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Here we argue in support of the human leukocyte antigen (HLA) supergene as a tumor suppressor. HLA is a recurring mutational target in a large and diverse group of malignancies. The tumor suppressor function of HLA is linked to an embryonic/stemness and drug resistance phenotype. A deeper understanding of the distinct roles of HLA, including immunosurveillance, stemness, and tumor suppressor functions, could illuminate the limited responses in cancer patients. Furthermore, it would provide guidelines for the design of new therapeutic strategies, including the potential of modulating HLA expression in the tumor stem cell compartment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.

Personalized or precision medicine as a diagnostic and therapeutic paradigm was introduced some 10-15 years ago, with the advent of biomarker discovery as a mechanism for identifying prognostic and predictive attributes associated with treatment indication and outcome. While the concept is not new, the successful development and implementation of novel 'companion diagnostics', especially in oncology, continues to represent a significant challenge and is currently at the forefront of smart trial design and therapeutic choice. The ability to determine patient selection for a specific therapy has broad implications including better chances for a positive outcome, limited exposure to potentially toxic drugs and improved health economics. Importantly, a significant step in this paradigm is the role of predictive pathology or the accurate assessment of morphology at the microscopic level. In breast cancer, this has been most useful where histologic attributes such as the classification of tubular and cribriform carcinoma dictates surgery while neoadjuvant studies suggest that patients with lobular carcinoma are not likely to benefit from chemotherapy. The next level of 'personalized pathology' at the tissue-cellular level is the use of 'protein biomarker panels' to classify the disease process and ultimately drive tumor characterization and treatment. The following review article will focus on the evolution of predictive pathology from a subjective, 'opinion-based' approach to a quantitative science. In addition, we will discuss the individual components of the precise pathology platform including advanced image analysis, biomarker quantitation with mathematical modeling and the integration with fluid-based (i.e. blood, urine) analytics as drivers of next generation precise patient phenotyping.

Unlabelled: We have recently recapitulated metastasis of human PTEN/TP53-mutant prostate cancer in the mouse using the RapidCaP system. Surprisingly, we found that this metastasis is driven by MYC, and not AKT, activation. Here, we show that cell-cell communication by IL6 drives the AKT-MYC switch through activation of the AKT-suppressing phosphatase PHLPP2, when PTEN and p53 are lost together, but not separately. IL6 then communicates a downstream program of STAT3-mediated MYC activation, which drives cell proliferation. Similarly, in tissues, peak proliferation in Pten/Trp53-mutant primary and metastatic prostate cancer does not correlate with activated AKT, but with STAT3/MYC activation instead. Mechanistically, MYC strongly activates the AKT phosphatase PHLPP2 in primary cells and prostate cancer metastasis. We show genetically that Phlpp2 is essential for dictating the proliferation of MYC-mediated AKT suppression. Collectively, our data reveal competition between two proto-oncogenes, MYC and AKT, which ensnarls the Phlpp2 gene to facilitate MYC-driven prostate cancer metastasis after loss of Pten and Trp53., Significance: Our data identify IL6 detection as a potential causal biomarker for MYC-driven metastasis after loss of PTEN and p53. Second, our finding that MYC then must supersede AKT to drive cell proliferation points to MYC inhibition as a critical part of PI3K pathway therapy in lethal prostate cancer., (©2015 American Association for Cancer Research.)

Acquired resistance has curtailed cancer survival since the dawn of the chemotherapy age more than half a century ago. Although the application of stem cell (SC) concepts to cancer captured the imagination of scientists for many years, only the last decade has yielded substantial evidence that cancer SCs (CSCs) contribute to chemotherapy resistance. Recent studies suggest that the functional and molecular properties of CSCs constitute therapeutic opportunities to improve the efficacy of chemotherapy. Here we review how these properties have stimulated combination strategies that suppress acquired resistance across a spectrum of malignancies. The clinical implementation of these strategies promises to rejuvenate the effort against an enduring challenge.

Tobacco smoke and its metabolites are carcinogens that increase tissue oxidative stress and induce target tissue inflammation. We hypothesized that genetic variation of inflammatory pathway genes plays a role in tobacco-related carcinogenesis and is modified by tobacco smoking. We evaluated the association of 12 single nucleotide polymorphisms of 8 inflammation-related genes with tobacco-related cancers (lung, oropharynx, larynx, esophagus, stomach, liver, bladder, and kidney) using 3 case-control studies from: Los Angeles (population-based; 611 lung and 553 upper aero-digestive tract cancer cases and 1,040 controls), Taixing, China (population-based; 218 esophagus, 206 stomach, 204 liver cancer cases, and 415 controls), and Memorial Sloan-Kettering Cancer Center (hospital-based; 227 bladder cancer cases and 211 controls). After adjusting for age, education, ethnicity, gender, and tobacco smoking, IL10 rs1800871 was inversely associated with oropharyngeal cancer (CT+TT vs. CC adjusted odds ratio [aOR]: 0.69, 95% confidence interval [CI]: 0.50-0.95), and was positively associated with lung cancer among never smokers (TT vs. CT+CC aOR: 2.5, 95% CI: 1.3-5.1) and inversely with oropharyngeal cancer among ever smokers (CT+TT vs. CC aOR: 0.63, 95% CI: 0.41-0.95). Among all pooled never smokers (588 cases and 816 controls), TNF rs1799964 was inversely associated with smoking-related cancer (CC vs. CT+TT aOR: 0.36, 95% CI: 0.17-0.77). Bayesian correction for multiple comparisons suggests that chance is unlikely to explain our findings (although epigenetic mechanisms may be in effect), which support our hypotheses, suggesting that IL10 rs1800871 is a susceptibility marker for oropharyngeal and lung cancers, and that TNF rs1799964 is associated with smoking-related cancers among never smokers.

Constituents of tobacco smoke can cause DNA double-strand breaks (DSBs), leading to tumorigenesis. The NBS1 gene product is a vital component in DSB detection and repair, thus genetic variations may influence cancer development. We examined the associations between NBS1 polymorphisms and haplotypes and newly incident smoking-related cancers in three case-control studies (Los Angeles: 611 lung and 601 upper aero-digestive tract (UADT) cancer cases and 1040 controls; Memorial Sloan-Kettering Cancer Center: 227 bladder cancer cases and 211 controls and Taixing, China: 218 esophagus, 206 stomach, 204 liver cancer cases and 415 controls). rs1061302 was associated with cancers of the lung [adjusted odds ratio (OR(adj)) = 1.6, 95% confidence interval (CI): 1.2, 2.4], larynx (OR(adj) = 0.56, 95% CI: 0.32, 0.97) and liver (OR(adj) = 1.7, 95% CI: 1.0, 2.9). Additionally, positive associations were found for rs709816 with bladder cancer (OR(adj) = 4.2, 95% CI: 1.4, 12) and rs1063054 with lung cancer (OR(adj) = 1.6, 95% CI: 1.0, 2.3). Some associations in lung and stomach cancers varied with smoking status. CAC haplotype was positively associated with smoking-related cancers: lung (OR(adj) = 1.7, 95% CI: 1.1, 2.9) and UADT (OR(adj) = 2.0, 95% CI: 1.1, 3.7), specifically, oropharynx (OR(adj) = 2.1, 95% CI: 1.0, 4.2) and larynx (OR(adj) = 4.8, 95% CI: 1.7, 14). Bayesian false-discovery probabilities were calculated to assess Type I error. It appears that NBS1 polymorphisms and haplotypes may be associated with smoking-related cancers and that these associations may differ by smoking status. Our findings also suggest that single-nucleotide polymorphisms located in the binding region of the MRE-RAD50-NBS1 complex or microRNA targeted pathways may influence tumor development. These hypotheses should be further examined in functional studies.

Recent genome-wide association studies identified key single nucleotide polymorphisms (SNPs) in the 8q24 region to be associated with prostate cancer. 8q24 SNPs have also been associated with colorectal cancer, suggesting that this region may not be specifically associated to just prostate cancer. To date, the association between these polymorphisms and tobacco smoking-related cancer sites remains unknown. Using epidemiologic data and biological samples previously collected in three case-control studies from U.S. and Chinese populations, we selected and genotyped one SNP from each of the three previously determined "regions" within the 8q24 loci, rs1447295 (region 1), rs16901979 (region 2), and rs6983267 (region 3), and examined their association with cancers of the lung, oropharynx, nasopharynx, larynx, esophagus, stomach, liver, bladder, and kidney. We observed noteworthy associations between rs6983267 and upper aerodigestive tract cancers [adjusted odds ratio (ORadj), 1.69; 95% confidence interval (95% CI), 1.28-2.24], particularly in oropharynx (ORadj, 1.80; 95% CI, 1.30-2.49) and larynx (ORadj, 2.04; 95% CI, 1.12-3.72). We also observed a suggestive association between rs6983267 and liver cancer (ORadj, 1.51; 95% CI, 0.99-2.31). When we stratified our analysis by smoking status, rs6983267 was positively associated with lung cancer among ever-smokers (ORadj, 1.45; 95% CI, 1.05-2.00) and inversely associated with bladder cancer among ever-smokers (ORadj, 0.35; 95% CI, 0.14-0.83). Associations were observed between rs16901979 and upper aerodigestive tract cancer among never-smokers and between rs1447295 and liver cancer among ever-smokers. Our results suggest variants of the 8q24 chromosome may play an important role in smoking-related cancer development. Functional and large epidemiologic studies should be conducted to further investigate the association of 8q24 SNPs with smoking-related cancers.

Cancer is a disease of aging. The accumulation of mutations in individual cells over a lifetime is thought to be the reason. In this work, we explored an additional hypothesis: could p53 function decline with age, which would contribute to an enhanced mutation frequency and tumorigenesis in the aging process? The efficiency of the p53 response to gamma-irradiation was found to decline significantly in various tissues of aging mice from several inbred strains, including lower p53 transcriptional activity and p53-dependent apoptosis. This decline resulted from a decreased stabilization of the p53 protein after stress. The function of the Ataxia-telangiectasia mutated (ATM) kinase declined significantly with age, which may then be responsible for the decline of the p53 response to radiation. Declining p53 responses to other stresses were also observed in the cultured splenocytes from aging mice. Interestingly, the time of onset of this decreased p53 response correlated with the life span of mice; mice that live longer delay their onset of decreased p53 activity with time. These results suggest an enhanced fixation of mutations in older individuals because of the declining fidelity of p53-mediated apoptosis or senescence in response to stress, and they suggest a plausible explanation for the correlation between tumorigenesis and the aging process.

Constitutive activation of extracellular signal-regulated kinases (Erk1/2) is frequently implicated in human cancers. Recently, aberrantly activated Erk was also found in brain lesions associated with tuberous sclerosis (TSC). We reported previously that Erk might contribute to tumorigenesis by phosphorylating TSC2 at specific residues, particularly S664. In our present study, 25 TSC-related cortical tubers or subependymal giant cell astrocytomas, as well as tissue microarrays of six types of human cancers, were analyzed for the expression of phospho-Erk (pErk) 1/2, S664-phospho-TSC2 (pTSC2), and phospho-S6 (pS6) by immunohistochemistry. We found that Erk-mediated TSC2 phosphorylation occurred at a high incidence and positively correlated with mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) activation in TSC-associated brain lesions as well as in various cancers. Interestingly, in certain types of cancers (e.g., breast carcinoma and colon carcinoma), S664-pTSC2 seemed to be a more sensitive marker than pErk. Furthermore, most of the pTSC2-positive samples ( approximately 75%) were positive for pS6, but only 40% to 55% of the pS6-positive tumors exhibited TSC2 phosphorylation. Our results show that S664 TSC2 phosphorylation is a marker for Erk-mediated (as opposed to Akt-mediated) mTOR activation in TSC and human cancer. On the basis of these findings, TSC2 phosphorylation at S664 can be used to identify patients that may benefit from antitumor therapy with MAPK and mTOR inhibitors. Importantly, our results indicate that Erk-mediated phosphorylation and inactivation of TSC2 can be critical in development of hamartomatous lesions in TSC and cancer pathogenesis.

Recent studies have established distinctive serum polypeptide patterns through mass spectrometry (MS) that reportedly correlate with clinically relevant outcomes. Wider acceptance of these signatures as valid biomarkers for disease may follow sequence characterization of the components and elucidation of the mechanisms by which they are generated. Using a highly optimized peptide extraction and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS-based approach, we now show that a limited subset of serum peptides (a signature) provides accurate class discrimination between patients with 3 types of solid tumors and controls without cancer. Targeted sequence identification of 61 signature peptides revealed that they fall into several tight clusters and that most are generated by exopeptidase activities that confer cancer type-specific differences superimposed on the proteolytic events of the ex vivo coagulation and complement degradation pathways. This small but robust set of marker peptides then enabled highly accurate class prediction for an external validation set of prostate cancer samples. In sum, this study provides a direct link between peptide marker profiles of disease and differential protease activity, and the patterns we describe may have clinical utility as surrogate markers for detection and classification of cancer. Our findings also have important implications for future peptide biomarker discovery efforts.

Purpose: Flavopiridol potently enhances the effect of irinotecan with cures in colorectal cancer xenografts, and is associated with modulation of several molecular targets, including p21, Differentiation-related gene 1 (Drg1), and p53. We initiated a phase I trial of the sequential combination of irinotecan followed by flavopiridol to determine the maximal tolerated dose of this combination therapy., Patients and Methods: Forty-five patients with advanced solid tumors were enrolled. Irinotecan was administered first (100 or 125 mg/m(2)) followed 7 hours later by escalating flavopiridol (10-70 mg/m(2)) given weekly over 1 hour for 4 of 6 weeks. At the maximal tolerated dose, the pharmacokinetic analysis was expanded and pre- and posttreatment tumor biopsies were done., Results: At irinotecan 100 mg/m(2), dose-limiting diarrhea and myelosuppression were observed with flavopiridol 70 mg/m(2). At irinotecan 125 mg/m(2), we observed dose-limiting hyperbilirubinemia, fatigue, and myelosuppression at flavopiridol 60 mg/m(2). Peak flavopiridol concentrations of >/=2 mumol/L were achieved above flavopiridol 50 mg/m(2). No significant pharmacokinetic interactions with irinotecan were noted. Baseline serum bilirubin significantly predicted cycle 1 dose-limiting toxicity and neutropenia. We observed partial responses in three patients and prolonged stable disease (i.e., >6 months) in 36% of patients including adrenocortical cancer and hepatocellular cancer. Patients with wild-type p53 and either no change or low posttreatment biopsy p21 and a decrease in Drg1 expression showed stable or responsive disease to the combination therapy., Conclusions: The recommended phase II dose with irinotecan 100 mg/m(2) is flavopiridol 60 mg/m(2) and with irinotecan 125 mg/m(2) is flavopiridol 50 mg/m(2). Toxicity can be predicted by baseline bilirubin. Clinical activity is encouraging and may correlate to changes in p21 and Drg1 levels in patients with wild type p53 tumors following therapy.

G3139 is a BCL-2 antisense oligonucleotide whose antitumor effects in preclinical models are enhanced when combined with taxane-based chemotherapy. This trial determined the safety and biologic activity of G3139 given with paclitaxel and docetaxel for the treatment of progressive solid tumors. Three cohorts of patients received weekly paclitaxel 100 mg/m2 on days 1, 8, and 15 concurrently with a 21-day continuous infusion of G3139 at 4.1, 5.3, and 6.9 mg/kg/d, depending on the cohort. Two subsequent cohorts received docetaxel (75 mg/m2) on day 5 of a 5-day infusion of G3139 at 5 or 7 mg/kg/d. Bcl-2 protein levels in peripheral blood mononuclear cells (PBMCs) were assayed on an exploratory basis. Fifteen patients were treated. Eight received a total of 14 cycles of G3139 and paclitaxel; seven received a total of 22 cycles of G3139 and docetaxel. Eight patients required dose modifications for either grade 4 neutropenia (6 patients) or grade 1-2 reversible transaminitis (2 patients). No radiographic responses were seen, although two of the six taxane-naive prostate cancer patients exhibited a prostate-specific antigen decline greater than 50%. Bcl-2 protein levels in PBMCs declined with treatment as assessed by immunohistochemistry. The authors conclude that G3139, whether given as a 5- or 21-day infusion, is well tolerated with taxane chemotherapy and is biologically active by immunohistochemistry at doses up to and including 7 mg/kg/d, using weekly paclitaxel (100 mg/m2) or docetaxel every 3 weeks (75 mg/m2). These data support the dose selection of ongoing phase 2 studies of G3139 at 7 mg/kg/d and docetaxel 75 mg/m2.

The cellular stress response pathway regulated by the p53 tumor suppressor is critical to the maintenance of genomic integrity and to the prevention of oncogenic transformation. Intracellular levels of p53 are tightly regulated by an autoregulatory feedback loop comprised of p53 and MDM2. It might be predicted that disruption of this loop, either through p53 mutation or overexpression of MDM2, would be a negative prognostic marker for cancer development, likelihood of relapse, or response to therapy. In fact, although MDM2 overexpression is common in cancer, it can be both a positive and a negative predictor of outcome in different tumors, and its significance as a biomarker remains controversial. Data from a number of different tumor types are reviewed for the predictive significance of MDM2 expression, along with evidence for different mechanisms of MDM2 overexpression in these different tumors. In light of the biological complexities underlying the p53-MDM2 loop, it is, perhaps, not surprising that no simple paradigm exists that is generally applicable. Much work remains to be done to elucidate the basic mechanisms underlying the physical interactions between the two proteins, the role of protein modifications in altering those interactions, and also the genetic and transcriptional deregulations by which protein levels are altered in human cancers. Only in this way will truly biologically relevant predictive factors emerge.

Purpose: To characterize the expression profile of p73 in human normal tissues by immunohistochemistry (IHC) and to analyze the correlation between p73 expression and bladder cancer progression., Experimental Design: CJDp73 was characterized for p73alpha detection in Western blot and IHC through its application to isoform-transfected 293 cells. Normal tissues were analyzed by IHC with the CJDp73 antiserum. Transitional cell carcinoma (TCC)-derived cell lines were subjected to reverse transcription-PCR and Western blot. TCC tissue microarrays were analyzed for p73alpha expression by IHC, and the results were statistically analyzed., Results: p73 immunostaining was nuclear and restricted to epithelial cells of certain organs such as squamous epithelium of the epidermis and transitional epithelium of the bladder. The expression was also observed in certain specialized glandular epithelia such as acinar cells of breast and parotid gland. Four of seven TCC-derived cell lines had low to undetectable p73alpha protein levels. We found undetectable or low p73alpha expression in 104 of 154 (68%) TCC cases, this phenotype being more frequently observed in invasive tumors when compared with superficial lesions. This association was statistically significant (P < 0.0001). We also observed a significant association between p53, p63, and p73alpha alterations with bladder cancer progression (P < 0.0001)., Conclusions: p73alpha plays a tumor suppressor role in bladder cancer, and its inactivation occurs through an epigenetic mechanism, most probably involving protein degradation.

Mutations in DKC1 cause dyskeratosis congenita (DC), a disease characterized by premature aging and increased tumor susceptibility. The DKC1 protein binds to the box H + ACA small nucleolar RNAs and the RNA component of telomerase. Here we show that hypomorphic Dkc1 mutant (Dkc1m) mice recapitulate in the first and second generations (G1 and G2) the clinical features of DC. Dkc1m cells from G1 and G2 mice were impaired in ribosomal RNA pseudouridylation before the onset of disease. Reductions of telomere length in Dkc1m mice became evident only in later generations. These results suggest that deregulated ribosome function is important in the initiation of DC, whereas telomere shortening may modify and/or exacerbate DC.

Purpose: To evaluate the safety and pharmacokinetics of BCL-2 antisense oligonucleotide (G3139) administered by prolonged i.v. infusion in patients with advanced cancer., Experimental Design: A total of 35 patients was treated in cohorts of 3-6 with 0.6-6.9 mg/kg/day of BCL-2 antisense oligonucleotide as a continuous infusion for 14 or 21 days. Plasma levels of intact antisense oligonucleotide were measured in all patients., Results: G3139 was generally well tolerated. At the highest dose level examined in this study (6.9 mg/kg/day), fatigue and transient reversible elevations of serum transaminases (grades 2-3) became apparent after >or=7 days of treatment. Both reactions were believed to be drug related. Pharmacokinetic analyses showed that steady-state plasma concentrations of G3139 were reached approximately 10 h after starting the infusion and increased linearly across the range of doses administered

The implementations of high-throughput genetic technologies, such as oligonucleotide microarrays, generate myriad points of data. The identified potential candidate genes need to be further characterized and selected using a large number of well-characterized tumors and stringent criteria. Tissue microarrays allow for such high-throughput expression profiling of tumor samples, providing, in addition, information at the microanatomical level. Different techniques could be applied for identification of specific phenotypic (immunohistochemistry and in situ hybridization) or genotypic (fluorescence in situ hybridization) alterations, holding strong potential for translational research. Tissue microarrays consisting of 0.6-mm biopsies of paraffin-embedded tissues are well validated and have been used for various clinicopathological studies. This review discusses the technical considerations for construction of such arrays from paraffin-embedded tissues and cell lines and outlines their potential for clinical research applications. The use of paraffin-embedded tissues has some limitations with regard to analysis of RNA or certain proteins. To overcome such limitations, we have developed a cryoarray strategy allowing for the processing of multiple frozen tissue specimens and/or cell lines on a single tissue block. These approaches offer the opportunity to conduct pilot and validation studies of potential targets using clinical samples linked to clinicopathological databases.

Solid tumor diagnosis is now entering an era in which molecular genetics plays an important role. Clinical trials have shown different responses to various therapies that correlate with molecular alterations. Biological determinants related to treatment response markers aimed at individualized therapies are being defined and implemented. Patients are now being treated based on the profile of molecular genetic alterations in individual tumors. Protocols based on molecular markers will increase the chances for cure by opting for the right management approach. They also will improve, in most situations, the quality of life of patients with cancer, for example by facilitating organ preservation strategies. The molecular characterization therefore has an important prognostic and practical role in diagnosis.

The relative expression of mucin antigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7 and glycoprotein antigens KSA, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2/neu, and human chorionic gonadotropin-beta on different cancers and normal tissues is difficult to determine from available reports. We have compared the distribution of these antigens by immunohistology on a broad range of malignant and normal tissues. MUC1 expression was most intense in cancers of breast, lung, ovarian, and endometrial origin; MUC2 was most intense in cancers of colon and prostate origin; and MUC5AC was most intense in cancers of breast and gastric origin. MUC4 was intensely expressed in 50% of cancers of colon and pancreas origin, and MUC3, MUC5B, and MUC7 were expressed in a variety of epithelial cancers, but not so intensely. KSA was intensely and uniformly expressed on all epithelial cancers; carcinoembryonic antigen was expressed in most cancers of breast, lung, colon, pancreas, and gastric origin; and PSMA was expressed only in cancers of prostate origin. Human chorionic gonadotropin-beta was expressed on the majority of sarcomas and cancers of breast, lung, and pancreas origin, although intense staining was not seen. Staining on normal tissues was restricted to one or many normal epithelial tissues ranging from MUC3, MUC4, and PSMA, which were expressed only on epithelia of pancreas, stomach, and prostate origin, respectively, to MUC1 and KSA, which were expressed on most normal epithelia. Expression was restricted to the secretory borders of these epithelia while stroma and other normal tissues were completely negative. These results plus the results of the two previous papers (S. Zhang et al, Int. J. Cancer, 73: 42-49, 1997; S. Zhang et al., Int. J. Cancer, 73: 50-56, 1997) in this series provide the basis for selection of multiple cell surface antigens as targets for antibody-mediated attack against these cancers.

Mxi1 belongs to the Mad (Mxi1) family of proteins, which function as potent antagonists of Myc oncoproteins. This antagonism relates partly to their ability to compete with Myc for the protein Max and for consensus DNA binding sites and to recruit transcriptional co-repressors. Mad(Mxi1) proteins have been suggested to be essential in cellular growth control and/or in the induction and maintenance of the differentiated state. Consistent with these roles, mxi1 may be the tumour-suppressor gene that resides at region 24-26 of the long arm of chromosome 10. This region is a cancer hotspot, and mutations here may be involved in several cancers, including prostate adenocarcinoma. Here we show that mice lacking Mxi1 exhibit progressive, multisystem abnormalities. These mice also show increased susceptibility to tumorigenesis either following carcinogen treatment or when also deficient in Ink4a. This cancer-prone phenotype may correlate with the enhanced ability of several mxi1-deficient cell types, including prostatic epithelium, to proliferate. Our results show that Mxi1 is involved in the homeostasis of differentiated organ systems, acts as a tumour suppressor in vivo, and engages the Myc network in a functionally relevant manner.

Blood group-related antigens have been attractive targets for immunotherapy of cancer since their initial identification as cancer-related antigens. However, available information on the relative expression of most of these antigens on human malignant and normal tissues has been insufficient for selecting optimal antigens and tumors for immune attack. In this study, the distribution of the blood group-related antigens TF, Tn, sTn, Le(a), sialyl Le(a), Le(b), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on 13 types of cancer and 16 normal tissues was compared. Our results show that sTn is strongly expressed on cancers of breast, colon, stomach, ovary, prostate and uterus; Tn on prostate cancer; TF on cancers of breast, colon, ovary, prostate and uterus; Le(y) on the cancers of colon, lung, pancreas and ovary; Le(a) and Le(x) on gastric cancer; and sialyl Le(a) and sialyl Le(x) on colon cancer. The complete absence of these antigens on cancers of neuroectodermal or mesodermal origin including melanoma, sarcoma, neuroblastoma and B cell lymphoma is as striking as their widespread presence on tumors of epithelial origin. Normal tissues were also tested. Tn and Le(b) were only detected on gastric and ovarian epithelia; sTn on Leydig cells of testis in addition to gastric and ovarian epithelia; Le(x) and sialyl Le(x) on polymorphonuclear leukocytes; and TF, Le(a), sialyl Le(a), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on epithelia from a variety of tissues.

Understanding the distribution of tumor-associated antigens on cancers and normal tissues is essential for selection of targets for cancer immunotherapy. Seven carbohydrate antigens, potential targets for immunotherapy, were studied using a panel of well-characterized MAbs by immunohistochemistry on cryostat-cut tissue sections of 13 types of cancers and 18 normal tissues. GD2 and GD3 were present on most cancers of neuroectodermal origin and GD2 was also present on B cell lymphomas. 9-O-acetyl-GD3 was detected only on melanoma while fucosyl GM1 was detected only on small cell lung cancers (SCLC). Surprisingly, GM2 was strongly expressed on all tested tumors, including cancers of neuroectodermal origin and cancers of epithelial origin. Polysialic acid was primarily expressed on SCLC and neuroblastomas. Globo H was present on most cancers of epithelial origin. These antigens were also identified in normal tissues. Fucosyl GM1 was not expressed significantly on any of the normal tissues analyzed. GD3, GD2, GM2 and polysialic acid were detected in normal brain to varying degrees. GM2 and Globo H were expressed on the luminal surface of epithelia of a variety of organs. The unexpected expression of GM2 on a broad range of cancers and normal epithelial tissues was confirmed by loss after methanol fixation and by immune thin layer chromatography.

In this chapter, we review major methodological and practical issues associated with the use of tumour markers. At this stage of development, studies with a combination of tumour, susceptibility and exposure markers are needed to illustrate the link between exposure and biological response and to assess the interactive effects of tumour susceptibility markers in this process. Several practical issues related to the application of tumour markers are discussed, including banking of tumour tissue, setting a laboratory strategy and performing etiological heterogeneity analysis.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

Neoplastic diseases are characterized by uncoordinated cell growth. Cellular proliferation follows an orderly progression through the cell cycle, which is governed by protein complexes composed of cyclins and cyclin-dependent kinases. These complexes exert their regulatory function by phosphorylation of key proteins involved in cell cycle transitions, such as the product encoded by the retinoblastoma gene (pRB). Mutations and overexpression of cyclins and cyclin-dependent kinases, mainly cyclin D1 and Cdk4, have been reported and proposed to be oncogenic events. More recently, a new family of negative regulators functioning as Cdk-inhibitory molecules has been identified. Because of their recessive nature in cell cycle control and the fact that some of them are mutated in human tumors, it has been suggested that they may also function as tumor suppressor genes. It appears that the molecular networking of these proteins and complexes impact on two fundamental cell cycle regulators: p53 and pRB. Cross-talk pathways between these two nuclear proteins are being delineated, implying potential links between p53 and pRB in cell cycle control, apoptosis, and tumor progression. In addition, the high rate and mutation pattern of TP53 and RB in primary tumors have rendered them prototype tumor suppressor genes. Furthermore, detection of TP53 and RB mutations and altered expression of their encoded products appear to be of clinical significance, often correlating with prognosis, when identified in specific cancers. Based on these findings, new strategies are being developed in the emerging field of gene replacement-therapy.

Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronal protein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system. In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also express the antigen.

The expression of monomorphic determinants of the histocompatibility leukocyte antigens (HLA) class I antigens by human malignant tumor cells was studied in tissue specimens of 70 primary tumor lesions obtained from patients with carcinoma of the breast (41 patients), colon (8 patients), urinary bladder (8 patients), and kidney (13 patients), and in samples of either synchronous or metachronous lymph node, lung, or liver metastases available in 44 of the patients. The frequencies of HLA class I expressor and nonexpressor tumor cells were determined by immunohistochemical staining of histological sections of fresh frozen tissue samples with the W6/32 monoclonal antibody. The tumor cell populations in the majority of the primary lesions consisted predominantly of HLA-immunoreactive cells (observed in 38 of 70 patients; 54%), especially in those patients who did not have clinical evidence of metastatic disease (8 of 11 patients; 73%). Various degrees of loss of reactivity were observed in other primary lesions, although in only 8 (12%) tumors (7 of which were obtained from patients with metastatic disease), the neoplastic cells were nearly exclusively HLA-nonreactive. In contrast, the majority of metastatic lesions consisted of either predominantly HLA-negative cells (33 of 44 specimens; 75%) or mixed populations (10 of 44 specimens; 23%), whereas only one metastatic lesion manifested HLA class I antigen staining in more than 70% of its tumor cells (P = 0.0005). Intravascular clusters of tumor cells consisted predominantly of HLA class I nonexpressors. The observed patterns of distribution of HLA expressors and nonexpressor tumor cells are compatible with the notion that HLA-negative cells in human carcinomas manifest a selective advantage with regard to metastatic progression and growth. The suppressed expression of major histocompatibility complex class I antigens on metastatic cells may lead to failure of presentation of cell surface tumor specific epitopes to host cytotoxic T-lymphocytes. Such a process would enable tumor cells to evade host immune responses and would promote and enhance cell dissemination and metastatic growth.