Randomized clinical trial of adjuvant fluorouracil, epirubicin, and cyclophosphamide chemotherapy for patients with fast-proliferating, node-negative breast cancer.

PURPOSE: The prospective applicability of new biologic tumor information to personalize adjuvant treatment of women with operable breast cancer remains to be demonstrated. The aim of the present study was to investigate whether patients with fast-proliferating, node-negative breast cancer could benefit from adjuvant chemotherapy with fluorouracil, epirubicin, and cyclophosphamide (FEC). PATIENTS AND METHODS: Beginning in November 1989, we analyzed the proliferative activity of primary tumors in a consecutive series of women with node-negative breast cancer to identify subgroups of patients with a worse prognosis and who were therefore suitable candidates for adjuvant systemic therapy. Proliferative activity was determined by means of the [3H]-thymidine incorporation assay using an autoradiographic technique. Women with fast-proliferating breast cancer ([3H]-thymidine labeling index, > 2.3%) were randomized to receive either six cycles of adjuvant FEC or no adjuvant therapy until disease progression. RESULTS: One-hundred twenty-five and 123 patients treated with radical surgery for pT1 to T2, N0, M0 breast cancer were randomized to the FEC and control arms, respectively. After a median follow-up of 70 months, 27 events (21.6%) were observed in the FEC arm and 39 (32.2%) in the control arm, with a significantly lower number of locoregional relapses in the FEC group. Five-year disease-free survival (DFS) was 81% in the FEC group and 69% in the control group (P <.02 by log-rank test). Cox multivariate analysis described the impact of adjuvant therapy with FEC on DFS as independent of the patients' main clinical-pathologic characteristics. CONCLUSION: FEC adjuvant polychemotherapy seems able to significantly improve the clinical outcome of patients with fast-proliferating, node-negative breast cancer.

[Biological-clinical significance of angiogenesis and mast cell infiltration in squamous cell carcinoma of the oral cavity]. / Significato biologico-clinico dell'angiogenesi ed infiltrato mastocitario nel carcinoma squamoso del cavo orale.

There are little data on the biological and prognostic role of neoangiogenesis in squamous cell carcinoma of the oral cavity (SCCOC). In particular, the role mast cells--reservoirs of angiogenetic peptides--play in neovascularization is not clear. In this work 50 cases of SCCOC T1-3 N0-1 M0 were studied, examining the microvasal density (MVD), mast cell density (MCD), relationship between these two parameters and their relationship with the pathological clinical features. Microvessels were identified with an immunohistochemical method using pan-endothelial anti-CD34 antibody while a histochemical method was used to label the mast cells with toluidine blue on adjacent sections for each tumor sample. MVD and MCD were characterized using an image analyzer. The mean MVD was 30 +/- 17 s.d. per sample while the average MCD was 8 +/- 6 s.d. per sample. Statistical analysis comparing MVD and MCD using the Pearson method showed a direct, significant correlation between the two variables (correlation coefficient = 0.496; p = 0.000). When the carcinomas were divided into subgroups with high and low MVD and MCD--using the median counts (27 and 7 respectively) as cutoff point--no association was found with the main clinical pathological features (age, sex, tumor diameter, lymph node status, cytopathological grading). As regards the correlation with prognosis, after an median 020 months of follow-up, the subgroup of patients with tumors with high MVD presented a better overall survival at 18 months from diagnosis than did the subgroup with tumors with a lower degree of vascularization (70% vs. 45%; p = 0.049 log rank test). The data obtained suggest that mast cells play an active role in angiogenetic processes in SCCOC and indicate that MVD is a favorable prognostic factor for SCCOC patients.

Primary tumours neoangiogenesis and P53 expression in oral carcinoma patients.

Paraffin embebbed tumour tissues from 47 T1-2 N0-1 M0 primary oral squamous carcinoma have been utilized for immunohistochemical analysis of p53 expression (moab DO-7) and microvessel density (MVD) analysis (moab CD34). Fifty percent of cases showed p53 immunostaining with an average of 21% of p53 positive cells. A strong trend for a longer survival in patients with tumor p53- versus p53+ was evidenced (median survival: 12 months versus not reached, respectively; p=0.08 by log-rank test). A mean value of 27 MVD was found. The probability of overall survival did not result significantly different in the subgroups of tumours with high and low MVD (median survival: 6 months versus 6 months, respectively; p=0.24). Cox multivariate analysis confirmed that the only prognostic factor significantly related to the overall survival was clinical nodal status (O.R.=2.7; 95% C.I. 1.09-6.9), while p53 status only approached the statistical significance (O.R.=2.5; 95% C.I. 0.96-6.5; p=0.06).

Mutations spanning P53 exons 5-9 detected by non-isotopic RNAse cleavage assay and protein expression in human colon cancer.

The non-isotopic assay (NIRCA), based on the observation that RNAse is able to specifically cleave a single mismatch in RNA/RNA duplexes, has been recently proposed to detect p53 mutations. To verify the use of this method as a valid screening for P53 mutations in a routinely collected cancer series, we used this assay on 3 cases with normal and 5 cases with abnormal P53 expression detected by Western blots. In all cases, P53 exons 5-6, 7 and 8-9 regions were analyzed. There were mutations only in the five overexpressed cases: two cases showed mutations in exon 5, one between intron 6 and exon 6 and two in the region spanning exons 8 and 9. Our experience showed NIRCA to be fast, reliable and providing the ability to study long target regions in a single step, thus making this assay useful for genetic screenings.

The expression pattern of the AML1 gene in non-Hodgkin's B-cell lymphomas and normal B lymphocytes.

We have studied the expression of the three human acute myeloid leukemia (AML) genes in primary samples of non-Hodgkin's B-cell lymphomas in which translocations involving these loci were not present. We found a widespread expression of the three AML genes in all the lymphoma samples as well as in the purified normal B-lymphocytes. Thus, the presence of the three mRNAs "per se" does not allow the identification of the pathological status. However, AML1 showed a different transcription pattern in the neoplastic tissues with respect to the normal B-cells. The AML1b isoform proved to be peculiar to this lymphoma. Our data support the idea that qualitative and quantitative alterations of AML1 gene expression deriving from deregulating mechanisms other than translocations may be involved in this malignancy. The usage of two differently regulated promoters driving the expression of the transcripts AML1b and AML1c may be one of these mechanisms. Finally, we report the presence of a new alternatively spliced transcript in normal B-cells.

GnRH receptors in human breast cancer and its contiguous not-involved breast tissue.

The present study was undertaken to evaluate the presence of GnRH receptors (GnRH-R) in breast cancer and not-involved breast tissue, and the relationships between GnRH-R and receptors for estrogen (ER) and progesterone (PgR) in the same tissues. Utilizing a tritiated natural GnRH in order to assay the native receptor binding we analyzed the level of binding sites for GnRH in membranes derived from 90 breast tumors and in 40 cases from neighboring, not-involved breast tissue. GnRH-R was found both in cancer and normal tissues. The prevalence for GnRH-R was higher in tumor than in not-tumor tissue (45% vs 39%, respectively), but the overall levels were not significantly different (15.9+/-24 fmol/mg protein vs 18.2+/-39 fmol/mg protein, respectively). The only statistically different content of GnRH-R we found concerned PgR negative vs PgR positive tumor tissues (mean content: 23 vs 11 fmol/mg protein, respectively in PgR- and PgR+ tumors, p=0.03 by t test); furthermore the proportion of GnRH-R positive cases in the tumor resulted significantly higher in premenopausal patients vs postmenopausal (56% vs 32%, by Chi square test, p<0.05). The GnRH receptors status of primary tumor and contiguous not-involved breast tissue resulted associated (overall agreement: 63%, p<0.05) but no specific steroid patterns for GnRH-R positivity was observed.

CEOP-B/VIMB vs. promace-CytaBOM in the treatment of intermediate or high grade non-Hodgkin's lymphoma: A randomised multicenter study of Southern Italy Cooperative Group.

From January 1992 to December 1995, 129 patients with previously untreated non-Hodgkin's lymphoma were randomised in a phase III multicenter trial to receive CEOP-B/VIMB or ProMACE-CytaBOM. Eligibility criteria included intermediate or high grade lymphoma (follicular large cell, diffuse small cleaved-cell, diffuse mixed, diffuse large-cell and immunoblastic) with an Ann Arbor stage II bulky, III or IV. All patients entered into the study were considered evaluable according to intent to treat analysis. At a median follow-up of 60 months there were no significant differences between the treatment response rates [82% (60%CR) for CEOP-B/VIMB vs. 81% (69% CR) for ProMACE-CytaBOM]. Conversely, with regard to disease-free survival, a significant difference was observed between the two treatment arms (42% for CEOP-B/VIMB vs. 24% for ProMACE-CytaBOM at 5 years; p=0.046). However, this difference did not translate in a significant difference in overall survival (45% vs. 39% at 5 years). Moreover, when response rates and outcome were analysed for different prognostic subgroups according to International Prognostic Index, no significant differences were observed between the treatment groups. It is important to note that neither regimen was able to improve outcome of poor risk patients who fared badly with both treatments (median survival 9 and 8 months respectively). Toxicity was also similar in both treatments with grade 3-4 leukopenia observed in 39% and 47% of cases and grade 3-4 thrombocytopenia in 24% and 27% of cases respectively. In conclusion, in this study CEOP-B/VIMB was not superior to ProMACE-CytaBOM in aggressive lymphomas and the alternating strategy failed to improve outcome of poor risk patients in which newer more aggressive treatments are needed.

Metastatic site and p53 primary tumor expression in previously untreated stage IV breast cancer patients.

p53 mutations have been reported to correlate with prognosis and response to therapy in patients with different tumor types. However, although p53 status is related to the primary tumor aggressiveness, an association between its expression and specific metastatic pattern has not yet been investigated. We immunohistochemically analyzed p53 (Pab1801) and ki67 (mib1) primary tumor expression in a series of advanced breast cancer patients presenting a selected pattern of distant metastases at the time of first diagnosis. Forty-eight percent of the overall series was classified as p53 positive while 22% as mib1 positive tumors. The overall agreement between p53 and mib1 expression was statistically significant (p = 0.03). While mib1 primary tumor expression did not show any association with the type of metastasis, p53 positivity was significantly higher in patients with soft tissue metastasis than in patients with bone or viscera metastasis (p = 0.002). No association with the probability of clinical response or different overall survival was found for patients with different p53 or mib1 status either in the overall series of patients or in subgroups of cases with different sites of distant metastasis.

DNA flow cytometry, p53 levels and proliferative cell nuclear antigen in human colon dysplastic, precancerous and cancerous tissues.

BACKGROUND: The cancerogenic process of colorectal cancer depends on a series of events involving oncogenes and inactivation of suppressor genes. This study concerns changes in DNA content, p53 and PCNA expression in human colon in dysplastic, precancerous and cancerous tissues. MATERIALS AND METHODS: These characteristics were analyzed in a series of hyperplastic polyps (HP), adenomas (AD), adenocarcinomas evolved within adenomas (AC-AD) and adenocarcinomas (AC) of the large bowel. DNA ploidy was analyzed by flow cytometry and PCNA and p53 expression was evaluated by immunohistochemistry using monoclonal antibodies PC10 and PAb 1801. RESULTS: Aneuploidy was found in 43/67 (64%) of AC and only occasionally in other subgroups (AC vs all other groups: 64% vs 99%; p = 0.00002). PCNA positivity gradually increased in the sequence from HP to AC and were significantly higher in AC compared to HP (90% vs 44%; p = 0.00007). p53 positive cells were found in 67% of AC while only occasionally in other groups (HP vs AC: p = 0.0002, AD (low dysplasia) vs AC: p = 0.001; AD (moderate dysplasia) vs AC: p = 0.001). CONCLUSIONS: These results demonstrated a progressive immunoreactivity for PCNA in the HP to AC sequence, while p53 positivity and aneuploidy seemed specific for colon carcinoma.

Complete response to chemotherapy in intra-abdominal desmoplastic small round cell carcinoma. A case report.

AIMS AND BACKGROUND: The authors report the case of a 23-year-old woman affected by intra-abdominal desmoplastic small round cell tumor (DSRCT) who obtained a complete response to multiagent chemotherapy. DSRCT is a rare, highly aggressive neoplasm generally arising in young people and seldom in females (about 20 cases described in the literature). METHODS: The patient underwent surgical resection of a large 15 x 15 cm mass located in the right lower abdominal quadrant, but after only 2 months later, two liver metastasis were noted. Thus, she was subjected to an aggressive antineoplastic treatment consisting of three groups of alternating non-cross resistant multiagent regimens administered every 21 days (cis-platin-etoposide-adriamycin-bleomicin; gemcitabine-ifosfamide-dacarbazine; methotrexate-5-fluorouracil-folinic acid) for a total of 9 administrations. RESULTS: After one cycle of treatment including the administration of all the three alternated schemes of chemotherapy, a complete disappearance of liver disease was noted. The treatment was relatively well-tolerated and the toxicity was acceptable. At present, after 15 months from diagnosis and 12 months after starting chemotherapy, the patient is disease-free and in good health. CONCLUSIONS: Even though this study regards only a single patient, it is noteworthy because of the rarity of this neoplasm and because of the infrequent complete responses reported in the literature. The efficacy and manageability of the treatment, suggests that both the timing and schedule used could constitute an important therapeutical option for this aggressive and poorly chemo-responsive tumor.

Role of prognostic factors in the therapeutic strategy of primary gastric non Hodgkin's lymphomas.

PURPOSE: To evaluate retrospectively in 64 gastric non-Hodgkin lymphoma (G-NHL) patients the role of some prognostic factors in the therapeutic strategy of this disease. PATIENTS AND METHODS: Sixty-four primary G-NHL patients (39 males and 25 females; median age: 57 years) were retrospectively evaluated. Treatment consisted of surgery alone (S) in 7 patients, chemotherapy alone (CT) in 15, CT + radiotherapy (RT) in 2, S + RT in 2, S + CT in 19, S + CT + RT in 16. Three patients had no treatment. Forty-four patients received sub-total gastrectomy (21) or total gastrectomy (23), and 20 were not submitted to surgery. RESULTS: After a median follow-up of 106 months (range 48-201), the four-year disease free survival (DFS) was 56% and overall survival (OS) was 59%. In the univariate analysis, tumor invasion depth (p = 0.007), stage (IIE1 vs IIE2: p = 0.007; I-IIE1 vs IIE2-IV: p = 0.0000009) and treatment (in stage IE-IIE1: p = n.s.; in stage IIE2-IV: p = 0.002) were significantly different. In the multivariate Cox regression model, stage was the only significant variable negatively influencing survival. CONCLUSIONS: Our study confirms the prognostic value of both the depth of invasion and the disease stage. In patients with early disease stages and disease localized to the gastric wall, a conservative approach can be recommended. No difference was found between the sub-total and total gastrectomy but surgery retains its fundamental role for G-NHL, even in advanced disease. Prospective trials are needed to confirm these results.

nm23 expression in human breast cancer: correlation with cell proliferation (S-phase) as observed with a double-labeling immunocytochemical-autoradiographic technique.

In 51 unselected breast cancer samples, a double-labeling immunocytochemical technique was utilized to observe the phenotypic expression of the nm23 gene during S-phase. The feasibility of the method was confirmed by comparison with routine evaluations for both thymidine-labeling index and nm23 (p<0.001). No correlation was found between the two parameters in the overall series or when subgroups regarding menopausal status, nodal involvement, tumor size, hormone receptor content were considered. Tumors with a higher nuclear grade showed a significant correlation only with TLI (p=0. 02). A trend for an inverse relationship between the two parameters was noted but was not statistically significant. A significant association between TLI and nm23 expression was found by Chi-square test using median values as cut-off. Our data based on morphological and in situ observations do not confirm a correlation between nm23 and cell proliferation, even if this correlation cannot be completely excluded. Multicentric trials of high power with these same techniques are still necessary to definitely establish the prognostic role of nm23 in breast cancer.

Neoadjuvant chemotherapy with accelerated CNF plus G-CSF in patients with breast cancer tumors larger than three centimeters: a pilot study.

From February 1992 to November 1993, forty patients with operable breast cancer tumors larger than three centimeters were enrolled in this study of accelerated neo-adjuvant chemotherapy. Thirty-seven patients are evaluable: one patient was excluded from the protocol and two refused to continue treatment after the first cycle. Chemotherapy consisted of three presurgical cycles of CNF [cyclophosphamide at 600 mg/m2, mitoxantrone (Novantrone) at 10 mg/m2 and 5-fluorouracil at 600 mg/m2] administered every 2 weeks, plus G-CSF (5 microg/kg s.c./day on days 7-12). Twenty-six of 37 patients (70%) achieved objective tumor response and were submitted to quadrantectomy. Toxicity was easily manageable. After a median 55-month follow-up (range 48-70), no locoregional recurrences were observed. Distant metastases occurred in 12/37 (32%) patients. The five-year disease-free (DFS) and overall (OS) survival were 58% and 80%, respectively. Accelerated CNF plus G-CSF proved to be a safe and tolerable regimen yielding a good clinical response thereby increasing the possibility of breast conservation surgery for patients otherwise candidates for mastectomy.

Polymerase chain reaction analysis of T-cell receptor gamma gene rearrangements in cutaneous B-cell lymphomas.

PCR analyses of T-cell receptor (TCR) gamma gene rearrangements in B-lymphoid neoplasms have shown lineage infidelity and double rearrangements involving both immunoglobulin heavy chain (igH) and TCRgamma genes. In order to investigate if this event is also a feature of cutaneous B-cell malignancies, we tested for clonal TCRgamma rearrangements a panel of immunophenotypically and genotypically well characterized cutaneous B cell lymphomas (CBCL). Fifteen samples of frozen CBCL biopsies were selected for the study. Diagnoses were established by routine histology and immunohistochemistry. Each of these cases displayed clonal igH gene rearrangement. Polymerase chain reaction (PCR) analysis of the TCRgamma rearrangements followed by high-resolution polyacrylamide gel electrophoresis was utilized for detection of clonal TCRgamma rearrangements. In our investigation, none of the cases of CBCL investigated by PCR showed the presence of clonal TCRgamma gene rearrangements. These data indicate that double rearrangements involving both igH and TCRgamma genes are not a feature of CBCL and confirm the B cell lineage specificity of this group of cutaneous lymphoid neoplasms.

Timing of breast cancer surgery within the menstrual cycle: tumor proliferative activity, receptor status and short-term clinical outcome.

We verified the variations of primary tumour steroid receptor status and proliferative activity at different times and phases (follicular vs luteal) of the menstrual cycle and their relationship with short clinical outcome in a cohort of 248 N- breast cancer patients. Steroid receptor content (ER and PgR) was evaluated by DCC assay and proliferative activity by 3H-Thymidine autoradiographic assay (TLI). Median age was 44 years, 60% of tumors were T1, and cytohistological grade was G1-2 in 54% of cases. At surgery, 57% were in the luteal phase while 43% were in the follicular phase. No significant variations were found in mean TLI or ER and PgR characteristics of the primary tumors surgically treated in different periods of the menstrual cycle; however, the ER level resulted significantly higher in 4th with respect to the 3rd week of menstrual cycle, while PgR level was higher in PgR+ cases treated during the 3rd week. The number of relapses and disease-free survival curves after 36 months median follow-up did not differ significantly for patients treated in different periods of the menstrual cycle (12% and 9% of disease relapses in luteal and follicular phases; p=n.s.). We can conclude, therefore, that TLI, ER and PgR expressions could vary significantly during menstrual cycle only in certain specific tumor subgroups.

1H NMR ganglioside ceramide resonance region on the differential diagnosis of low and high malignancy of brain gliomas.

1. The high-resolution 1H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3-5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 3). The broad resonance was identified as ceramide, a sphingosine-fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3-5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal. 2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity. 3. The properties of tumor gangliosides include antigenic specificity and immunosuppressive activity and the ceramide, a sphingosine-fatty acid combination, noticeably influences the ganglioside immunosuppressive activity. 4. The observation of 1H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberant gangliosides and ceramide precursors on the grade of malignancy and invasiveness. 5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.

Comparison of formalin, ethanol, and Histochoice fixation on the PCR amplification from paraffin-embedded breast cancer tissue.

The polymerase chain reaction (PCR) has been successfully employed for the laboratory analyses of genetic and infectious disorders using DNA extracted from paraffin-embedded tissues. However, fixative type and fixation time influenced PCR reactions and in some circumstances amplification fragments could not be efficiently generated. In this study, we determined the effects of three commonly used fixatives including ethanol, formalin and Histochoice, on the PCR amplification of DNA from paraffin-embedded breast cancer tissue. The effect of fixatives and fixation times was measured by the ability of the extracted DNA to serve as a template for the amplification of 280 and 530 base pair DNA fragments. On amplifying DNA, positive reactions were uniformly seen in the ethanol specimens. The next best fixative was Histochoice with positive results almost constantly observed in the PCR reactions performed. Formalin fixation sometimes compromised DNA amplification. Our results are consistent with previous reports investigating the effect of ethanol and formalin fixation on DNA amplification by PCR. Moreover, this is the first study showing that paraffin-embedded tissues fixed with Histochoice can be efficiently used for PCR gene amplification.

Analysis of clonal antigen receptor gene rearrangements in T-cells involved with Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multifocal neoplasm of unknown origin. All forms of KS are composed of spindle-shaped cells with elongated nuclei and sheets of endothelial-like cells. The proliferation of spindle cells is accompanied by the presence of an inflammatory infiltrate composed predominantly of T-cells. It has been suggested that this infiltrate might consist of a virally stimulated clonal population of T-lymphocytes which can produce growth factors initiating and substaining the proliferation of spindle-shaped cells. In this study we analyzed for clonal T-cell receptor gama gene rearrangements the T-cell populations present in the cutaneous infiltrate of seven cases of classical Kaposi's sarcoma using a polymerase chain reaction-based approach. Our data demonstrate the lack of a significant clonal population of T-cells in the cutaneous infiltrates of KS. This finding is indicative of a reactive polyclonal response of T-cells to the spindle-shaped cells and supports the contention that spindle-shaped cells are pathogenetically the central cell type in the disease. Our data also indicate that the anti-KS T-cell response, being polyclonal in nature, does not result from clonal expansion of T-cells targeting tumor-associated antigenic peptides.

The effect of fixation type on DNA extracted from paraffin-embedded tissue for PCR studies in dermatopathology.

BACKGROUND: Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is widely used to detect viral genomes, clonal gene rearrangements and oncogene mutations in skin specimens. Fixation with embedding of skin tissue is a procedure that has a profound effect on its molecular arrangement. OBJECTIVE: The aim of this study was to determine the effect of different fixatives on the PCR amplification of DNA. METHODS: We fixed randomly chosen fresh pathologic skin specimens in formalin, ethanol and Histochoice for 24 and 72 h and then embedded the tissue in paraffin. DNA was extracted from the paraffin-embedded tissues and used as template for amplification, producing 530- and 760-bp fragments of the phosphoglycerokinase gene. RESULTS: Our results indicate that PCR can be performed with excellent results on ethanol- and Histochoice-fixed, paraffin-embedded skin tissue with a rate of success comparable to that using fresh tissues; formalin-fixed tissue gave slightly less satisfactory results. CONCLUSION: This investigation corroborates previous reports investigating the effect of ethanol and formalin fixation on DNA amplification by PCR. Moreover, this is the first study showing that DNA extracted from tissue fixed with Histochoice is suitable for PCR gene amplification.

Nuclear grade and DNA ploidy in stage IV breast cancer with only visceral metastases at initial diagnosis.

AIMS AND BACKGROUND: The presence of early metastases to distant sites in breast cancer patients is an infrequent event whose mechanisms are still not clear. The aim of this study was to evaluate the biologic and clinical role of DNA ploidy and cell nuclear grade of primary tumors in the metastatic process of a series of stage IV previously untreated breast cancer patients with only visceral metastases. METHODS: DNA flow cytometry analysis on paraffin-embedded material and cell nuclear grading of primary tumors was performed on a series of 50 breast cancer patients with only visceral metastases at the time of initial diagnosis. RESULTS: Aneuploidy was found in 28/46 (61%) of evaluable cases and was independent of site of involvement, clinical response, time of progression and overall survival of patients. Of the 46 cases evaluable for nuclear grade, 5 (11%), 16 (35%) and 25 (54%) were classified as G1 (well-differentiated) G2 and G3, respectively. Nuclear grade also was unrelated to response to therapy and overall survival, whereas time to progression was significantly longer in G1-2 than G3 tumors with the logrank test (P < 0.03) and multivariate analysis. CONCLUSIONS: Our results seem to stress the difficulty to individualize different prognostic subsets from a series of breast cancer patients with only visceral metastases at initial diagnosis according to DNA flow cytometry and nuclear grade.