Jordi Estapé

Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis

PURPOSE Circulating melanoma cells can be detected in peripheral blood by means of tyrosinase mRNA amplification by reverse-transcriptase polymerase chain reaction (RT-PCR). We conducted a prospective study to evaluate the clinical significance of the presence of circulating neoplastic cells in the blood of patients with malignant melanoma (MM). METHODS A sensitive RT-PCR assay was used to detect tyrosinase mRNA in the peripheral blood of patients with stages I to IV melanoma. Healthy subjects or patients with other malignancies were used as negative controls. RESULTS Ninety-one assessable patients were included in the study. There was a statistically significant association between RT-PCR positivity and clinical stage. Circulating melanoma cells were detected in 36% of patients with localized disease (stages I and II), in 45% of patients with regional nodal involvement (stage III), and in 94% of patients with metastatic disease (stage IV) (P < .001). In stage II-III patients who were RT-PCR-positive for mRNA tyrosinase in blood, the recurrence rate and disease-free survival were significantly worse than patients who were RT-PCR-negative. In multivariate analysis, RT-PCR was an independent prognostic factor for recurrence in patients with nonmetastatic disease (P = .002). CONCLUSION The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.

The diagnostic value of a new tumor marker, c-erbB-2, was studied in the sera of 50 healthy subjects, 58 patients with benign breast diseases, and 413 patients with breast cancer (186 locoregional, 185 with advanced disease, and 42 with no evidence of disease). Using 15 U/ml as the cut-off, no healthy subjects or patients with benign diseases and only 2.4% of no evidence of disease patients had elevated serum levels. Abnormal c-erbB-2 levels were found in 29% (101/370) of the patients with breast carcinoma (locoregional 9%, metastases 45.4%). CEA (cut-off 5 U/ml) and CA 15.3 (cut-off 35 U/ml) sensitivity was 18% and 16% in patients with locoregional disease and 61% and 70% in those patients with advanced disease, respectively. A trend toward higher serum levels of all three tumor markers in patients with nodal involvement or greater tumor size was found, but was statistically significant only with CEA (p < 0.01). By contrast, c-erbB-2 was related to steroid receptors, in both locoregional and metastatic tumors. When the prognostic value of these markers was evaluated, patients with abnormally high presurgical CEA and c-erbB-2 had a worse prognosis than those patients with normal values, in both node-negative (p < 0.05 and p < 0.001, respectively) and node-positive patients (p < 0.556 and p < 0.001, respectively). By contrast, no relationship was found between CA 15.3 values and prognosis. Multivariate analysis showed that CEA and c-erbB-2 were also prognostic factors. The correlation between serum and tissue levels of c-erbB-2 was studied in the tumors of 161 patients. Significantly higher c-erbB-2 serum levels were found in patients with overexpression in tissue by immunohistochemistry, in both locoregional and advanced disease (p=0.0001). Serum concentrations in patients with advanced disease were related to the site of recurrence, with significantly higher values in patients with metastases (mainly in those with liver metastases) than in those with locoregional recurrence. In summary, c-erbB-2 serum levels seem to be a useful tumor marker in the prognosis of patients with breast cancer. Using all three tumor markers, sensitivity was 35% in patients with locoregional breast cancer and 88% in patients with recurrence.

Treatment of chronic lymphocytic leukemia in advanced stages. A randomized trial comparing chlorambucil plus prednisone versus cyclophosphamide, vincristine, and prednisone

Ninety‐six patients with advanced chronic lymphocytic leukemia (CLL) (Stage C; anemia and/or thrombocytopenia of nonimmune origin) were randomized to receive either chlorambucil (CLR) (0.4 mg/kg orally, day 6) plus prednisone (PDN) (60 mg/m2 orally, days 1–5) every 2 weeks or cyclophosphamide (600 mg/m2 intravenously, day 6), vincristine (1 mg/m2 intravenously, day 6), and prednisone (60 mg/m2 orally, days 1–5) (COP) each month for 5 months. Complete remission (CR) was defined as the total disappearance of signs and symptoms related to the disease. Partial remission (PR) was considered to be achieved when, after treatment, the clinical stage changed to a less advanced one. Thirty (59%) responses (8% CR) with CLR plus PDN and 14 (31%, 2% CR) with COP were observed (P < 0.01). The survival was not significantly different for the two groups. Patients previously treated had a lower number of responses (11/35, 31%) than those with no previous treatment (33/61, 54%) (P < 0.05). Patients who attained a CR or a good PR had longer survivals (median not reached) than those with a poor PR (median, 25.2 months) or those who did not respond to treatment (median, 11.5 months) (P <0.005).

Survival of multiple myeloma patients who are potential candidates for early high-dose therapy intensification/ autotransplantation and who were conventionally treated.

PURPOSE To analyze the outcome of patients with multiple myeloma (MM) who were potential candidates for early high-dose therapy (HDT) intensification followed by autotransplantation from a series treated with conventional chemotherapy. PATIENTS AND METHODS From January 1985 through December 1989, 487 patients with symptomatic MM were entered onto a randomized study to compare melphalan and prednisone (MP) versus vincristine, cyclophosphamide, melphalan, and prednisone (VCMP) /vincristine, carmustine (BCNU), doxorubicin, and prednisone (VBAP). The sub-group of 77 patients who could have been candidates for early intensification with HDT followed by stem-cell support (ie, < 65 years of age, stage II or III disease, performance status < 3, and objective or partial response to initial chemotherapy) are the subjects of this report. RESULTS Seventy-seven of 487 patients could have been candidates for early intensification. The median age was 56 years (range, 27 to 64). At diagnosis, 12% had abnormal renal function, 16% hypercalcemia, and 42% serum beta 2-microglobulin level > or = 6 mg/L; 62% had stage III disease at diagnosis. Thirty-six patients were initially treated with MP and 41 with VCMP/VBAP. The median response duration to initial chemotherapy was 22 months, and the actuarial probability of being in continued first response at 5 years was 14%. After a median follow-up time of 58 months, 59 patients have died, one was lost to follow-up evaluation, and 17 are still alive 69 to 119 months after initial chemotherapy. The median survival time from initiation of treatment was 60 months and from the time when autotransplantation would be considered, 52 months. The only independent prognostic parameter for survival was renal function at diagnosis. CONCLUSION The median survival time of patients with MM who are less than 65 years of age and who respond to initial chemotherapy is 5 years. This survival duration is similar to that reported in selected series of patients given early HDT and stresses the importance of ongoing randomized trials to determine the role of HDT in the treatment of younger myeloma patients.

Cyclin D1 and retinoblastoma gene expression in human breast carcinoma : Correlation with tumour proliferation and oestrogen receptor status

Cyclin D1 (CCND1) and retinoblastoma (Rb) genes are cell cycle regulators which are altered in some breast carcinomas. However, the possible cooperation between CCND1 and Rb, as well as the influence and coincidence of their abnormalities in the proliferative capacity of mammary carcinoma cells in vivo, is still unknown. In order to assess both the significance of the CCND1 gene and Rb alterations in breast carcinomas and their relationship with the proliferative capacity of the tumours and other clinico‐pathological factors, CCND1 mRNA expression was studied in 46 cases of primary breast carcinomas and matched normal tissue, 45 of which were also studied immunohistochemically. Rb expression was analysed in the same cases by immunohistochemistry, whereas the proliferative activity of the carcinomas was evaluated by flow cytometry. CCND1 mRNA was overexpressed in 19 tumours (41 per cent). Sixteen cases showed diffuse immunohistochemical expression, ten carcinomas had few positive cells, and 19 were absolutely negative. CCND1 mRNA and protein overexpression was associated with oestrogen receptor (ER) expression by the tumour. Interestingly, lack of ER expression was associated with a decreased CCND1 mRNA signal in non‐overexpressed tumours. No association was observed between CCND1 mRNA or protein overexpression and tumour proliferation or other clinico‐pathological parameters. Loss of Rb expression was observed in 26 per cent of the tumours. This abnormality was significantly associated with increased mean S‐phase (P=0·017) and decreased CCND1 mRNA expression in non‐overexpressed tumours, supporting in vivo the postulated regulatory loop between Rb and CCND1 in vitro. We conclude that CCND1 up‐regulation is not associated with increased proliferative activity in breast carcinomas, whereas its expression might be regulated in vivo by hormones and Rb. Loss of Rb expression is significantly associated with an increased proliferation of tumour cells, suggesting an important role in the progression of a subset of breast carcinomas, regardless of CCND1 abnormalities.

Papillary-cystic neoplasm of the pancreas. Report of two cases and review of the literature

Two cases of papillary‐cystic neoplasm of the pancreas are reported in women aged 22 and 23 years. The patient in the first case presented with acute abdominal pain and hemoperitoneum. This form of presentation has not been previously reported. This type of pancreatic tumor is very rare, exclusively affecting young women, and has a good prognosis despite its various histologic features, which suggest a malignant appearance. The authors consider this neoplasm as having an acinar origin because in the cells of one of the patients we observed ultrastructurally the presence of abundant rough endoplasmatic reticulum with formation of annulate lamellae and a few prezymogen granules.

p21WAF1/Cip1 is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma.

p21WAF1/Cip1 is an inhibitor of cdk/cyclin complexes, and thus regulates the cell cycle. p21 is also related to cell differentiation and is regulated by wild‐type p53, although p53‐independent regulatory pathways have been proposed. In order to analyse p21 expression as well as its relationship with p53 in human breast cancer, an immunohistochemical analysis was undertaken of 77 breast carcinomas, 16 of them with an in situ component; 30 adjacent normal tissue samples; and five non‐neoplastic specimens. Forty‐four infiltrating carcinomas (57 per cent) were p21‐positive. Expression of p21 was also observed in pre‐invasive lesions, whereas normal ducts were negative or focally and weakly positive. p21 expression was associated with high histological grade (II+III) (P‐0·017) and poor tubule formation (P‐0·002), and was significantly less frequent in lobular carcinomas (P‐0·0001). p21 positivity also correlated with increased proliferation, but this seemed to be dependent on the histological grade. Twenty carcinomas (26 per cent) showed p53 overexpression, but this was not associated with p21 negativity, suggesting the existence of p53‐independent mechanisms for p21 regulation in vivo. Cyclin D1CCND1 expression was analysed in the same series and an association between p21 and cyclin D1 expression was found, since 23 of 26 cyclin D1‐positive carcinomas were p21‐positive (P<0·001 …). In conclusion, p21 is frequently overexpressed in breast carcinomas and this occurs in the early stages of neoplastic progression. This overexpression seems to be independent of p53 status and might be involved in cyclin D1 modulation.

Tyrosinase mRNA in blood of patients with melanoma treated with adjuvant interferon.

PURPOSE To evaluate the clinical significance of the detection of circulating melanoma cells in patients treated with adjuvant interferon and to determine their potential value as a marker of interferon response. PATIENTS AND METHODS We prospectively analyzed 616 peripheral-blood samples from 120 melanoma patients with stage IIA (n = 33), IIB (n = 22), III (n = 50), or IV (surgically resected) (n = 15) disease receiving adjuvant interferon alfa-2b therapy. Tyrosinase mRNA was assayed by reverse transcriptase polymerase chain reaction (RT-PCR) as a marker of circulating melanoma cells before the start of interferon and every 2 to 3 months thereafter. RESULTS With a median follow-up time of 32.3 months (range, 7.1 to 77.5 months), 47 patients (39.8%) relapsed and 31 (26%) died. During adjuvant interferon treatment, 76 patients (64%) had undetected circulating melanoma cells and 44 patients (36%) had a positive RT-PCR result in at least one sample. Actuarial 5-year disease-free survival was 62% in patients with persistently negative RT-PCR during interferon treatment and 38% for patients with positive RT-PCR during interferon (P =.02). Actuarial 5-year overall survival was 75% and 50%, respectively (P =.03). CONCLUSION Patients with melanoma and tyrosinase mRNA detected in the blood during adjuvant interferon therapy had a worse prognosis compared with patients with undetected tyrosinase mRNA during treatment. Further investigation into the detection of circulating melanoma cells as a surrogate marker of response to adjuvant interferon therapy is warranted.

Positive Results of Adjuvant Mitomycin-C in Resected Gastric Cancer: A Randomised Trial on 134 Patients

In order to evaluate the results on successful adjuvant chemotherapy in resected gastric cancer we performed a randomised trial on 134 patients in two arms: a control one with no further treatment after surgery versus a treatment arm given mitomycin-C (MMC), 20 mg/m2 intravenously one day every 6 weeks for four courses, starting before the sixth week after surgery. The median follow-up was 105 months. In the control arm, 49 out of 66 patients died due to recurrence, versus 40 out of 68 patients in treatment arm. Actuarial survival curve was statistically significant (P < 0.025) in favour of the treatment group. Liver metastases were lower in adjuvant group than in the control group (8/68 versus 19/66). Toxicity was mild. Main toxic effects were thrombocytopenia, leukopenia, nausea and vomiting. A pelvis renal cancer as a second malignancy 8 years after gastric cancer was observed. In that particular case MMC was given after surgery. We conclude that adjuvant chemotherapy based on MMC given in the early period after surgery, improves survival rate in gastric cancer resected patients.

CYFRA 21-1 in lung cancer: comparison with CEA, CA 125, SCC and NSE serum levels

CYFRA 21–1, CEA, CA 125, SCC and NSE serum levels were determined in 50 healthy subjects and in 189 patients with primary lung cancer (101 with locoregional disease, 68 with recurrence and 20 patients with no evidence of residual disease (NED). Abnormal CYFRA 21–1 serum levels were found in 53.6% (90/168) of the patients with active cancer. Neither healthy subjects nor NED patients had abnormal serum levels. CYFRa 21–1 serum concentrations were significantly higher in patients with active cancer than in healthy subjects or in NED patients (p < 0.0001). CYFRA 21–1 sensitivity was related to tumor histology with abnormal levels in 64.7% of patients with NSCLC and in 30% of patients with SCLC (P <0.0001). In NSCLC, serum CYFRA 21–1 concentrations were also related to histological type, the highest values being found in squamous cell carcinomas and LCLC and the lowest in adenocarcinomas (p < 0.04). There was also a clear relationship between CYFRA 21–1 and tumor extension, with significantly higher values in patients with metastases than in those without metastases (p < 0.0001). Abnormal CEA values were found in 49.1%, CA 125 in 39%, SCC in 27.8% and NSE in 21.3% of the patients with active cancer. With respect to histological type, CYFRA was elevated in 68.3% of squamous cell carcinomas (CEA: 46.7%, SCC: 50%, CA 125:31.7%, NSE: 11.7%), in 54.8% of adenocarcinomas (CEA: 62%, SCC: 26.2%, CA 125: 59.5%, NSE: 9.5%), in 78.6% of LCLC (CEA: 64.3%, SCC: 28.6%, CA 125: 78.6%, NSE: 7.1%) and in 30% of SCLC (CEA: 37.7%, SCC: 3.8%, CA 125:20.8%, NSE: 45.3%). In summary, CYFRA 21–1 is the most sensitive tumor marker in patients with lung cancer, especially in NSCLC patients.