GARP inhibits allergic airway inflammation in a humanized mouse model.

BACKGROUND: Regulatory T cells (Treg) represent a promising target for novel treatment strategies in patients with inflammatory/allergic diseases. A soluble derivate of the Treg surface molecule glycoprotein A repetitions predominant (sGARP) has strong anti-inflammatory and regulatory effects on human cells in vitro as well as in vivo through de novo induction of peripheral Treg. The aim of this study was to investigate the immunomodulatory function of sGARP and its possible role as a new therapeutic option in allergic diseases using a humanized mouse model. METHODS: To analyze the therapeutic effects of sGARP, adult NOD/Scidγc(-/-) (NSG) mice received peripheral blood mononuclear cells (PBMC) derived from allergic patients with sensitization against birch allergen. Subsequently, allergic inflammation was induced in the presence of Treg alone or in combination with sGARP. RESULTS: In comparison with mice that received Treg alone, additional treatment with sGARP reduced airway hyperresponsiveness (AHR), influx of neutrophils and macrophages into the bronchoalveolar lavage (BAL), and human CD45(+) cells in the lungs. Furthermore, the numbers of mucus-producing goblet cells and inflammatory cell infiltrates were reduced. To elucidate whether the mechanism of action of sGARP involves the TGF-ß receptor pathway, mice additionally received anti-TGF-ß receptor II (TGF-ßRII) antibodies. Blocking the signaling of TGF-ß through TGF-ßRII abrogated the anti-inflammatory effects of sGARP, confirming its essential role in inhibiting the allergic inflammation. CONCLUSION: Induction of peripheral tolerance via sGARP is a promising potential approach to treat allergic airway diseases.

Expression of microRNAs in basal cell carcinoma.

BACKGROUND: Perturbations in the expression profiles of microRNAs (miRNAs) have been reported for a variety of different cancers. Differentially expressed miRNAs have not been systematically evaluated in basal cell carcinoma (BCC) of the skin. OBJECTIVES: To initiate a microarray-based miRNA profiling study to identify specific miRNA candidates that are differentially expressed in BCC. METHODS: Patients with BCC (n = 7) were included in this study. Punch biopsies were harvested from the tumour centre (lesional, n = 7) and from adjacent nonlesional skin (intraindividual control, n = 7). Microarray-based miRNA expression profiles were obtained on an Agilent platform using miRBase 16 screening for 1205 Homo sapiens (hsa)-miRNA candidates. To validate the microarray data, the expression of seven dysregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction. RESULTS: We identified 16 significantly upregulated (hsa-miR-17, hsa-miR-18a, hsa-miR-18b, hsa-miR-19b, hsa-miR-19b-1*, hsa-miR-93, hsa-miR-106b, hsa-miR-125a-5p, hsa-miR-130a, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-455-3p, hsa-miR-455-5p and hsa-miR-542-5p) and 10 significantly downregulated (hsa-miR-29c, hsa-miR-29c*, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-145, hsa-miR-378, hsa-miR-572, hsa-miR-638, hsa-miR-2861 and hsa-miR-3196) miRNAs in BCC compared with nonlesional skin. Data mining revealed connections to many tumour-promoting pathways, such as the Hedgehog and the mitogen-activated protein kinase/extracellular signal-regulated kinase signalling cascades. CONCLUSIONS: This study identified several miRNA candidates that may play a role in the molecular pathogenesis of BCC.

Clinical and genetic analysis of 18 pancreatic carcinoma/melanoma-prone families.

Families with both melanoma and pancreatic cancer are extremely rare and some are affected with the autosomal dominant inherited familial atypical multiple mole melanoma-pancreatic cancer (FAMMM-PC) syndrome. The phenotypic and genotypic expressions of such pancreatic cancer-melanoma prone families are not well defined. The National Case Collection of Familial Pancreatic Cancer of the Deutsche Krebshilfe includes 110 pancreatic cancer families, 18 of which (16%) show an association of pancreatic cancer and melanoma. These 18 families were analysed regarding their phenotype and the prevalence of germline mutations in the candidate genes CDKN2A, BRCA2, CHEK2, NOD2, ARL11 and Palladin (PALLD). There were two types of families: five families with the FAMMM-PC phenotype and 13 PC/melanoma families without the multiple mole phenotypes (PCMS). The prevalences of PC and melanoma in the two types of families were similar. The prevalence of other tumour types, especially breast carcinoma, was higher (11%) in PCMS- than in FAMMM-PC families (2.4%, p = 0.02). CDKN2A mutations were identified in 2 of 18 (11%) PCMS families. A cosegregating BRCA2 mutation was detected in one PCMS family without breast cancer. None of the reported germline mutations in the NOD2, Palladin, ARL11 or CHEK2 genes were detected in either type of family. In conclusion, families with an accumulation of PC and melanoma show a large variety of phenotypic expression, which is not always consistent with the FAMMM-PC phenotype. More PC/melanoma-prone families need to be analysed to clarify whether such families represent variations of the FAMMM-PC syndrome or two distinct hereditary cancer syndromes.

Clonal T-cell populations are frequent in the skin and blood of patients with systemic sclerosis.

BACKGROUND: Pilot studies were suggestive for a role of clonal T cells in the pathogenesis of systemic sclerosis (SSc). OBJECTIVES: To investigate the presence of clonal T cells in both peripheral blood and skin of a large collection of patients with SSc. METHODS: Polymerase chain reaction and high-resolution capillary electrophoresis for detecting T-cell clonality were performed in a series of 126 patients with SSc. RESULTS: Seventy-seven (61%) of 126 patients had clonal T cells in their peripheral blood. In contrast, a clonal T-cell population was present in only four of 29 (14%) age-matched healthy controls (P = 0.03). Older patients were more likely to have clonal T cells than younger patients with SSc (P < 0.0001). Clonal T cells were more commonly detected in the blood of patients with limited cutaneous SSc (48 of 65 patients, 74%) than in those with diffuse cutaneous disease (29 of 61, 48%; P = 0.0002). Lesional skin specimens from 20 of 44 patients (45%) had detectable clonal T-cell populations. There was no correlation between the presence of circulating clonal T cells and lesional clonal T cells, sex, disease duration, extent of skin sclerosis, digital ulcers, organ involvement (e.g. interstitial lung disease, kidney disease, oesophagus involvement), treatment of SSc, or autoantibody profile. CONCLUSIONS: Many patients with SSc have expanded clonal T cells in their peripheral blood and skin. These clonal T cells could play a critical role in the pathogenesis of SSc, especially in limited cutaneous disease.

Predictive and prognostic value of microsatellite instability in patients with advanced colorectal cancer treated with a fluoropyrimidine and oxaliplatin containing first-line chemotherapy. A report of the AIO Colorectal Study Group.

BACKGROUND AND AIMS: Microsatellite instability (MSI) is a potential indicator of prognosis in patients with colorectal cancer (CRC). To date, there are a limited number of studies which investigated its role in advanced CRC. Our study investigated the value of high degree of MSI (MSI-H) in patients treated with 5-FU/oxaliplatin-based chemotherapy which has been done by only one further study recently. PATIENTS AND METHODS: In this study, we investigated tumour tissues from 108 patients with metastatic CRC who were treated in a prospective, randomised trial comparing two oxaliplatin and 5-FU-based therapy regimens (FUFOX vs. CAPOX) involving a total of 474 patients. We determined the incidence and prognostic value of a high degree of microsatellite instability. The specimens were analysed by PCR corresponding to the National Institute of Health reference panel. In addition, immunostaining of the mismatch repair proteins MLH1, MSH2 and MSH6 was performed. RESULTS AND FINDINGS: The incidence of MSI-H was 4%. MSI-H was correlated with a lower rate of disease control compared to non-MSI-H patients (p = 0.02). However, there was no correlation between MSI-H and progression-free survival or overall survival. INTERPRETATION AND CONCLUSION: MSI-H incidence in metastatic CRC was low. Our data suggest that MSI-H may be correlated with a poorer response to a 5-FU/oxaliplatin treatment. This finding needs confirmation in a larger cohort.

Maximal force is unaffected by emphysema-induced atrophy in extensor digitorium longus.

Patients with chronic obstructive pulmonary disease (COPD) demonstrate a limited exercise capacity. It is unknown whether muscle fiber atrophy and subsequent decrease in force production contributes to this functional limitation. Therefore, the purpose of this investigation was to determine whether emphysema-induced muscle fiber atrophy leads to a reduction in locomotory muscle force production. Maximal muscle force production and fiber cross-sectional area were measured in the almost exclusively fast-twitch extensor digitorium longus muscles at 4 and 8 months following saline (control, n=8/time period) or elastase (emphysema, n=15/time period) instillation in the lungs of hamsters. Excised lung volume increased 145 and 161% with emphysema at 4 and 8 months, respectively (both P<0.01). Muscle mass, maximal force, and fiber cross-section were unaltered at 4 months. However, absolute mass (-15%) and fiber cross-sectional area (-18%) were reduced at 8 months (both P<0.01). Surprisingly, maximal force was preserved in emphysema animals. These data demonstrate that maximal muscle force may be preserved in the face of emphysema-induced fiber atrophy.

MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known for its very poor overall prognosis. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. We used 377 feature microRNA (miRNA) arrays to investigate miRNA expression in normal pancreas, chronic pancreatitis, and PDAC tissues as well as PDAC-derived cell lines. A pancreatic miRNome was established comparing the data from normal pancreas with a reference set of 33 human tissues. The expression of miR-216 and -217 and lack of expression of miR-133a were identified as characteristic of pancreas tissue. Unsupervised clustering showed that the three pancreatic tissues types can be classified according to their respective miRNA expression profiles. We identified 26 miRNAs most prominently misregulated in PDAC and a relative quantitative reverse transcriptase-polymerase chain reaction index using only miR-217 and -196a was found to discriminate normal pancreas, chronic pancreatitis and cancerous tissues, establishing a potential utility for miRNAs in diagnostic procedures. Lastly, comparing differentially expressed genes from PDAC with predicted miRNA target genes for the top 26 miRNAs, we identified potential novel links between aberrant miRNA expression and known target genes relevant to PDAC biology. Our data provides novel insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development and offers new candidate targets to be exploited both for diagnostic and therapeutic strategies.

Downhill treadmill running trains the rat spinotrapezius muscle.

There are currently no models of exercise that recruit and train muscles, such as the rat spinotrapezius, that are suitable for transmission intravital microscopic investigation of the microcirculation. Recent experimental evidence supports the concept that running downhill on a motorized treadmill recruits the spinotrapezius muscle of the rat. Based on these results, we tested the hypothesis that 6 wk of downhill running (-14 degrees grade) for 1 h/day, 5 days/wk, at a speed of up to 35 m/min, would 1) increase whole body peak oxygen uptake (Vo(2 peak)), 2) increase spinotrapezius citrate synthase activity, and 3) reduce the fatigability of the spinotrapezius during electrically induced 1-Hz submaximal tetanic contractions. Trained rats (n = 6) elicited a 24% higher Vo(2 peak) (in ml.min(-1).kg(-1): sedentary 58.5 +/- 2.0, trained 72.7 +/- 2.0; P < 0.001) and a 41% greater spinotrapezius citrate synthase activity (in mumol.min(-1).g(-1): sedentary 14.1 +/- 0.7, trained 19.9 +/- 0.9; P < 0.001) compared with sedentary controls (n = 6). In addition, at the end of 15 min of electrical stimulation, trained rats sustained a greater percentage of the initial tension than their sedentary counterparts (control 34.3 +/- 3.1%, trained 59.0 +/- 7.2%; P < 0.05). These results demonstrate that downhill running is successful in promoting training adaptations in the spinotrapezius muscle, including increased oxidative capacity and resistance to fatigue. Since the spinotrapezius muscle is commonly used in studies using intravital microscopy to examine microcirculatory function at rest and during contractions, our results suggest that downhill running is an effective training paradigm that can be used to investigate the mechanisms for improved microcirculatory function following exercise training in health and disease.

Muscle microvascular oxygenation in chronic heart failure: role of nitric oxide availability.

AIM: To test the hypothesis that diminished vascular nitric oxide availability might explain the inability of individuals with chronic heart failure (CHF) to maintain the microvascular PO(2)'s (PO(2mv) proportional, variant O(2) delivery-to-uptake ratio) seen in healthy animals. METHODS: We superfused sodium nitroprusside (SNP; 300 microm), Krebs-Henseleit (control, CON) and L-nitro arginine methyl ester (L-NAME; 1.5 mM) onto the spinotrapezius muscle and measured PO(2mv) by phosphorescence quenching in female Sprague-Dawley rats (n = 26) at rest and during twitch contractions (1 Hz). Seven rats served as controls (Sham) while CHF was induced by myocardial infarction. CHF rats were grouped as moderate (MOD; n = 15) and severe CHF (SEV; n = 4) according to morphological data and baseline PO(2mv). RESULTS: In contrast to Sham and MOD, L-NAME did not affect the PO(2mv) response (dynamics and steady-state) of SEV when compared with CON. SNP restored the PO(2mv) profile of SEV to that seen in Sham animals during CON. Specifically, the effect of L-NAME expressed as Delta(L-NAME - CON) were: Baseline PO(2mv) [in mmHg, DeltaSham = -7.0 +/- 1.6 (P < 0.05); DeltaSEV =-1.2 +/- 2.1], end-contractions PO(2mv) [in mmHg, DeltaSham = -5.0 +/- 1.0 (P < 0.05); DeltaSEV = -2.5 +/- 0.5] and time constant of PO(2mv) decrease [in s, DeltaSham = -6.5 +/- 3.0 (P < 0.05); DeltaSEV = -3.2 +/- 1.8]. CONCLUSION: These data provide the first direct evidence that the pathological profiles of PO(2mv) associated with severe CHF can be explained, in part, by a diminished vascular NO availability.

German national case collection of familial pancreatic cancer - clinical-genetic analysis of the first 21 families.

BACKGROUND: The observation of a familial accumulation of ductal pancreatic adenocarcinoma (PC) and the increased risk for PC in certain hereditary tumor syndromes point to a genetic predisposition for PC. In order to evaluate the characteristics of familial PC, a German national case collection for familial pancreas cancer (FaPaCa) was established. PATIENTS AND METHODS: In FaPaCa, families of patients with PC are being collected, who have at least 1 first-degree relative with PC or with malignant melanoma. Histopathologic verification of tumor diagnoses, acquisition of clinical data, and full genetic counselling are prerequisites for the enrollment of PC families in FaPaCa. RESULTS: So far, 21 families fulfilled the criteria for partaking in FaPaCa. In 11 families, PC represented the sole tumor entity. Additional tumors included malignant melanoma in 5, breast cancer in 3, and prostatic, colon or lung cancer in 2 families. Compared to the preceding generation, a younger age at diagnosis of PC was observed in the offspring of PC patients (offspring median 53 years vs. parents median 75.5 years). CONCLUSION: The association of PC and breast cancer, and of PC and malignant melanoma suggests predisposing mutations in the BRCA2 or CDKN2A genes in about one third of the FaPaCa families. Mutational analyses in both candidate genes may help to identify individuals who are at an increased risk for developing PC. A shift towards a younger age at diagnosis in our PC families may indicate genetic anticipation and/or changes of patterns of exogenous risk factors.

Allelic loss is often the first hit in the biallelic inactivation of the p53 and DPC4 genes during pancreatic carcinogenesis.

The presumed precursor lesions of pancreatic ductal adenocarcinoma were recently classified according to their increasing grade of dysplasia and were designated as pancreatic intraepithelial neoplasia (PanIN) 1 through 3. In this study, we tested whether molecular genetic alterations can be correlated with this classification and may help to further categorize the various PanIN grades. We determined the frequencies of allelic loss at chromosomal arms 9p, 17p, and 18q in 81 microdissected duct lesions of various PanIN grades, using a combination of whole genome amplification and microsatellite analysis. In addition we examined the p53 and Dpc4 protein expression patterns by immunohistochemical analysis. In PanIN-1, we did not detect allelic losses. In PanIN-2, allelic losses were found in increasing frequency, and were particularly high in those lesions with moderate-grade dysplasia (low grade, 20, 33, and 17%, loss at 9p, 17p, and 18q, respectively; moderate grade, 46, 77, and 58%). PanIN-3 and invasive carcinomas exhibited abundant losses. Abnormal p53 and Dpc4 protein expression was only rarely identified in PanIN-2 lesions, but occurred frequently in PanIN-3 lesions and invasive carcinomas. The combined genetic and protein expression data support a model in which allelic loss is the first hit in the biallelic inactivation of the p53 and DPC4 tumor suppressor genes. In addition, our data indicate that allelic loss analysis may be useful in separating PanIN-2 lesions with low-grade dysplasia from those PanIN-2 lesions with moderate-grade dysplasia, each potentially representing a distinct progression step toward invasive carcinoma.

Update of familial pancreatic cancer in Germany.

BACKGROUND/AIMS: The prevalence of familial pancreatic cancer (FPC) and the characteristics of FPC have not yet been well investigated in the German population. Therefore, a German case collection for FPC was established in July 1999 to collect and evaluate data on FPC families. METHODS: The prevalence of pancreatic cancer (PC) as well as other tumours and diseases was studied in families with at least 2 first-degree relatives with histologically confirmed PC, and in families of patients with PC and a first-degree relative with malignant melanoma. All participating family members were genetically counselled and evaluated by a standardised questionnaire. RESULTS: In an 18-month period, 73 independent kindreds with potential FPC contacted the national case collection. So far, 20 kindreds have fulfilled the criteria for FPC and have undergone complete workups. Most families revealed an autosomal dominant pattern of inheritance. Twelve families revealed an isolated accumulation of PC. Importantly, in 8 of 20 (35%) families, additional tumour types such as melanoma, breast and prostate cancer occurred. CONCLUSION: The observed phenotypic heterogeneity indicates an association with predisposing tumour suppressor genes p16 and BRCA2 in up to 30% of FPC families. Mutation analysis of these candidate genes might lead to the identification of the predisposing gene defect in a proportion of FPC families.

Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis.

Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs. Smad4 encodes a key intracellular messenger in the transforming growth factor beta (TGF-beta) signaling cascade. TGF-beta is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation in vivo, yet it did not restore sensitivity to TGF-beta. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors in vivo, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.

Mutations of the DPC4/Smad4 gene in neuroendocrine pancreatic tumors.

Tumors of the endocrine pancreas are extremely rare, and molecular mechanisms leading to their development are not well understood. A candidate tumor suppressor gene, DPC4, located at 18q21, has recently been shown to be inactivated in half of pancreatic adenocarcinoma xenografts. The close anatomical relationship of the exocrine and endocrine pancreas prompted us to determine the role of DPC4 in the tumorigenesis of 25 pancreatic islet cell tumors (11 insulinomas, nine non-functioning endocrine carcinomas, three gastrinomas, two vipomas). A mutation screening of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational variant (SSCP) analysis and a PCR-based deletion assay. Five of nine (55%) non-functioning endocrine pancreatic carcinomas revealed either point mutations, small intragenic deletions or homozygous deletion of DPC4 sequences compared to none of the insulinomas, gastrinomas or vipomas. These results suggest that DPC4 is an important target gene promoting tumorigenesis of non-functioning neuroendocrine pancreatic carcinomas.

DPC4/SMAD4 mediated tumor suppression of colon carcinoma cells is associated with reduced urokinase expression.

We recently identified DPC4/Smad4 as a candidate tumor suppressor gene mutated or lost in one half of pancreatic carcinomas and in a subset of colon and biliary tract carcinomas. DPC4 plays a key role in signal transduction of the TGF-beta superfamily of molecules and inactivation of TGF-beta mediated growth inhibition is supposed to be the driving force for DPC4 inactivation in human tumors. However, DPC4 mediated tumor suppression by reconstitution of defective cells has not yet been reported. Here we show suppression of tumorigenicity in nude mice by stable reexpression of DPC4 in SW480 colon carcinoma cells. In vitro growth of DPC4-transfected cells was not affected and resistance towards TGF-beta mediated growth inhibition was retained. Instead, cells exhibited morphological alterations and adhesion and spreading were accelerated. These phenotypic changes were associated with reduced expression levels of the endogenous urokinase-type plasminogen activator (uPA) and plasminogen-activator-inhibitor-1 (PAI-1) genes, the products of which are implicated in the control of cell adhesion and invasion. In patients, high expression levels of uPA and PAI-1 correlate with poor prognosis. Thus, reduced expression of uPA and PAI-1 is consistent with suppression of tumorigenicity in DPC4 reconstituted cells. These results demonstrate DPC4's tumor suppressive function and suggest a potential role for DPC4 as a modulator of cell adhesion and invasion.

Recent discoveries in cancer genetics of exocrine pancreatic neoplasia.

Within the past 4 years major advances in our understanding of pancreatic carcinogenesis have been made. The discovery of a high frequency of mutations in the tumor suppressor genes p16 and p53 together with an extraordinary high rate of K-ras mutations have shed light on how the disturbance of cell cycle control is a major hallmark in this tumor type. Furthermore, another very recently identified tumor suppressor gene, DPC4 (deleted in pancreatic carcinoma, locus 4), revealed that the TGFbeta-Smad signalling pathway is also likely to contribute to the development of this tumor type. It is now hoped that our improved knowledge of the molecular profile of pancreatic carcinoma will also translate into better diagnostic and therapeutic options to deal with this dismal disease.

Mutations of the DPC4/Smad4 gene in biliary tract carcinoma.

A candidate tumor suppressor gene, DPC4, located at 18q21.1, has recently been shown to be inactivated in half of pancreatic adenocarcinomas. The close developmental relationship of the pancreas and biliary tract prompted us to determine the role of DPC4 in the multistep carcinogenesis of biliary tract carcinoma. A search for mutations in the genomic sequence of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational polymorphism analysis. Five of 32 (16%) primary biliary tract carcinomas had point mutations in the DPC4 sequence. Interestingly, inactivation of DPC4 was especially common in carcinomas originating from the common bile duct (four of eight specimens analyzed), suggesting an important role for DPC4 in the development of this subtype of biliary tract tumor.

Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas.

The Rb/p16 tumor-suppressive pathway is abrogated frequently in human tumors, either through inactivation of the Rb or p16INK4a/CDKN2/MTS1 tumor-suppressor proteins, or through alteration or overexpression of the cyclin D1 or cyclin-dependent kinase 4 oncoproteins. We reported previously that the p16 gene was genetically inactivated in 82% of pancreatic carcinomas. Nearly half of these inactivations were by intragenic mutation of p16, and the remainder were by homozygous deletion of the gene. Here, we analyzed pancreatic carcinomas for additional mechanisms by which the Rb/p16 pathway might be inactivated. Transcriptional silencing of the p16 gene in association with methylation of its 5'-CpG island was examined by methylation-specific PCR in 18 pancreatic carcinomas. Nine of these were known to harbor an intragenic mutation in p16, and nine had a wild-type p16 coding sequence. Seven of the 18 tumors were hypermethylated, and all 7 were p16 wild-type (P = 0.001). Complete silencing of transcription from methylated wild-type gene sequences was demonstrated. Immunohistochemical analysis revealed normal expression levels of the Rb protein in all carcinomas studied. None of the carcinomas had genomic amplification of the cyclin D1 or CDK4 genes, and none had mutation of the p16-binding domain of CDK4. An additional p16 mutation was identified. In total, the Rb/p16 pathway was abrogated in 49 of the 50 carcinomas (98%) studied, all through inactivation of the p16 gene. Similar results were obtained in an independently analyzed series of 19 pancreatic carcinomas. These data demonstrate the central role of the Rb/p16 pathway in the development of pancreatic carcinoma.

Tumor-suppressive pathways in pancreatic carcinoma.

During tumorigenesis, positive selection is exerted upon those tumor cells that alter rate-limiting regulatory pathways. A corollary of this principle is that mutation of one gene abrogates the need for alteration of another gene in the same pathway and also that the coexistence in a single tumor of mutations in different genes implies their involvement in distinct tumor-suppressive pathways. We studied 42 pancreatic adenocarcinomas for genetic alterations in the K-ras oncogene and the p16, p53, and DPC4 tumor suppressor genes. All of them had the K-ras gene mutated. Thirty-eight % of the tumors had four altered genes, another 38% had three altered genes, 15% had two altered genes, and 8% of the tumors had one altered gene. Interestingly, we noted a high concordance of DPC4 and p16 inactivations (P = 0.007), suggesting that the genetic inactivation of p16 increases the selective advantage of subsequent mutation in DPC4. No statistically significant association was identified between the alteration of these cancer genes and pathological or clinical parameters. This type of multigenic analysis in human tumors may serve to substantiate experimental tumor models and thus increase our understanding of the truly physiologically relevant tumor-suppressive pathways that are abrogated during human tumorigenesis.

The structural basis of molecular genetic deletions. An integration of classical cytogenetic and molecular analyses in pancreatic adenocarcinoma.

Molecular genetic alterations are known to be important in human carcinoma, but the structural basis of these changes is largely unknown. To examine the basis of these changes, we compared the karyotypic chromosomal abnormalities of primary pancreatic adenocarcinomas with the molecular changes identified in these same cancers. In 14 cancers with abnormal karyotypes, 65% (123 of 188) of the chromosomal arms with molecular loss of heterozygosity (LOH) were associated with karyotypic structural anomalies. Karyotypic changes accounting for these molecular allelic losses included 83 chromosome losses, 18 partial deletions, nine isochromosomes, eight additions, and five translocations. Eight bomozygous deletions were also identified by molecular analyses. Of the three homozygous deletions identified at 9p21, the only karyotypic change was a single case in which one entire copy of chromosome 9 was deleted. Of the four homozygous deletions identified at 18q21.1, one showed a loss of both copies of chromosome 18, two showed a loss of one copy of chromosome 18, and the fourth had two structurally normal copies of chromosome 18. One homozygous deletion was identified at 13q12.3, and the karyotype revealed the loss of one entire copy of chromosome 13. The second copy of chromosome 13 in this carcinoma was structurally normal. These results indicate that chromosomal structural anomalies can account for two-thirds of the LOH in pancreatic adenocarcinomas and that most homozygous deletions are likely to be interstitial chromosomal deletions that are below the detection limit of conventional karyotypic analyses. Some of the molecular deletions detected as LOH on chromosomes with karyotypically normal structure can be explained by chromosomal loss with reduplication of the remaining chromosome.