Benefit from anthracyclines in relation to biological profiles in early breast cancer.

There are no validated predictors of benefit from anthracyclines. We compared cyclophosphamide, methotrexate, 5-fluorouracil (CMF), and epirubicin in different sequences with CMF alone in a phase III trial on operable breast cancers. Outcomes were analyzed in relation to tumor biological profiles to identify potential predictors of the efficacy of different treatments/drug combinations. Patients with N- or 1-3N+ tumors, were randomized to receive (a) epirubicin (4 cycles) followed by CMF (4 cycles); (b) CMF (4 cycles) followed by epirubicin (4 cycles), or (c) CMF (6 cycles) alone. Immunohistochemical assessments of estrogen (ER) and progesterone (PgR) receptors, HER2 and Ki67 were available for 705 patients (arm A/B/C: 276/269/160). Prognostic and predictive relevance was analyzed by log-rank tests and Cox models. Ki67 > 20 % and absent/low expression of ER and PgR were associated with worsen disease-free (DFS) and overall survival (OS). In patients with triple negative tumors (ER-, PgR-, HER2-), epirubicin-containing regimens yielded better DFS (HR 0.33, 95 % CI 0.17-0.62, P = 0.0007) and OS (HR 0.24, 95 % CI 0.10-0.57, P = 0.001) compared with CMF alone, whereas no differences were found in patients with HER2-positive (HER2+, ER-, PgR-) subtype. Treatment by subtype interaction (HER2-positive vs. others) was significant for DFS (χ (2) = 6.72, P = 0.009). In triple unfavorable (ER-, PgR-, Ki67 > 20 %) tumors, the use of epirubicin yielded better DFS (HR 0.45,95 % CI 0.26-0.78, P = 0.005) and OS (HR 0.30, 95 % CI 0.15-0.63, P = 0.001). Epirubicin-containing regimens seem to be superior to CMF alone in patients with highly proliferating, triple negative or triple unfavorable tumors.

Genetic heterogeneity by comparative genomic hybridization in BRCAx breast cancers.

The chromosomal changes in eight familial BRCAx breast cancers (i.e., negative for BRCA1 or BRCA2) were analyzed by comparative genomic hybridization (CGH) to investigate intratumor heterogeneity. This was the first step in a study of most frequent chromosomal aberrations in BRCAx familial breast cancers. Laser microdissection analysis of paraffin tissue samples was followed by whole-genome amplification. CGH was performed on DNA isolated from two to three different cell groups per case to detect any cytogenetic aberrations in important clones that might have been missed when analyzing DNA extracted from large numbers of cells. The results were compared, to evaluate the influence of tumor heterogeneity on CGH, and the heterogeneity was confirmed comparing CGH with fluorescence in situ hybridization results. Different chromosomal aberrations were detected between adjacent clones within the same section, which highlights the utility of microdissection in addressing the problem of heterogeneity in whole-genome studies. Some chromosomal regions were more frequently altered in the eight BRCAx tumors; loss of 2q, 3p, 3q, 8p, 9p, and 15q and gains of 1p, 4p, 4q, 5p, 6q, 12q, and 19p were the most common. Further studies focusing on specific genes and sequences with more sensitive approaches, such as array-CGH, are warranted to confirm these findings.

RhoA protein expression in primary breast cancers and matched lymphocytes is associated with progression of the disease.

RhoA protein is over-expressed in breast cancer and other solid tumors and has been used in tumor biopsies as a quantitative tumor marker for progression, stage and prognosis in molecular detection strategies. Measuring protein markers in plasma or blood cells is preferred to tumor biopsies as it represents a minimally invasive, repeatable measurement that can be followed over time. In this study we evaluated the hypothesis that quantitative RhoA protein expression in circulatory lymphocytes is identically associated with the same tumor clinico-pathological features found in biopsies. RhoA protein levels were analyzed by Western blotting in circulating lymphocytes isolated from 52 consecutive patients with breast cancer and in 34 paired breast tumor biopsies from the same case study, and compared with the following clinico-pathological features of the patients: histological grade, tumor size, steroid receptor status, lymphonode status, proliferative activity and prognosis [Nottingham Prognostic Index (NPI)]. We observed that the level of circulatory, peripheral lymphocyte RhoA expression reflected that found in the matched biopsy of the same patient. Furthermore, similarly to previous reports regarding breast cancer tissue biopsies, the level of RhoA protein expression in both biopsies and in circulatory lymphocytes was positively associated with tumor size, grade, proliferative activity of the tumor biopsy and NPI, while there was no significant association of RhoA protein expression with either estrogen- or progesterone-receptor expression. Our study demonstrated that the association of lymphocyte RhoA protein expression with classical clinico-pathological parameters closely corresponded with that observed for RhoA protein expression in the tumor biopsies. We propose that measurement of RhoA expression in the circulatory lymphocytes of breast cancer patients can be used to predict breast cancer occurrence, progression and prognosis and may prove valuable in the management of cancer patients.

FISH testing of HER2 immunohistochemistry 1+ invasive breast cancer with unfavorable characteristics.

Diagnostic assays for human epidermal growth factor receptor 2 (HER2) expression have a high predictive value because patients with HER2-positive tumors could benefit from HER2-targeted therapy. The aim of the present study is to analyze the incidence of HER2 gene amplification in selected tumors with adverse features that scored 1+ by immunohistochemistry (IHC). For that purpose, 331 consecutive invasive breast cancers (IBCs) were tested by IHC for HER2 expression between January and December 2013, 102 of which (31%) scored 1+. Of these 102 women with IBC who underwent surgery, 75 entered the study (73.5%). A total of 48 out of 75 (64%) IBC samples (patients' median age, 60.75 years) were selected according to ≥1 unfavorable tumor characteristics, and tested by fluorescence in situ hybridization (FISH). Of these 48 IBC samples scoring 1+ by IHC, 22 (46%) exhibited high histological grade (G3), 23 (48%) had a high proliferative index (Ki-67, >30%), 27 (56%) showed vascular invasion and 32 out of 41 evaluable cases (78%) were node-positive. Regarding hormone receptor expression, 3 (6%) and 10 (21%) cases were negative for estrogen and progesterone receptors expression, respectively. FISH was performed on 48 IBC cases scoring 1+ by IHC, and 7 infiltrating ductal carcinomas (IDCs) (14.6%) demonstrated HER2 amplification with a high proliferative index. In 42 IDC samples, statistical analysis evidenced a significant association between histological grade and high proliferative index (P=0.0200). In addition, in 48 HER2 scoring 1+ IBCs, Fisher's exact test evidenced a significant association between the presence of gene amplification and high proliferative index (P=0.0033). Based on these biopathological parameters, particularly a high proliferative index, the present results indicate that it is possible to of identify tumors scoring 1+ by IHC with HER2 amplification by FISH, thus aiding the selection of patients who are suitable for HER2-targeted therapy according to an acceptable cost/benefit ratio.

Metachronous primary uterine cancer surgically resected during Crizotinib treatment in a ALK-rearranged advanced lung adenocarcinoma.

Rearrangements of the anaplastic lymphoma kinase (ALK) gene are present in 3% to 7% of non-small-cell lung cancers (NSCLCs). Patients harboring ALK rearrangements show very favourable outcomes if treated with targeted agents, among which crizotinib is the first and best studied. Crizotinib, an oral small-molecule tyrosine kinase inhibitor of ALK, MET, and ROS1 kinases, is a very active and well tolerated drug. Nevertheless, the optimal therapy management with this new drug is still partially unknown, especially with regard to the safety of combined treatments. Recently, the integration of locoregional treatments has been proposed as a feasible multimodality strategy in selected patients with good clinical conditions and slow-growing or oligoprogressive disease. In this report, a case of advanced lung adenocarcinoma, progressed after first line chemotherapy and re-biopsied detecting ALK rearrangement, is described. During crizotinib treatment the primary lung tumor showed an excellent regression; meanwhile a major surgery for a metachronous uterine cancer was safely and successfully carried out.

P16(INK4a) protein expression in endocervical, endometrial and metastatic adenocarcinomas of extra-uterine origin: diagnostic and clinical considerations.

Determining the primary site of uterine adenocarcinoma (ADC) may be problematic, especially with small specimens. This is particularly important in light of the increase of endocervical and endometrial adenocarcinoma and the decrease in incidence of squamous cell carcinoma. P16(INK4a) , a member of the INK4 family of cell cycle regulatory proteins, plays a critical role. It functions as a negative regulator of cell cycle progression and differentiation by controlling the activity of the tumor-suppressor protein retinoblastoma (pRb), which regulates the cell cycle. Its expression is variable according to the tumoral histotype and in metastasis. The aim of this study was to investigate P16(INK4a) expression in endocervical, endometrial, and metastatic ADCs of extra-uterine origin. Fifty gynaecological biopsies (cervix or endometrium) comprised the study for P16(INK4a) determination. Cases were classified as (1) diffuse positive (P), in intense nuclear immunostaining and/or cytoplasmic in > 30% of neoplastic cells; (2) focal positive (FP), in intense immunostaining in 10% to 30% in isolated cells or small groups; and (3) negative (N), in absence of immunostaining or weak, sporadic immunostaining in < 10% of neoplastic cells. Included in the study were the following: 6 endocervical ADCs, 11 endometrioid-type endometrial ADCs, 5 endometrial serous papillary ADCs, 7 ovarian ADCs, 4 large intestine ADCs, 1 breast ADC, 12 not-otherwise-specified (NOS) ADCs, and 4 endocervical biopsy without atypia (as control). Diffuse, strong positivity with P16(INK4a) suggests an endocervical rather than an endometrial or metastatic ADC. In fact, a P16(INK4a) positive immunostaining pattern was prevalent in endocervical (83%) and serous papillary ADCs of endometrial or ovarian origin, whereas endometrioid ADCs such as metastatic non-ovarian lesions generally presented only focal or negative immunostaining. 10/12 cases of ADC-NOS were reclassified using P16(INK4a) immunostaining: 2 as endocervical ADCs (2 P), 4 as endometrioid-type endometrial ADCs (2 FP, 2 N), 3 as endometrial serous papillary ADCs (3 FP), and 1 as ovarian serous papillary ADC (1 FP).

Centromere 17 copy number alteration: negative prognostic factor in invasive breast cancer?

CONTEXT: Chromosome 17 polysomy has been identified in 5% to 50% of invasive breast cancers; even though a relationship with human epidermal growth factor receptor 2 (HER2/ neu ) status has been reported, other studies have shown that coincident centromere 17 (Cep17) amplification may be the cause of an overestimation of chromosome 17 polysomy in fluorescence in situ hybridization (FISH) testing. OBJECTIVE: To evaluate polysomy/amplification of Cep17 in invasive breast cancer with relation to proliferative activity (Ki-67), estrogen receptor, progesterone receptor, and HER2/ neu status, in an attempt to identify a subgroup of patients with a worse prognosis. DESIGN: A total of 647 cases of invasive ductal breast cancer were collected and subjected to FISH analysis for HER2/neu gene and centromere 17 alteration, HercepTest for HER2/ neu protein expression, and routine immunohistochemistry for Ki-67 and hormone receptor status. RESULTS: Copy number gain of Cep17 was observed in 27.3% of cases. Within this group, HER2/neu gene amplification was detected in 14.1% of cases, whereas HER2/ neu expression was scored 3+ in 20.1% of cases; about half of the HER2/ neu overexpressing cases (9.8%) did not show amplification by FISH. Moreover, 69% of polysomic cases showed high Ki-67 index. CONCLUSIONS: (1) Centromere 17-altered cases are frequently HER2/ neu overexpressing but not amplified, resulting in HercepTest/FISH disagreement; (2) HER2/neu amplification is seen at a higher incidence in cases without Cep17 copy number alterations, which are therefore not necessarily due to chromosome 17 disorder; (3) proliferation index is significantly higher in aneusomic tumors. These data suggest that the presence of Cep17 alterations could identify a subset of breast cancers with more aggressive biological and clinical behavior, which may show nonresponsiveness to conventional therapy independently of HER2/neu amplification status.