, the accuracy of these parameters for predicting indolent cancer is limited by a prohibitive rate of false-negative findings.

The theory that poorly differentiated prostate carcinoma develops a neuroendocrine (NE) phenotype is controversial. Supportive data is variable with NE expression being observed in anywhere from 5% to 83% of prostate cancers. These percentages are derived from standard immunohistochemistry studies, which make no attempt to quantify the results. High-density tissue microarrays (TMAs), represent a novel method for evaluating up to 1000 tissue samples with a 0.6 mm diameter on a single glass slide. This high throughput technology for screening antibodies, however, requires validation to determine if TMAs are useful in evaluating heterogeneously expressed proteins such as the NE markers chromogranin A (CGA) and synaptophysin (SYN). This study compares results from standard slides to TMAs in 50 primary and metastatic prostate tumors taken from 12 rapid autopsies from men with hormone refractory prostate cancer. One hundred standard and 2 TMA slides were immunostained for CGA and SYN. Using standard slides, focal NE expression was seen in 1/12 primary prostate tumors. Overall, 13/100 (13%) standard slides showed focal NE expression for both primary and metastatic prostate tumors; NE expression was observed in 4/12 autopsy cases (33%) when all tumor sites per case were considered. 458 tissue elements (tumor and normal) were arrayed into one paraffin block. Seventy-three percent (332/458) of the elements placed into the TMA were confirmed histologically to represent tumor. Seventy-five percent (250/332) and 66% (218/332) could be evaluated for CGA and SYN expression, respectively. Six of the metastatic tumors expressed CGA and SYN or 2.4% (6/250; 95% CI = 0.9% to 5.2%) and 2.3% (6/218; 95% CI = 0.8% to 5.3%), respectively. In conclusion, only focal NE expression was observed by both methods (eg, standard and TMA slides). The focal expression in these advanced prostate tumors was unexpected given data from prostate tumor cell lines and animal models suggesting that progression to the NE phenotype parallels tumor progression. This study also supports the use of high density TMAs to screen for protein expression, even when expression is focal.

Tissue microarray technology promises to enhance tissue-based molecular research by allowing improved conservation of tissue resources and experimental reagents, improved internal experimental control, and increased sample numbers per experiment. Organized, well-validated collection and analysis of the voluminous image data produced by tissue microarray technology is critical to maximize its value. Web-based technology for visual analysis and searchable storage of microarray image data could provide optimal flexibility for research groups in meeting this goal, but this approach has not been examined scientifically. Toward this goal, a prostate tissue microarray block containing 432 tissue cores (0.6 mm diameter) was constructed. Moderately compressed (200 kb).jpg images of each tissue spot were acquired and were saved using a naming convention developed by the SPORE Prostate Tissue Microarray Collaborative Group. Four hundred three tissue array spot images were uploaded into a database developed for this study and were converted to.fpx format to decrease Internet transmission times for high-resolution image data. In phase I of the image analysis portion of the study, testing and preliminary analysis of the Web technology was performed by 2 pathologists (M.A.R. and G.S.B.). In phase II, 2 pathologists (J.I.E. and T.M.W.) with no previous exposure to this technology and no knowledge of the structure of the study were presented a set of 130 sequential tissue spot images via the Web on their office computers. In phase III, the same pathologists were presented a set of 193 images, including all 130 from phase II and 63 others, with image presentation order randomized. With each zoomable tissue spot image, each pathologist was presented with a nested set of questions regarding overall interpretability of the image, presence or absence of cancer, and predominant and second most frequent Gleason grade. In phases II and III of the study, 319 of 323 (99%) image presentations using this Web technology were rated interpretable. Comparing the 2 pathologists' readings in phases II and III, Gleason grade determinations by each pathologist were identical in 179 of 221 (81%) determinations and were within 1 point of each other in 221 of 221 (100%) determinations, a performance rate similar to if not better than that previously reported for direct microscopic Gleason grading. Interobserver comparison of Gleason score determinations and intraobserver comparisons for Gleason grade and score also showed a pattern of uniformity similar to those reported in direct microscope-based Gleason grading studies. Interobserver (7.5%) and intraobserver (5% and 3%) variability in determining whether diagnosable cancer was present point out the existence of a “threshold effect” that has rarely been studied but may provide a basis for identification of features that are most amenable to improved diagnostic standardization. In summary, storage and analysis of tissue microarray spot images using Web-based technology is feasible and practical, and the quality of images obtained using the techniques described here appears adequate for most tissue-based pathology research applications. H UM P ATHOL 32:417-427. Copyright © 2001 by W.B. Saunders Company

Objectives. Elevation of serum prostate-specific antigen (PSA) after radical prostatectomy for prostate cancer is considered a surrogate marker of therapeutic failure. The most likely explanation for early PSA failure is considered to be due to local recurrent disease, provided the patient had a nondetectable PSA level after radical prostatectomy. Others have recently suggested that benign prostatic glands located on the surgical margins may often lead to detectable PSA levels. We examined the frequency of benign prostatic glands at the surgical margins of radical prostatectomy specimens and the factors associated with this finding. Methods. One hundred nineteen consecutive radical prostatectomies were performed by two experienced oncologic surgeons. Whole-mount sectioning of the prostatectomy specimens was performed at 3-mm intervals. Bivariate and multivariate analyses were used to determine which clinical and pathologic factors were associated with benign glands on inked surgical margins. Results. Of the 119 cases, 13 (11%) had benign glands on the inked surgical margins. Four of these 13 had tumor on the inked margins. The remaining 9 cases (8%) were organ confined (pT2), with negative surgical margins. Benign glands were most often seen to involve the apex focally (7 of 9 cases). On bivariate and multivariate analyses, a high Gleason score and prostate gland volume were significantly associated with finding benign glands on the surgical margins. Only 2 of 86 patients with follow-up had PSA recurrence at 59 and 67 days and neither had benign glands on the inked surgical margins. Conclusions. The presence of benign prostatic glands identified on inked surgical margins was an infrequent occurrence in this consecutive series of 119 whole-mount prostatectomy specimens. When benign glands were identified, they most often consisted of 1 to 3 glands at the apex margin. These findings suggest that benign glands on surgical margins are an unusual cause of postoperative detectable PSA.