Pathological and molecular characteristics distinguishing contralateral metastatic from new primary breast cancer.

BACKGROUND: Breast cancer patients have a cumulative lifetime risk of 2%-15% of developing a contralateral metastatic or ex novo primary cancer. From prognostic and therapeutic viewpoints, it is important to differentiate metastatic from second primary. To distinguish these entities, we investigated whether the pattern of X chromosome inactivation could determine whether the two tumors derived from different progenitor cells. MATERIALS AND METHODS: The clonality of bilateral breast cancer was evaluated through the X-inactivation analysis using the human androgen receptor gene (HUMARA) polymorphism and the histopathologic and molecular results were compared. A different or an identical pattern of X inactivation was considered as indicator of a second primary cancer or not informative, respectively. We considered morphological indicators of a new primary cancer the absence of concordance in the histological type or a better histological differentiation. RESULTS: Ten patients with bilateral breast cancer were evaluated. Morphological criteria indicated that eight were second primary, a conclusion confirmed by the X-inactivation analysis. Two cases classified as recurrence according to morphological criteria were classified as second tumor by molecular analysis. CONCLUSION: Our results show that the HUMARA clonality assay can improve the histological parameters in differentiating metastatic cancer from second primary cancer.

Epidermal growth factor receptor gene copy number, K-ras mutation and pathological response to preoperative cetuximab, 5-FU and radiation therapy in locally advanced rectal cancer.

BACKGROUND: Cetuximab improves activity of chemotherapy in metastatic colorectal cancer (mCRC). Gene copy number (GCN) of epidermal growth factor receptor (EGFR) has been suggested to be a predictive factor of response to cetuximab in patients (pts) with mCRC; on the contrary, K-ras mutation has been associated with cetuximab resistance. PATIENTS AND METHODS: We have conducted a phase II study with cetuximab administered weekly for 3 weeks as single agent and then with 5-fluorouracil and radiation therapy as neo-adjuvant treatment for locally advanced rectal cancer (LARC). EGFR immunohistochemistry expression, EGFR GCN and K-ras mutation were evaluated on diagnostic tumor biopsy. Dworak's tumor regression grade (TRG) was evaluated on surgical specimens. RESULTS: Forty pts have been treated; 39 pts are assessable. TRG 3 and 4 were achieved in nine (23.1%) and three pts (7.7%) respectively; TRG 3-4 rate was 55% and 5.3% in case of high and low GCN, respectively (P 0.0016). Pts with K-ras mutated tumors had lower rate of high TRG: 11% versus 36.7% (P 0.12). In pts with wild-type K-ras, TRG 3-4 rate was 58.8% versus 7.7% in case of high or low GCN, respectively (P 0.0012). CONCLUSIONS: In pts with LARC, EGFR GCN is predictive of high TRG to cetuximab plus 5-FU radiotherapy. Moreover, our data suggest that a wild-type K-ras associated with a high EGFR GCN can predict sensitivity to cetuximab-based treatment.

Detection of tissue abnormalities in normal mucosa surrounding colorectal cancer using an endocytoscopy system.

The endocytoscopy system is a novel diagnostic technique that provides extremely high-magnification imaging of the gastrointestinal mucosa. We are currently using a prototype Olympus endocytoscope for clinical research in gastrointestinal tumors. In one surgical specimen obtained after resection of a cancer of the transverse colon, focal abnormalities of colonic glands were detected 7 cm away from the primary tumor, within macroscopically normal mucosa. Our finding, which was confirmed by histopathological examination, suggests the need for further clinical investigation to assess whether endocytoscopy is able to identify premalignant changes in apparently normal mucosa. This could potentially be useful for accurate evaluation before planning minimally invasive therapy.

Pancreatic intraductal sampling during ERCP in patients with chronic pancreatitis and pancreatic cancer: cytologic studies and k-ras-2 codon 12 molecular analysis in 47 cases.

BACKGROUND: A preoperative tissue diagnosis of pancreatic cancer is desirable but difficult to obtain. METHODS: Pancreatic brush cytology, salvage cytology, and collection of pancreatic juice were attempted prospectively during ERCP in 34 patients with pancreatic cancer and 11 with chronic pancreatitis. K-ras-2 codon 12 was analyzed for presence and type of point mutations. RESULTS: Brush cytology coupled with salvage cytology had a sensitivity of 74%. The addition of cytologic analysis of pancreatic juice did not substantially improve sensitivity (76%). K-ras-2 was mutated in both cancer (87%) and pancreatitis (40%). The specificity for cytology was 100% and for K-ras-2 mutations 60%. Combining cytology with mutation analysis increased sensitivity to 93% but reduced the positive predictive value. The negative predictive value never exceeded 75%. None of the patients with chronic pancreatitis had cancer develop (median follow-up 60 months). CONCLUSIONS: Pancreatic ductal brushing with salvage cytology is useful in the diagnosis of cancer, whereas cytologic analysis of pancreatic juice can be abandoned. At present, K-ras-2 mutation is not useful for differentiating pancreatic cancer from chronic pancreatitis or the identification of patients with chronic pancreatitis at risk for malignant transformation.

Randomized trial of fenretinide in superficial bladder cancer using DNA flow cytometry as an intermediate end point.

Retinoids have shown a potential activity in preventing tumor recurrence in superficial bladder cancer. We assessed the activity of the synthetic retinoid fenretinide in superficial bladder cancer using DNA flow cytometry and conventional cytology as surrogate biomarkers. A total of 99 subjects with resected superficial bladder cancer (pTa, pT1) were randomized to either fenretinide (200 mg day p.o. for 24 months) or no intervention. Cystoscopy and bladder washing for DNA flow cytometry end points (proportion of DNA aneuploid histograms, hyperdiploid fraction, and percentage of apoptotic cells) and proportion of abnormal cytological examinations were repeated every 4 months for up to 36 months. The primary study end point was the proportion of DNA aneuploid histograms after 12 months. This figure was 48.9% in the fenretinide arm and 41.9% in the control arm (odds ratio, 1.16; 95% confidence interval, 0.44-3.07). There was no difference in any other response biomarker between the two groups up to 36 months, nor was any biomarker able to predict recurrence risk. Recurrence-free survival was comparable between the arms (27 events in the fenretinide arm versus 21 in the control arm; P = 0.36). Twelve subjects in the fenretinide arm complained of diminished dark adaptability, and nine subjects in the fenretinide arm versus one control subject had mild dermatological alterations. We conclude that fenretinide showed a lack of effect on the DNA content distribution and the morphology of urothelial cells obtained in serial bladder washings. Recurrence-free survival was comparable between groups. Because our data are hampered by the lack of predictivity of the selected biomarkers, additional studies are necessary to assess the activity of fenretinide in preventing bladder cancer.

Assessment of DNA flow cytometry as a surrogate end point biomarker in a bladder cancer chemoprevention trial.

Although conventional cytology represents the most widely performed cytometric analysis of bladder cancer cells, DNA flow cytometry has, over the past decade, been increasingly used to evaluate cell proliferation and DNA ploidy in cells from bladder washings. We have investigated whether DNA flow cytometry and conventional cytology of epithelial cells obtained from bladder washings provide reliable surrogate endpoint biomarkers in clinical chemoprevention trials. We used cytometric and clinical data from a chemoprevention trial of the synthetic retinoid Fenretinide on 99 patients with superficial bladder cancer. A total of 642 bladder washing specimens obtained from the patients at 4 month intervals was analyzed. Intra-individual agreement and correlation of flow cytometric DNA ploidy (diploid vs. aneuploid), DNA Index, Hyper-Diploid-Fraction (proportion of cells with DNA content higher than 2C), and conventional cytologic examination, as assessed by kappa statistics and Spearman's correlation test, were poor from baseline through 24 months. Moreover, no correlation was found between DNA ploidy and cytology at each time point. The same results were obtained when the analyses were stratified by treatment group. In addition, the association between the results of bladder washing (by either DNA flow cytometry or cytology) and concomitant tumor recurrence was significant only for abnormal cytology, while neither biomarker was predictive of tumor recurrence at the subsequent visit. During the time of this study only four patients progressed to muscle-invasive bladder cancer, indicating the "low-risk" features of the patient population. We conclude that DNA flow cytometry and conventional cytology on epithelial cells obtained from bladder washings do not appear to provide suitable surrogate endpoint biomarkers during the early stages of bladder carcinogenesis.

Distal hyperplastic polyps do not predict proximal adenomas: results from a multicentric study of colorectal adenomas.

BACKGROUND: The association between distal hyperplastic polyps and proximal adenomas is still a matter of debate. We investigated this association while taking into account patient characteristics. METHODS: After exclusion of patients with inflammatory bowel diseases, familial adenomatous polyposis, or any cancer, 3088 eligible consecutive subjects aged 18 to 69 years underwent total colonoscopy in four gastroenterology units. The odds ratios (OR) of having proximal adenomas according to patient characteristics (age, sex, medical center, year of endoscopy, reasons for referral, and distal findings) were estimated in univariate and multivariate analyses. RESULTS: Patients with distal polyps of any type showed an adjusted OR of 2.5 (95% CI [1.9, 3.1] p < .001) of having proximal adenomas as compared with those without distal polyps. When distal adenomas and distal hyperplastic polyps were included in the multivariate model as independent factors, the presence of adenomas significantly increased the risk of proximal adenomas (OR = 2.8: 95% CI [2.2, 3.6] p < .001), whereas the presence of hyperplastic polyps did not (OR = 1.1: 95% CI [0.8, 1.5] p = .64). No association with number, size, or location of distal hyperplastic polyps was seen. CONCLUSIONS: Our data show that the presence of hyperplastic polyps should not be the sole indication for total colonoscopy because they are not associated with proximal adenomas when adjusting for patient characteristics and presence of distal adenomas.

Endoductal tissue sampling of biliary strictures through endoscopic retrograde cholangiopan creatography (ERCP).

AIM AND BACKGROUND: Pathological proof of malignant in biliary strictures is useful in the preoperative setting as it helps define therapeutic planning and prognosis, and reduces the length of the subsequent surgical intervention. However, it is difficult to obtain. The aim of this study was to evaluate the yield of histological and cytological examination of endobiliary samples obtained during endoscopic retrograde cholangiopancreatography (ERCP). METHODS: Endobiliary forceps biopsy and brush cytology were performed during ERCP examination in 52 consecutive patients, 36 with malignant and 16 with benign strictures. RESULTS: Histology and cytology turned out to have the same sensitivity (53%). The gain in sensitivity achieved by combining the two techniques was limited, reaching a value of 61%. The specificity, however, was 100% for both methods. Most of the few complications observed were due to sphincterotomy and subsided spontaneously or with medical treatment. However, one patient experienced a serous complication and chose to be treated by surgical intervention. The complication was caused by forceps biopsy. CONCLUSIONS: This study shows that 1) sampling of biliary strictures during ERCP is the primary approach to tissue diagnosis; 2) brush cytology alone is sufficient in clinical practice; 3) forceps biopsy must always be used to sample intra-ampullary strictures but should be considered as a secondary step to sample strictures located more proximally, in the bile ducta, if previous cytology was negative.

Effect of tamoxifen on endometrial proliferation.

PURPOSE: An increase in the incidence of endometrial cancer and a potential increase in related mortality has been associated with the administration of 20 mg tamoxifen, the dose adopted in breast cancer chemoprevention trials, thus urging studies on intermediate markers of risk. PATIENTS AND METHODS: Thirty-three women who received 20 mg tamoxifen as adjuvant breast cancer treatment underwent endometrial biopsy. Samples were divided for histologic examination, including a quantitative analysis of stromal:epithelial ratio, and an assessment of DNA ploidy and proliferation by flow cytometry. Results were compared with 37 symptomatic subjects. RESULTS: All histograms were DNA diploid. Compared with controls, a significant increase in the risk of proliferation as measured by the hyperdiploid fraction was associated with tamoxifen duration (< or = 36 months: cumulative odds ratio = 16.5, 95% confidence interval, 1.85 to 146.5; > 36 months: cumulative odds ratio = 28.2, 95% confidence interval, 2.56 to 310.6, P for trend < .05). Tamoxifen-induced risk was significantly reduced by the extent of menopausal status. No cases of cancer or epithelial hyperplasia were observed in the tamoxifen group, whereas seven cases of epithelial hyperplasia without atypia were observed in the control group. The effect of tamoxifen on proliferation was associated with an increase in the stromal component. CONCLUSION: Tamoxifen at 20 mg/d exerts a time-dependent proliferative effect on the endometrium, particularly in premenopausal and early postmenopausal women. This effect appears to be mediated by the stromal component, which accounts for the discrepancy between flow cytometry and histology. Our study provides preliminary evidence that the DNA flow cytometric hyperdiploid fraction may be a useful tool for monitoring endometrial cell proliferation in women exposed to tamoxifen.

Endoscopic retrograde forceps biopsy and brush cytology of biliary strictures: a prospective study.

BACKGROUND: Nonsurgical pathologic confirmation of malignant bile duct strictures is desirable for defining subsequent treatment and prognosis. Endoscopic retrograde cholangiopancreatography is frequently performed in patients suspected of having pancreaticobiliary obstruction, but there exists no standardized method for defining the nature of obstructing lesions by ERCP. METHODS: We prospectively evaluated the yields of endoscopic retrograde brush cytology and biopsy for the diagnosis of malignant bile duct strictures. Fluoroscopically guided endobiliary biopsy and brush cytology (52) or cytology alone (42) were performed during endoscopic retrograde cholangiopancreatography in 94 consecutive patients, 64 with malignant strictures and 30 with benign strictures. A single cytopathologist classified the results of these studies as positive or negative for malignancy. RESULTS: The sensitivities of the two procedures were identical (53%) and the gain achieved by combining the two techniques (61%) was small. Specificity proved excellent for both methods. One major complication that occurred was perforation of the common hepatic duct with leakage of bile, which was managed by surgical oversewing. This complication was ascribed to biopsy and untimely removal of the nasobiliary drain by the patient herself. CONCLUSIONS: This study indicates that endoscopic retrograde brush cytology alone may be sufficient in daily practice, at least in centers that have access to experienced cytopathologists. We recommend use of forceps biopsy in selected cases where brush cytology is negative.

Mixed pleomorphic-osteoclast-like tumor of the pancreas. Light microscopical, immunohistochemical, and molecular biological studies.

The morphological, immunohistochemical, and molecular biological features of a case of giant cell tumor of the pancreas are described. This neoplasm showed mononuclear and multinucleated tumor giant cells as well as numerous osteoclast-like cells with multiple foci of osteoid-osseous metaplasia. The pleomorphic and osteoclastic giant cells displayed extensive homologies in their immunohistochemical profiles. Neither the pleomorphic nor osteoclast-like portion of the tumor showed neither c-Ki-ras nor p53 mutation and did not express the mutated p53 protein. The results suggest that the pleomorphic and osteoclast-like components are histogenetically related and that this rare neoplasm originates from a precursor cell capable of differentiating along divergent cell type.

Evaluation of cathepsin D as prognostic predictor in breast cancer.

Cathepsin D is an acidic lysosomal protease expressed in all cells. Some studies have shown correlations between high levels of tissue cathepsin D and poor prognosis. This paper deals with 158 cases of breast cancer in which tissue concentrations in cathepsin D, age, estrogen and progesterone receptor content, and pathological characteristics of the tumor were investigated. Tumors were considered to be cathepsin D+ when a concentration > 40 pmol/mg protein (median value in our samples) was determined. The expression of cathepsin D appears to be related to grading (p = 0.04) and lymph node status (p = 0.05). We found no significant associations among cathepsin D levels, patient age, steroid receptors and histological type. Moreover, the levels of cathepsin D have been evaluated in 9 samples of recurring or metastatic neoplasia and 11 cases of benign breast lesions. We conclude that cathepsin D may be a useful prognostic predictor in breast cancer. Further investigations are required to improve and extend the applications of this assay.

Activity of 4-HPR in superficial bladder cancer using DNA flow cytometry as an intermediate endpoint.

The ability of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) to affect the outcome of previously resected superficial bladder cancer was investigated in a pilot study using DNA content flow cytometry and conventional cytology as intermediate endpoints. Twelve patients were treated with oral 4-HPR (200 mg daily) and compared with 17 non-randomized, untreated controls. The median interval between transurethral resection and 4-HPR administration was 5.5 months (range 0-36). The median follow-up period was 12 months (range 3-31) in the 4-HPR group and 9 months (range 2-22) in the control group. The proportion of patients with DNA aneuploid stemlines in bladder-washed cells decreased from 7/12 (58%) to 5/11 (45%) in the 4-HPR group, but increased from 7/17 (41%) to 10/17 (59%) in the control group. In patients with stable diploid profiles, mean (+/- SE) S-phase and G2+M-phase fractions decreased in the course of retinoid treatment from basal levels of 15.2 +/- 4.1% to 7.5 +/- 3.3% and 10.3 +/- 2.2% to 5.2 +/- 0.4%, respectively. The same parameters in the control group changed from basal levels of 14.6 +/- 3.4% to 12.4 +/- 2.7% and 9.8 +/- 1.6% to 12.6 +/- 1.6%, respectively. Positive or suspicious cytologic examinations were present in 3/12 (25%) treated cases prior to 4-HPR administration and all subsequently reverted to normal. The same parameter in the control group increased from 4/17 (24%) to 6/17 (35%) during follow-up. Impaired adaptation to darkness was recorded in 4 patients, and transient dermatologic alterations were observed in one-third of the patients, requiring dose reduction in one case.(ABSTRACT TRUNCATED AT 250 WORDS)

Univariate and multivariate analyses of the relationship between adenocarcinoma and solitary and multiple adenomas in colorectal adenoma patients.

In 584 consecutive patients with no previous history of cancer or polypectomy, 769 adenomas were excised endoscopically and classified retrospectively according to the following parameters: macroscopic growth pattern, size, histological type, grade of dysplasia, anatomical site, presence of adenocarcinoma (ADK), number of adenomas and sex and age of the patient. A multivariate logistic analysis confirmed that size and histological type are the 2 most important predictors of ADK, both in solitary and in multiple adenomas. Adenomas located in the sigmoid portion and in the rectum have an increased probability of ADK, independent of size and histology. Multiple polyps, when compared to solitary polyps, were more frequent in males (p less than 0.01) and were more often larger than 2 cm. Significant similarities in histology, morphology and degree of dysplasia were observed among multiple adenomas from the same patient.

Endocervical metastases secondary to breast carcinoma. A case report.

Only 24 cases of breast cancer metastases to the uterine cervix have been reported to date. The present case present secondary cervical involvement in the absence of concomitant extragenital metastases. The patient did not present with abnormal vaginal discharge and the cervical lesion responded to conventional combination chemotherapy for breast cancer.

Value of multiple forceps biopsies in assessing the malignant potential of colonic polyps.

Fifty-nine colo-rectal polyps were detected at endoscopy and repeatedly biopsied before removal by endoscopic snare polypectomy. The aim of the present paper was to evaluate the reliability of multiple forceps biopsies in assessing both the malignant potential and the presence or absence of invasive cancer (IC) in colo-rectal adenomas (CRA). In order to achieve the first objective, the histologic types and the degree of dysplasia have been defined. The data obtained by means of multiple biopsies examination, compared with those of polyp in toto study, show that fractional biopsies were of value in the histologic classification of only the smallest 41 polyps (agreement 88.09%), whilst no reliability of biopsies was demonstrated in the 18 largest polyps (agreement 27.68%). In the field of dysplasia grading, the agreement was 55% and 61% for the smallest and the largest CRA respectively. These last figures are hardly acceptable. Biopsies examination gave also under- and overestimation of the histologic severity and of dysplasia as well as a significant incidence of false negative results in IC detection. It is concluded that polypectomy is the only method which provides adequate material for precise diagnosis, no matter how large a polyp. Therefore it should be performed whenever possible. Finally the authors discuss the management of small sessile adenomas.