DNA aneuploidy identifies a subset of Luminal subtype breast carcinoma patients with worse clinical outcome.

AIM: In breast carcinoma (BC), the relationship between the ploidy pattern and molecular subtyping is still unknown. We aim to investigate the prognostic impact of DNA ploidy within molecular subtypes of a large cohort of BC patients. METHODS: The series involved 520 BC patients with no neoadjuvant therapy and a median follow-up of 115.5 months. Immunohistochemical assessment of hormonal receptors, ERBB2 (HER2) status and Ki67 proliferative activity was the basis of the surrogate molecular subtyping. Ploidy was evaluated by DNA flow cytometry in fresh/frozen tumour samples. Kaplan-Meier (K-M) survival estimation was used for prognostic statistical analysis. RESULTS: Luminal A subtype had the lowest prevalence of disease recurrences (23.6 %) and deaths from disease (18.3 %), while Luminal B (42.2 %/37.9 %), Triple-negative (46.4 %/40.6 %), and HER2-positive (55.9 %/50.0 %) subtypes had the highest. The Triple-positive subtype shows an intermediate/low frequency of adverse events (29.4 % of disease recurrences and 17.6 % of deaths from disease). Luminal A tumours were mostly diploid (55.3 %), while Triple-negative and HER2-positive tumours showed a high incidence of aneuploidy (82.6 % and 88.2 %, respectively). Luminal B and Triple-positive tumours had an intermediate percentage of aneuploidy (63.8 % and 70.6 %, respectively). K-M survival curves showed that DNA aneuploidy is significantly associated with shorter disease-free survival and overall survival in Luminal A and Luminal B molecular subtypes. CONCLUSION: DNA aneuploidy identifies a subset of Luminal BC patients with worse clinical outcome, potentially eligible for more aggressive adjuvant therapy.

S-phase fraction, lymph node status and disease staging as the main prognostic factors to differentiate between young and older patients with invasive breast carcinoma.

The influence of age on the outcome of patients with invasive breast carcinoma (IBC) has not yet been fully established. The present study investigated two subgroups of patients either side of the age spectrum, and evaluated cytometric, histopathological and molecular characteristics. The series involved 219 patients with IBC that had long-term follow-up, which were divided into two subgroups: Young (≤45 years; n=103) and old patients (≥75 years; n=116). Immunohistochemical evaluation of hormonal receptors, Ki67 index and HER2 status (plus HER2 silver in situ hybridization in equivocal cases) were used as the basis for surrogate molecular subtyping. Ploidy and S-phase fraction (SPF) were analysed by DNA flow cytometry. Differences between the subgroups' characteristics were assessed by the two proportion Z test. Kaplan-Meier estimation and log-rank test were applied for survival analyses. The median age in the subgroups were 40 years (range, 19-45 years) in the young group and 78 years (range, 75-91 years) in the older subgroup. Young patients exhibited higher lymph node involvement, more advanced disease staging, higher SPF tumour proliferative activity, and a trend of lower incidence of Luminal A and higher incidence of Luminal B tumours. The median SPF value was significantly higher in young patients [7.1% (range, 1.5-23.7%) vs. 4.5% (range, 0.7-26.4%)], whereas the ploidy pattern showed no significant difference. In the whole series, as within IBC of no special type, young patients had a higher rate of recurrence (46.6 vs. 22.4%; P<0.001) and deaths from disease (35.9 vs. 20.7%; P=0.030), with a statistically significant difference for disease-free survival. In conclusion, the present study indicated that young patients with IBC exhibited more aggressive disease, with an increased risk of recurrence and shorter disease-free survival. SPF, lymph node status and staging appeared to be the main prognostic factors to differentiate young from older patients with IBC.

Male Breast Cancer-Immunohistochemical Patterns and Clinical Relevance of FASN, ATF3, and Collagen IV.

BACKGROUND: Male breast carcinoma (male BC) is an uncommon neoplasia without individualized strategies for diagnosis and therapeutics. Low overall survival (OS) rates have been reported, mostly associated with patients' advanced stage and older age. Intratumoral heterogeneity versus homogeneity of malignant epithelial cells seems to be an important factor to consider for the development of combination therapies with curative intention. OBJECTIVE: In this preliminary study, we aim to provide valuable insight into the distinct clinicopathologic features of male BC. MATERIAL AND METHODS: In a series of 40 male BC patients, we evaluated by immunohistochemistry androgen receptor; activating transcription factor 3 (ATF3); p16; cyclin D1; fatty acid synthase (FASN); fatty acid transport protein 1 (FATP1); ß1, ß3, ß4, and ß6 integrins; collagen I and collagen IV; and their interactions. Kaplan-Meier survival curves and log-rank tests were assessed for statistical analysis. RESULTS: Homogeneous epithelial staining of p16, ATF3, ß6 integrin, FASN, and FATP1 was found to be significantly intercorrelated, and associated with high Ki67. These markers also stained tumor stromal fibroblasts. The prognostic analysis showed statistically significant associations of FASN with disease-free survival (DFS) and OS, as well as of ATF3 with OS and collagen IV with DFS. CONCLUSIONS: This study highlights, as a novel finding, the relevance of FASN, ATF3, and collagen IV immunophenotypes, which may have innovative application in the clinical management of male BC.

Male breast cancer: Specific biological characteristics and survival in a Portuguese cohort.

Male breast cancer (BC) represents an individual subtype of BC, with therapeutic procedures based on female BC therapy results. The present study evaluated the parameters currently used for the characterization and therapy of male BC, and their association with disease-free (DFS) and overall survival (OS), aiming to obtain a comprehensive basis to improve the personalized care of male BC. A total of 196 patients from March 1970 to March 2018 (mean follow-up, 84.9 months) were profiled, using clinicopathological review, molecular assessment [BRCA1/2, DNA repair associated (BRCA1/2) status, immunohistochemistry, fluorescence in situ hybridization and DNA flow cytometry] and Cox regression statistical analysis. The median age of patients was 66.5 years. At presentation, 39.2% of patients with invasive carcinomas were in anatomic stage (AS) I. Patients exhibited primarily invasive carcinomas of no special type, histological grade 2, estrogen receptor α-(ERα) and progesterone receptor (PR)-positive, receptor tyrosine kinase erbB-2-negative, high Ki-67, Luminal B-like and aneuploid tumors. A total of 13 of the 44 (29.5%) BRCA-evaluated patients exhibited BRCA2 mutations, significantly associated with family history (FH), bilaterality, high Ki-67 expression, absence of PR and Luminal B-like tumors. Bilaterality was associated with the occurrence of non-breast primary neoplasms (NBPN). The 5 and 10-year DFS rates, excluding patients with distant metastasis, NBPN and in situ carcinomas (n=145) were 65.9 and 58.2%, respectively, and the 5 and 10-year OS rates were 77.5 and 59.2%, respectively. In the univariate analysis, Luminal B-like subtype, BRCA2 mutations, high Ki-67 expression, and AS II and III were significantly associated with shorter DFS and OS. In addition, age >70 years was associated with low OS. In the multivariate analysis, FH, AS II and III, and Luminal B-like subtypes were associated with poorer OS. In conclusion, the data from the present study emphasize the high incidence of BRCA2 mutation in male BC, and its association with FH, bilaterality, high Ki-67 expression, negative PR expression and Luminal B-like subtypes, and with shorter DFS and OS in univariate analysis.

Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis.

Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.

Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas.

OBJECTIVE: The genes causing familial nonmedullary thyroid carcinoma (FNMTC) identified to date are only involved in a small fraction of the families. Recently, somatic mutations in TERT promoter region and in EIF1AX gene were reported in thyroid tumours of undefined familial status. The aim of this study was to investigate the role of TERT and EIF1AX mutations in familial thyroid tumours. DESIGN: The promoter region of TERT was sequenced in leucocyte DNA of the probands from 75 FNMTC families. In thyroid tumours from 54 familial cases, we assessed somatic TERT promoter, RAS and BRAF hotspot mutations, and the whole EIF1AX gene. RESULTS: No potentially pathogenic germline variants were identified in TERT in the 75 FNMTC families' probands. In the 54 carcinomas, we identified five cases (9%) with hotspot somatic TERT promoter mutations. BRAF mutations were found in 41% of the tumours. All TERT-positive samples were also positive for BRAF p.Val600Glu, and this co-occurrence was found to be statistically significant (P=.008). RAS mutations were detected in four tumours wild-type for TERT (7%). Evaluation of tumour mutation data together with the patients' clinicopathological features revealed a significant correlation between TERT plus BRAF mutations and advanced tumour stage (T4) (P=.020). No mutations were identified in EIF1AX. CONCLUSIONS: The results of this study suggest that TERT promoter and EIF1AX mutations are not frequently involved in FNMTC aetiology. However, we show for the first time that TERT alterations are associated with familial thyroid tumour progression. Our data also suggest that TERT mutations are more often found in concomitance with BRAF mutations in advanced stages of FNMTC.

Prognostic relevance of DNA flow cytometry in breast cancer revisited: The 25-year experience of the Portuguese Institute of Oncology of Lisbon.

The potential prognostic significance of DNA flow cytometric measurements (DNA ploidy and S-phase fraction) in breast cancer remains in dispute. Inconclusive data, primarily due to the lack of consistent standardization and quality control programs, have limited its translation into clinical practice. The aim of the present review, based on the 25-year experience of the Portuguese Institute of Oncology of Lisbon, is to assess the clinical relevance and application of DNA flow cytometry for the prognosis of breast cancer. Overall, data from Portuguese Institute of Oncology of Lisbon indicate that DNA flow cytometry provides significant prognostic information that is biologically relevant and clinically useful for the management of patients with breast cancer. Furthermore, this data has demonstrated the independent value of DNA aneuploidy as a prognostic indicator of poor clinical outcome in various subgroups of patients with early or locally advanced breast cancer at short- and long-term follow-up. Notably, aneuploidy identifies subsets of patients with grade (G)1 or G2 tumours who exhibit a poor clinical outcome. These patients may benefit from adjuvant chemotherapy, particularly those with luminal A and luminal B/human epidermal growth factor-2-negative endocrine-responsive breast cancer. In conclusion, data from Portuguese Institute of Oncology of Lisbon reinforces the clinical importance and utility of DNA flow cytometric analysis, particularly DNA ploidy, in the prognostic assessment and therapeutic planning for patients with breast cancer.

Aneuploidy identifies subsets of patients with poor clinical outcome in grade 1 and grade 2 breast cancer.

OBJECTIVE: Histological grade is a well-established prognostic/predictive factor in breast cancer. However, mainly within intermediate categories, patients may have unpredictable outcome. We hypothesised whether ploidy status can distinguish different prognostic groups among breast cancer patients with similar tumour grade. MATERIAL AND METHODS: The study involved 684 patients with invasive breast carcinoma, and median follow-up of 134.5 months. Pathological staging was evaluated according to WHO classification. Tumour differentiation was assessed using the Nottingham grading system. Ploidy was determined prospectively by DNA flow cytometry. Disease-free survival (DFS) and overall survival (OS) were estimated by the Kaplan-Meier method. RESULTS: There were 179 (26.2%) deaths and 239 (33.3%) disease recurrences. For grading, tumours were classified as follows: 163 (23.8%) G1, 356 (52.1%) G2 and 165 (24.1%) G3, while 389 (56.9%) tumours presented aneuploidy. Ploidy and grading are strongly associated (P < 0.001). Patients with aneuploid G2 tumours showed worse DFS (P = 0.001) and OS (P < 0.001), as well as those with aneuploid G1 tumours in relation to OS (P = 0.013). When a subset analysis was performed in early breast cancer patients (n = 451) with Stage I/IIA of disease, it remained the same significant associations of aneuploid G1 (to OS) and G2 tumours (to DFS and OS) with unfavourable prognosis. CONCLUSIONS: Aneuploidy identifies subsets of breast cancer patients with G1 and G2 tumours who showed poor clinical outcome. The finding has therapeutic implications, as these patients are potential candidates to risk-adapted adjuvant therapy.

Fibromatosis-like metaplastic carcinoma of the breast has a claudin-low immunohistochemical phenotype.

Fibromatosis-like metaplastic carcinoma (FLMCa) of the breast is a rare low-grade spindle cell carcinoma, of which the biological characteristics have not been well studied. This study aims to assess, in FLMCa, immunohistochemical expression of claudins (CLDN) and features connected with the claudin-low subtype, such as the presence of tumor initiating cells (TIC), epithelial-mesenchymal transition (EMT) phenotype, as well as EGFR activating mutations. Three cases of FLMCa were retrieved from our hospital archives. Histological and immunohistochemical characteristics were reviewed. Expression of CLDN-1, CLDN-3, CLDN-4 and CLDN-7, CD44 and CD24 (TIC phenotype), and vimentin and E-cadherin (EMT features) were studied. EGFR mutations on exons 18, 19, 20, and 21 were investigated by real-time PCR. In all cases, the low-grade spindle cell component was predominant, with two cases presenting <5 % of epithelioid and squamous areas. The tumors expressed basal cytokeratins and vimentin and were hormone receptor and ERBB2 negative. CLDN membrane expression was negative in the spindle cell component. The epithelioid areas were CLDN-1 positive. Nuclear/cytoplasmatic expression of CLDN-4 was observed in all components, except in one case in which it was strongly expressed in the non-spindle areas. All three cases were CD44+/CD24-. E-cadherin was focally expressed in epithelioid cells, only in the squamous areas. Activating EGFR mutations were not found. One patient developed local recurrences, metastases and died. FLMCa have the immunohistochemical profile of claudin-low breast tumors, with low expression of adhesion molecules, presence of TIC and EMT phenotype. No EGFR activating mutations were found.

Familial vs sporadic papillary thyroid carcinoma: a matched-case comparative study showing similar clinical/prognostic behaviour.

OBJECTIVE: Familial non-medullary thyroid cancer has been proposed as an aggressive clinical entity. Our aim in this study is to investigate potential distinguishing features as well as the biological and clinical aggressiveness of familial vs sporadic papillary thyroid carcinoma (PTC). We assessed clinicopathological characteristics, outcome measures and DNA ploidy. DESIGN: A matched-case comparative study. METHODS: A series of patients with familial PTC (n=107) and two subgroups, one with three or more affected elements (n=32) and another including index cases only (n=61), were compared with patients with sporadic PTC (n=107), matched by age, gender, pTNM disease extension and approximate follow-up duration. Histological variant, extrathyroidal extension, vascular invasion, tumour multifocality and bilateral growth were evaluated. Ploidy pattern was analysed in available samples by DNA flow cytometry. The probabilities of disease-free survival (DFS) and overall survival (OS) were estimated according to the Kaplan-Meier (K-M) method. RESULTS: No patient with familial PTC died of disease during follow-up (median, 72 months), contrarily to five patients (4.7%) (P=0.06) with sporadic PTC (median, 90 months). There was a significantly higher tumour multifocality in familial PTC (index cases subgroup) vs sporadic PTC (P=0.035), and a trend, in the familial PTC cohort with three or more affected elements, to show extrathyroidal extension (P=0.054) more frequently. No difference was observed in DNA ploidy status. The K-M analyses showed no significant differences between both entities in relation to DFS or OS. CONCLUSION: Apart from multifocality, familial PTC appears to have similar clinical/prognostic behaviour when compared with sporadic forms of the disease.

Clinical relevance of the reappraisal of negative hormone receptor expression in breast cancer.

BACKGROUND: Accurate assessment of estrogen (ER) and progesterone (PR) receptors is critical in predicting the response to endocrine therapies in breast cancer. MATERIAL AND METHODS: From a series of 360 patients with breast invasive carcinoma assessed for hormone receptors by immunohistochemistry (IHC) in the 90's, we re-analysed, on the same tumour material, the cases considered negative (n = 164), i.e., ER-/PR- (n = 95), ER+/PR- (n = 63) and ER-/PR+ (n=6), and 16 of 196 ER+/PR+ tumours with unfavourable outcome. Concordance between the previous IHC (Streptavidin-Biotin-Peroxidase) method and the current one (Peroxidase-Indirect-Polymer) was determined by the McNemar's test. Relapse-free (RFS) and overall survival (OS) were estimated by the Kaplan-Meier method. RESULTS: From 101 ER- and 158 PR- cases, 38 (37.6%) and 58 (36.7%) became positive, increasing ER and PR expression from 71.9% and 56.1% to 82.5% and 72.2%, respectively (P<0.001). All 16 ER+/PR+ cases maintained their co-positivity, while all ER-/PR+ tumours changed to ER positive. Kaplan-Meier survival curves showed significant differences related to RFS and OS for PR, either in the whole series or in the subset (n = 151) submitted to hormonal treatment. The patients' subgroup with ER+/PR- tumours exhibited the worst prognosis. CONCLUSION: The current IHC method improves the clinical usefulness of ER/PR assessment by decreasing the rate of false negative results.

DNA ploidy is an independent predictor of survival in breast invasive ductal carcinoma: a long-term multivariate analysis of 393 patients.

PURPOSE: To evaluate "classic" prognostic parameters, as well as DNA ploidy and S-phase fraction (SPF), in relation to disease-free (DFS) and disease-specific (DSS) survival in breast invasive ductal carcinoma (IDC) with long-term follow-up study. METHODS: The study involved 393 patients with IDC and median follow-up of 134 months (50-240). Histological grading, tumor size, axillary nodal involvement, pathological staging and hormone receptor status were considered as established prognostic markers. Ploidy and SPF were determined prospectively by DNA flow cytometry using fresh/frozen tissue. A Cox regression model was used for statistical analysis of the prognostic variables. RESULTS: There were 105 (26.7 %) deaths and 140 (35.6 %) disease recurrences during follow-up. Two hundred thirty-one (58.8 %) tumors were aneuploid. High SPF and aneuploidy were associated with tumors with higher grade of differentiation, greater size and negative hormone receptors. Higher SPF and advanced disease stage are correlated. In univariate analysis, all the clinicopathological and cytometric features, including patients <40 years and a subgroup presenting hypertetraploid/multiploid tumors, are significantly correlated with clinical outcome, apart from SPF and estrogen receptors for DFS. In multivariate analysis, nodal involvement, DNA aneuploidy and lack of progesterone receptors (for DSS) retained statistically significant association with shorter survival. In node-negative patients, ploidy (for DFS) and estrogen receptors (for DSS) significantly predicted survival. In both subgroups of node-positive patients and those (n = 195) with intermediate differentiation tumors (G2), aneuploidy was an indicator of worse prognosis. CONCLUSIONS: Along with nodal status and hormone receptor expression, DNA ploidy is an independent predictor of long-term survival in IDC.

S-phase fraction and ploidy as predictive markers in primary disease and recurrence of papillary thyroid carcinoma.

OBJECTIVE: To investigate the prognostic influence of DNA ploidy and S-phase fraction (SPF) on disease-free (DFS) and overall survival (OS) of patients with primary disease and loco-regional lymph node recurrence of papillary thyroid carcinoma (PTC). DESIGN: A large prospective study with long-term follow-up (median, 117 months). PATIENTS: Two series of patients with primary PTC (n = 305) and lymph node recurrence metastasis (LNM) (n = 39) were involved in the study. MEASUREMENTS: Patient's age and gender, histological variant, pathological tumour-node-metastasis (pTNM) staging, extrathyroidal extension, vascular and lymphatic invasion and tumour bilateral growth were the clinical and pathological characteristics evaluated. DNA flow cytometry was performed on fresh/frozen surgical tumour samples. Cox regression models were estimated for prognostic analyses. RESULTS: Seventeen (5·6%) primary tumours and five (12·8%) LNMs were aneuploid, while mean SPF was 2·7% and 3·7%, respectively (P = 0·022). High SPF was significantly associated with lymphatic invasion and tall cell and diffuse sclerosing variants. In univariate analysis, all the clinico-pathological variables, but tumour bilateral growth and gender, were significantly correlated with survival. SPF showed borderline significance (P = 0·051) in relation to OS. In multivariate analysis, older age (≥48 years), lymph node spread and high SPF were significantly adverse prognostic factors. Extrathyroidal extension and distant metastasis for OS, as well as tumour size for DFS, were also found as unfavourable prognostic features. In LNM, the Kaplan-Meier curves showed significant differences for older age and DNA aneuploidy (recurrence; P = 0·011). CONCLUSION: The results indicate that SPF and ploidy can provide additional predictive information in patients with PTC.

[DNA ploidy is an independent prognostic biomarker in breast invasive ductal carcinoma]. / A ploidia de ADN é um biomarcador prognóstico independente no carcinoma ductal invasivo da mama.

OBJECTIVE: To evaluate 'classic' prognostic parameters, as well as DNA ploidy and S-phase fraction, in relation to disease-free and overall survival in breast invasive ductal carcinoma with long-term follow-up. MATERIAL AND METHODS: The study involved 400 patients with breast invasive ductal carcinoma and median follow-up of 134 months (50-240). Histological grading, tumour size, axillary nodal involvement, pathological staging and hormone-receptor status were assessed as established prognostic markers. Ploidy and S-phase fraction were determined prospectively by DNA flow cytometry using fresh/frozen tissue. A Cox regression model was used for statistical analysis of the prognostic variables. RESULTS: There were 106 deaths (26.5%) and 141 disease recurrences (35.2%) during follow-up. Two hundred thirty-five (58.7%) tumours were aneuploid. High S-phase fraction and aneuploidy were associated with tumours with higher grade of differentiation, greater size and negative hormonal receptors. In univariate analysis, all the clinicopathological and cytometric features (including patients < 40 years and a subgroup presenting hipertetraploid/multiploid tumours), but S-phase fraction and estrogen receptors for disease free survival, significantly correlated with clinical outcome. In multivariate analysis, advanced disease stage, DNA aneuploidy and lack of progesterone receptors retained statistically significant association with shorter survival. In the subgroup of patients with intermediate differentiation tumours (G2), aneuploidy associated with worse prognosis. In the subset of node-negative patients, only estrogen receptors showed significant correlation with disease evolution. In node-positive patients, greater size tumours and aneuploidy (in relation to overall survival) were indicators of worse prognosis. CONCLUSION: Along with disease staging and hormone-receptor expression, DNA ploidy is an independent prognostic biomarker of long-term survival in breast invasive ductal carcinoma.

Ploidy and S-phase fraction as predictive markers of response to radiotherapy in cervical cancer.

The aim of this study was to investigate the potential clinical utility of DNA flow cytometry biomarkers, ploidy, and S-phase fraction (SPF) in predicting overall survival in cervical cancer. This prospective study involved 159 patients with cervical carcinoma (median follow-up, 48 months). Pretreatment clinical staging was done according to the FIGO 2009 update classification. Biopsy tumor samples were used for flow cytometry analysis and histological examination. A prognostic study was performed using both Cox and Bayesian Weibull regression models. Eighty (50.3%) tumors presented DNA aneuploidy, mostly observed in adenosquamous (AS) cell carcinoma (8 of 9 cases) and adenocarcinoma (AC) (12 of 17 cases). The median SPF value (8.6%) was used for discriminating low vs. high tumor cell proliferation. High SPF significantly correlated with aneuploidy (p<0.001). All AS carcinomas had SPF>15%, while all ACs presented SPF<10% (p<0.001). Forty-three (27%) patients died of the disease during follow-up. Log-rank tests revealed significant differences between survival curves for older patients (≥44 years) (p=0.029), advanced clinical staging (p<0.001), and DNA diploidy in stage IIB of disease (p=0.039). Both regression analyses showed that advanced clinical staging and low SPF independently predict worse overall survival of patients. The results suggest that DNA flow cytometry parameters can provide additional predictive information in cervical cancer management.

High prevalence of RAS mutations in RET-negative sporadic medullary thyroid carcinomas.

CONTEXT: Sporadic medullary thyroid carcinomas (MTC) frequently harbor mutations in the RET protooncogene. We have earlier reported a series of 51 sporadic MTC with 64.7% of RET-positive and 35.3% of RET-negative cases. OBJECTIVE: In the present study, we investigated the possible involvement of RAS and BRAF protooncogenes in the development of sporadic RET-negative MTC. PATIENTS AND DESIGN: We performed PCR amplification and sequencing analysis of the three mutational hotspots (codons 12, 13, and 61) of the H-, K-, and N-RAS genes, and of the mutational hotspot (codon 600) and exon 11 of the BRAF gene in 65 sporadic MTC, of which 40 were RET positive and 25 were RET negative. RESULTS: Somatic H-RAS and K-RAS mutations were detected in 14 of 25 (56.0%) and three of 25 (12.0%) of RET-negative sporadic MTC, respectively. On the other hand, only one of 40 (2.5%) RET-positive sporadic MTC had a RAS mutation, namely in H-RAS. One of the H-RAS mutations was novel (c.32_37dupCCGGCG). No mutations of N-RAS or BRAF were detected in all assessed tumor samples. CONCLUSIONS: Overall, our results showed that RAS mutations were present in 68.0% (17 of 25) of the RET-negative MTC and in only 2.5% of the RET-positive MTC (P < 0.0001), suggesting that activation of the protooncogenes RAS and RET represents alternative genetic events in sporadic MTC tumorigenesis.

BCL-6 oncoprotein in breast cancer: loss of expression in disease progression.

OBJECTIVE: To investigate the biological role of BCL-6 oncoprotein in breast cancer disease progression (recurrence and metastasis). METHODS: The series consisted of 93 consecutive female patients with primary breast cancer and median follow-up of 10 years. BCL-6 expression was assessed in vivo by immunohistochemistry and real-time PCR. Breast cancer cell lines and some metastasis-related genes (CXCR4, Itgbeta-3 and FLT-1) were also analysed by molecular techniques. Prognostic evaluation was performed by fitting a multivariate Cox regression model. RESULTS: BCL-6 immunoexpression was positive in 22 (23.7%) tumours and negative in 71 (76.3%). All axillary lymph node metastases of 47 node-positive patients were negative, including 12 cases showing BCL-6-positive primary tumours. Likewise, in 9 recurrence cases, BCL-6 expression was similar or decreased compared with primary tumours. No correlation between immunoexpression and gene expression of BCL-6 was observed. BCL-6 was significantly reduced both in derived metastases of a breast cancer cell line (M435) and when the latter was treated with a demethylation agent (5-azacytidine). However, BCL-6-transfected breast cancer cell lines expressed significantly higher levels of CXCR4, Itgbeta-3 and FLT-1. Co-expression of the 4 genes was found in 4 of 17 tumours evaluated, but lacking prognostic significance. BCL-6 oncoprotein revealed no significant influence on outcome. CONCLUSION: The results strongly suggest the loss of BCL-6 expression in breast cancer progression, which might be related with methylation status alterations of still unknown partner gene(s).

Clinical implications of molecular markers in follicular cell-derived thyroid cancer.

The increasing use/applications of molecular biology techniques have provided new insights on the genetic changes that underlie carcinogenesis and tumor progression in thyroid cancer. Molecular analysis may improve the histopathologic evaluation of follicular cell-derived thyroid carcinoma, not only elucidating some unresolved problems related to the diagnosis and disease prognosis, but also by improving patient management. Besides increasing our comprehension of cancer biology, either genetic alterations or gene expression profiles implicated in thyroid carcinogenesis shed new light on innovative diagnostic procedures as well as on targeted therapies.

Underexpression of PPARgamma is associated with aneuploidy and lower differentiation of thyroid tumours of follicular origin.

Peroxisome proliferator-activated receptor gamma (PPARgamma) gene is a nuclear receptor that is involved in thyroid tumourigenesis. Recently, our group has shown that follicular carcinomas underexpressing PPARgamma protein are more prone to develop distant metastases, to invade locally, to present poorly differentiated areas and to persist after surgery. Aneuploidy is also observed in some thyroid tumours, particularly in the more advanced cases. The aim of the present study was to investigate the association of PPARgamma expression with the degree of differentiation and ploidy status of benign and malignant thyroid neoplasias. DNA cytometric studies, ploidy and S-phase fraction (SPF) determination, and quantitative RT-PCR analysis of molecular markers specific for thyroid follicular cells, namely Tg (thyroglobulin), TSHR (TSH receptor) and NIS (Na+/I- symporter) were compared between thyroid lesions with positive or negative PPARgamma protein expression. We observed that PPARgamma-negative tissues expressed lower levels of Tg mRNA [4.66 x 10(6) a.u. (arbitrary units) +/- 1.49 x 10(6)], and were more frequently aneuploid (36%), and presented higher SPF (3.1%+/-0.4) than PPARgamma-positive samples (Tg mRNA = 2.54 x 10(7) a.u. +/- 9.72 x 10(6), P=0.0006; aneuploidy=8%, P=0.0031; SPF=2.2%+/-0.2, P=0.0430). A similar trend was also observed for TSHR and NIS mRNA, although not reaching statistical significance. This study showed that underexpression of PPARgamma is associated with poor tumour differentiation, aneuploidy and higher cell proliferative activity. Therapies designed to modulate expression of PPARgamma may have an impact on the growth of thyroid neoplasias.

Aneuploidy and high S-phase as biomarkers of poor clinical outcome in poorly differentiated and anaplastic thyroid carcinoma.

This study aimed to investigate the prognostic influence of DNA flow cytometry and RAS gene mutations in patients with poorly differentiated (PDTC) and anaplastic thyroid carcinoma (ATC). The series consisted of 26 patients with PDTC and ATC, and a median follow-up of 10 months (range 1-138). DNA ploidy and S-phase fraction (SPF) were assessed by flow cytometry on frozen samples. RAS point mutations were detected using PCR techniques. Disease staging and tumour angioinvasion were included as prognostic parameters for survival analysis. Nineteen patients (73.1%) succumbed to the disease (median time 5 months; range 1-45). Eighteen tumours (69.2%) were classified as DNA aneuploid. Median SPF was 5.6% (range 1.9-23.1), which was used as a cut-off value to distinguish between low versus high cell proliferation. Three of 20 (15%) patients presented N-RAS gene mutations in codon 61. DNA aneuploidy was most frequently found in female patients (p=0.034). Kaplan-Meier and Cox regression analyses showed that only DNA aneuploidy (p=0.044 and p=0.055, respectively) and high SPF (p=0.001 and p=0.006, respectively) significantly correlated with worse survival. The results indicate that aneuploidy and high SPF are biomarkers of poor clinical outcome in PDTC and ATC, which may provide useful prognostic information with a potentially therapeutic impact in patient management.