Angelo M. DeMarzo

Pathological and molecular aspects of prostate cancer

This review focuses on new findings and controversial issues in the the pathology and molecular biology of adenocarcinoma of the prostate. Since management of high-grade prostatic intraepithelial neoplasia on needle biopsy--the most common precursor lesion to prostate cancer--is the crucial issue with this lesion, we discuss the risk of cancer subsequent to this histological diagnosis and the issue of whether such neoplasia should be regarded as carcinoma-in-situ. We also look at prostate cancer itself, starting with its diagnosis, reporting on needle biopsy, and reviewing how the most frequently used grading system, the Gleason grading system, affects treatment. The molecular basis of prostate cancer includes inheritable and somatic genetic changes (tumour suppressor genes, loss of heterozygosity, gene targets and regions of chromosomal gain, CpG island promoter methylation, invasion and metastasis suppressor genes, telomere shortening, and genetic instability). Changed gene expression (eg, proliferation-related genes, changes in the androgen receptor, apoptosis and stress-response genes) have potential as biomarkers and therapeutic targets in prostate cancer.

Germline mutations and sequence variants of the macrophage scavenger receptor 1 gene are associated with prostate cancer risk

Deletions on human chromosome 8p22–23 in prostate cancer cells and linkage studies in families affected with hereditary prostate cancer (HPC) have implicated this region in the development of prostate cancer. The macrophage scavenger receptor 1 gene (MSR1, also known as SR-A) is located at 8p22 and functions in several processes proposed to be relevant to prostate carcinogenesis. Here we report the results of genetic analyses that indicate that mutations in MSR1 may be associated with risk of prostate cancer. Among families affected with HPC, we identified six rare missense mutations and one nonsense mutation in MSR1. A family-based linkage and association test indicated that these mutations co-segregate with prostate cancer (P = 0.0007). In addition, among men of European descent, MSR1 mutations were detected in 4.4% of individuals affected with non-HPC as compared with 0.8% of unaffected men (P = 0.009). Among African American men, these values were 12.5% and 1.8%, respectively (P = 0.01). These results show that MSR1 may be important in susceptibility to prostate cancer in men of both African American and European descent.

Phenotypic Analysis of Prostate-Infiltrating Lymphocytes Reveals TH17 and Treg Skewing

Purpose: Pathologic examination of prostate glands removed from patients with prostate cancer commonly reveals infiltrating CD4+ and CD8+ T cells. Little is known about the phenotype of these cells, despite accumulating evidence suggesting a potential role for chronic inflammation in the etiology of prostate cancer. Experimental Design: We developed a technique that samples the majority of the peripheral prostate through serial needle aspirates. CD4+ prostate-infiltrating lymphocytes (PIL) were isolated using magnetic beads and analyzed for subset skewing using both flow cytometry and quantitative reverse transcription-PCR. The transcriptional profile of fluorescence-activated cell sorted prostate-infiltrating regulatory T cells (CD4+, CD25+, GITR+) was compared with naïve, peripheral blood T cells using microarray analysis. Results: CD4+ PIL showed a paucity of TH2 (interleukin-4–secreting) cells, a surprising finding given the generally accepted association of these cells with chronic, smoldering inflammation. Instead, CD4+ PIL seemed to be skewed towards a regulatory Treg phenotype (FoxP3+) as well as towards the TH17 phenotype (interleukin-17+). We also found that a preponderance of TH17-mediated inflammation was associated with a lower pathologic Gleason score. These protein level data were reflected at the message level, as analyzed by quantitative reverse transcription-PCR. Microarray analysis of pooled prostate-infiltrating Treg revealed expected Treg-associated transcripts (FoxP3, CTLA-4, GITR, LAG-3) as well as a number of unique cell surface markers that may serve as additional Treg markers. Conclusion: Taken together, these data suggest that TH17 and/or Treg CD4+ T cells (rather than TH2 T cells) may be involved in the development or progression of prostate cancer.

Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors

Poorly differentiated neuroendocrine carcinomas (NECs) of the pancreas are rare malignant neoplasms with a poor prognosis. The aim of this study was to determine the clinicopathologic and genetic features of poorly differentiated NECs and compare them with other types of pancreatic neoplasms. We investigated alterations of KRAS, CDKN2A/p16, TP53, SMAD4/DPC4, DAXX, ATRX, PTEN, Bcl2, and RB1 by immunohistochemistry and/or targeted exomic sequencing in surgically resected specimens of 9 small cell NECs, 10 large cell NECs, and 11 well-differentiated neuroendocrine tumors (PanNETs) of the pancreas. Abnormal immunolabeling patterns of p53 and Rb were frequent (p53, 18 of 19, 95%; Rb, 14 of 19, 74%) in both small cell and large cell NECs, whereas Smad4/Dpc4, DAXX, and ATRX labeling was intact in virtually all of these same carcinomas. Abnormal immunolabeling of p53 and Rb proteins correlated with intragenic mutations in the TP53 and RB1 genes. In contrast, DAXX and ATRX labeling was lost in 45% of PanNETs, whereas p53 and Rb immunolabeling was intact in these same cases. Overexpression of Bcl-2 protein was observed in all 9 small cell NECs (100%) and in 5 of 10 (50%) large cell NECs compared with only 2 of 11 (18%) PanNETs. Bcl-2 overexpression was significantly correlated with higher mitotic rate and Ki67 labeling index in neoplasms in which it was present. Small cell NECs are genetically similar to large cell NECs, and these genetic changes are distinct from those reported in PanNETs. The finding of Bcl-2 overexpression in poorly differentiated NECs, particularly small cell NEC, suggests that Bcl-2 antagonists/inhibitors may be a viable treatment option for these patients.

In vivo Therapeutic Responses Contingent on Fanconi Anemia/BRCA2 Status of the Tumor

Purpose:BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor. Experimental Design: Two retrovirally complemented pancreatic cancer cell lines having defects in the Fanconi anemia pathway, PL11 (FANCC-mutated) and Hs766T (FANCG-mutated), as well as several parental pancreatic cancer cell lines with or without mutations in the Fanconi anemia/BRCA2 pathway, were assayed for in vitro and in vivo sensitivities to various chemotherapeutic agents. Results: A distinct dichotomy of drug responses was observed. Fanconi anemia–defective cancer cells were hypersensitive to the cross-linking agents mitomycin C (MMC), cisplatin, chlorambucil, and melphalan but not to 5-fluorouracil, gemcitabine, doxorubicin, etoposide, vinblastine, or paclitaxel. Hypersensitivity to cross-linking agents was confirmed in vivo; FANCC-deficient xenografts of PL11 and BRCA2-deficient xenografts of CAPAN1 regressed on treatment with two different regimens of MMC whereas Fanconi anemia–proficient xenografts did not. The MMC response comprised cell cycle arrest, apoptosis, and necrosis. Xenografts of PL11 also regressed after a single dose of cyclophosphamide whereas xenografts of genetically complemented PL11FANCC did not. Conclusions: MMC or other cross-linking agents as a clinical therapy for pancreatic cancer patients with tumors harboring defects in the Fanconi anemia/BRCA2 pathway should be specifically investigated.

Cyclophosphamide Augments Antitumor Immunity: Studies in an Autochthonous Prostate Cancer Model

To study the immune response to prostate cancer, we developed an autochthonous animal model based on the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse in which spontaneously developing tumors express influenza hemagglutinin as a unique, tumor-associated antigen. Our prior studies in these animals showed immunologic tolerance to hemagglutinin, mirroring the clinical situation in patients with cancer who are generally nonresponsive to their disease. We used this physiologically relevant animal model to assess the immunomodulatory effects of cyclophosphamide when administered in combination with an allogeneic, cell-based granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapy. Through adoptive transfer of prostate/prostate cancer-specific CD8 T cells as well as through studies of the endogenous T-cell repertoire, we found that cyclophosphamide induced a marked augmentation of the antitumor immune response. This effect was strongly dependent on both the dose and the timing of cyclophosphamide administration. Mechanistic studies showed that immune augmentation by cyclophosphamide was associated with a transient depletion of regulatory T cells in the tumor draining lymph nodes but not in the peripheral circulation. Interestingly, we also noted effects on dendritic cell phenotype; low-dose cyclophosphamide was associated with increased expression of dendritic cell maturation markers. Taken together, these data clarify the dose, timing, and mechanism of action by which immunomodulatory cyclophosphamide can be translated to a clinical setting in a combinatorial cancer treatment strategy.

Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma.

The histologic distinction between high-grade prostate cancer and infiltrating high-grade urothelial cancer may be difficult, and has significant implications because each disease may be treated very differently (ie, hormone therapy for prostate cancer and chemotherapy for urothelial cancer). Immunohistochemistry of novel and established prostatic and urothelial markers using tissue microarrays (TMAs) were studied. Prostatic markers studied included: prostate-specific antigen (PSA), prostein (P501s), prostate-specific membrane antigen (PSMA), NKX3.1 (an androgen-related tumor suppressor gene), and proPSA (pPSA) (precursor form of PSA). “Urothelial markers” included high molecular weight cytokeratin (HMWCK), p63, thrombomodulin, and S100P (placental S100). TMAs contained 38 poorly differentiated prostate cancers [Gleason score 8 (n=2), Gleason score 9 (n=18), Gleason score 10 (n=18)] and 35 high-grade invasive urothelial carcinomas from radical prostatectomy and cystectomy specimens, respectively. Each case had 2 to 8 tissue spots (0.6-mm diameter). If all spots for a case showed negative staining, the case was called negative. The sensitivities for labeling prostate cancers were PSA (97.4%), P501S (100%), PSMA (92.1%), NKX3.1 (94.7%), and pPSA (94.7%). Because of PSAs high sensitivity on the TMA, we chose 41 additional poorly differentiated primary (N=36) and metastatic (N=5) prostate carcinomas which showed variable PSA staining at the time of diagnosis and performed immunohistochemistry on routine tissue sections. Compared to PSA, which on average showed 18.8% of cells with moderate to strong positivity, cases stained for P501S, PSMA, and NKX3.1 had on average 42.5%, 53.7%, 52.9% immunoreactivity, respectively. All prostatic markers showed excellent specificity. HMWCK, p63, thrombomodulin, and S100P showed lower sensitivities in labeling high-grade invasive urothelial cancer in the TMAs with 91.4%, 82.9%, 68.6%, and 71.4% staining, respectively. These urothelial markers were relatively specific with only a few prostate cancers showing scattered (≤2%) weak-moderate positive cells. In summary, PSA can be used as the first screening marker for differentiating high-grade prostate adenocarcinoma from high-grade urothelial carcinoma. Immunohistochemistry for P501S, PSMA, NKX3.1, and pPSA are useful when high-grade prostate cancer is suspected based on the morphology or clinical findings, yet shows negative or equivocal PSA staining. HMWCK and p63 are superior to the novel markers thrombomodulin and S100P.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy

A prostate-specific membrane antigen–activated prodrug selectively kills cancer cells and is being tested in patients with advanced cancer. An Old Approach Is New Again In the 1995 film The Last Supper, a group of graduate students invite a diverse cast of characters for a series of Sunday dinners. After one guest threatens the lives of several of the students, subsequent dinners turn deadly. If the guest holds views that the group considers toxic to society, then the house wine is made poisonous and served only to the unwanted houseguest, who promptly dies. In a related scenario, Denmeade et al. use a prodrug to seek out and selectively poison unsavory guests that are toxic to the body—namely, cancer cells. The new work describes the development of a thapsigargin (TG) prodrug that is activated in the vasculature of solid tumors by tumor endothelial cells. The carboxypeptidase prostate-specific membrane antigen (PSMA)—which is selectively expressed on the surface of prostate cancer cells, including metastatic ones, and tumor, but not normal, endothelial cells—cleaves and activates the prodrug extracellularly in the tumor microenvironment. The activated cytotoxic moiety then poisons neighboring cancer cells within sites of metastases by entering the cells and inhibiting the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump, which is essential to the function of all normal and tumor cell types. The authors showed that treatment with the prodrug caused significant tumor regression in two mouse xenograft models of human prostate cancer and one model of human breast cancer with relatively little toxicity—less than that of the maximally tolerated dose of the widely used cancer drug docetaxel. Although the targeted prodrug concept is not new, the current approach has several features that make it superior to many previous ones. First, unlike most cytotoxic cancer drugs, TG is not cell cycle–dependent and thus can kill nondividing cancer cells. Furthermore, drug toxicity is expected to be low, because the PSMA substrate in the prodrug is cleaved primarily by prostate cancer cells and in the vicinity of tumor endothelial cells. In fact, the authors report that studies in cynomolgus monkeys showed minimal toxic effects except in the kidney, and even that renal toxicity was minimal to mild and reversible at the low drug dose. As with all cancer drugs, the new findings will require clinical validation in ongoing studies. However, this unusual therapeutic approach has the potential to be an effective and selective ouster of unwanted invaders that threaten their hosts. Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

Expanding the histologic spectrum of mucinous tubular and spindle cell carcinoma of the kidney

Mucinous tubular and spindle cell carcinomas (MTSCs) are polymorphic neoplasms characterized by small, elongated tubules lined by cuboidal cells and/or cords of spindled cells separated by pale mucinous stroma. Nonclassic morphologic variants and features of MTSC have not been well studied. We identified 17 previously unreported MTSCs from Surgical Pathology and consultative files of the authors and their respective institutions and studied their morphologic features. A total of 10/17 cases were considered “classic,” as described above, with 5/10 showing at least focal (20% to 50%) tubular predominance without apparent mucinous matrix. Alcian blue staining revealed abundant (>50%) mucin in all classic cases. Seven of 17 MTSCs were classified as “mucin-poor,” with little to no extracellular mucin appreciable by hematoxylin and eosin. Four of these cases showed equal tubular and spindled morphology, 2 cases showed spindle cell predominance (70%; 95%), and 1 case showed tubular predominance (90%). In 5/7 mucin-poor cases, staining for Alcian blue revealed scant (<10%) mucin in cellular areas with the other 2 cases having 30% mucin. Unusual histologic features identified in the 17 cases were: foamy macrophages (n=8), papillations/well formed papillae (n=6/n=1), focal clear cells in tubules (n=3), necrosis (n=3), oncocytic tubules (n=2; 40%, 5%), numerous small vacuoles (n=2), heterotopic bone (n=1), psammomatous calcification (n=1), and nodular growth with lymphocytic cuffing (n=1). An exceptional case contained a well-circumscribed, HMB45-positive angiomyolipoma within the MTSC. MTSCs may be “mucin-poor” and show a marked predominance of either of its principal morphologic components, which coupled with the presence of other unusual features such as clear cells, papillations, foamy macrophages, and necrosis, may mimic other forms of renal cell carcinoma. Pathologists must be aware of the spectrum of histologic findings within MTSCs to ensure their accurate diagnosis.

Individual and cumulative effect of prostate cancer risk-associated variants on clinicopathologic variables in 5,895 prostate cancer patients.

More than a dozen single nucleotide polymorphisms (SNPs) have been associated with prostate cancer (PCa) risk from genome‐wide association studies (GWAS). Their association with PCa aggressiveness and clinicopathologic variables is inconclusive.