Galgo: a bi-objective evolutionary meta-heuristic identifies robust transcriptomic classifiers associated with patient outcome across multiple cancer types.

MOTIVATION: Statistical and machine-learning analyses of tumor transcriptomic profiles offer a powerful resource to gain deeper understanding of tumor subtypes and disease prognosis. Currently, prognostic gene-expression signatures do not exist for all cancer types, and most developed to date have been optimized for individual tumor types. In Galgo, we implement a bi-objective optimization approach that prioritizes gene signature cohesiveness and patient survival in parallel, which provides greater power to identify tumor transcriptomic phenotypes strongly associated with patient survival. RESULTS: To compare the predictive power of the signatures obtained by Galgo with previously studied subtyping methods, we used a meta-analytic approach testing a total of 35 large population-based transcriptomic biobanks of four different cancer types. Galgo-generated colorectal and lung adenocarcinoma signatures were stronger predictors of patient survival compared to published molecular classification schemes. One Galgo-generated breast cancer signature outperformed PAM50, AIMS, SCMGENE and IntClust subtyping predictors. In high-grade serous ovarian cancer, Galgo signatures obtained similar predictive power to a consensus classification method. In all cases, Galgo subtypes reflected enrichment of gene sets related to the hallmarks of the disease, which highlights the biological relevance of the partitions found. AVAILABILITY AND IMPLEMENTATION: The open-source R package is available on www.github.com/harpomaxx/galgo. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Heat shock proteins (HSPs) based anti-cancer vaccines.

The importance of HSPs themselves in antigen presentation and cross-presentation remains controversial. Most studies agree that as part of their molecular chaperone function, HSPs can bind and present tumor associated antigens to professional antigen presenting cells through MHC class I and class II molecules, leading to the activation of anti-tumor CD8+ and CD4+ T cells. The regulation of the innate and adaptive immune responses by HSPs is still a matter of intense research. HSPs are seen as important anticancer vaccine adjuvants. They are used through different delivery systems: HSPs/antibodies, peptide/protein-HSP complexes, tumor antigen/HSP gene fusion, viral peptides/HSP complexes or gene fusion, viral proteins/bacterial HSP fusion. In preclinical models different administration routes, subcutaneous, intradermal, intramuscular or even peroral (under special conditions) can be used, and the animal toxicities are non-significant. The HSP-based vaccines can induce specific and non-specific cellular immune responses all of which are important to induce tumor rejection. In addition, the antibodies generated after vaccination are emerging as important protagonist in the antitumoral response. This response is significantly enhanced when the suppressive tumor microenvironment and the immune suppressing effector cells are blocked. Several clinical studies have been carried out and are ongoing, immunizing cancer patients with autologous tumor derived HSP-peptide complexes (HSPPCs). The most promising results have been observed in patients with melanoma and renal clear cell cancer without advanced disease. There are clinical trials with HSP-based anticancer vaccines other than with HSPPCs (including patients with non-Hodgkin lymphoma, high-grade transitional cell carcinoma of the bladder, high-grade cervical dysplasia, etc).

Heat shock factor 1 represses estrogen-dependent transcription through association with MTA1.

Heat shock factor 1 (HSF1), the transcriptional activator of the heat shock genes, is increasingly implicated in cancer. We have shown that HSF1 binds to the corepressor metastasis-associated protein 1 (MTA1) in vitro and in human breast carcinoma samples. HSF1-MTA1 complex formation was strongly induced by the transforming ligand heregulin and complexes incorporated a number of additional proteins including histone deacetylases (HDAC1 and 2) and Mi2alpha, all components of the NuRD corepressor complex. These complexes were induced to assemble on the chromatin of MCF7 breast carcinoma cells and associated with the promoters of estrogen-responsive genes. Such HSF1 complexes participate in repression of estrogen-dependent transcription in breast carcinoma cells treated with heregulin and this effect was inhibited by MTA1 knockdown. Repression of estrogen-dependent transcription may contribute to the role of HSF1 in cancer.

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer.

Using the clinically relevant 4T1-derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease-free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

Predictive value of apoptosis, proliferation, HER-2, and topoisomerase IIalpha for anthracycline chemotherapy in locally advanced breast cancer.

PURPOSE: Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase IIalpha (topo IIalpha) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation (Ki-67), apoptosis (TUNEL), and expression of HER-2 and topo IIalpha are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness. EXPERIMENTAL DESIGN: Thirty-three women with primary breast tumors > or =3 cm received either doxorubicin (75 mg/m(2)) or epirubicin (120 mg/m(2)) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle taken before treatment (D0), at 24-48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy. RESULTS: The overall response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate (4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIalpha at baseline (D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl (p = 0.06) while median Ki-67 decreased (p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo IIalpha had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03. CONCLUSIONS: In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIalphaa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIalpha levels declined in responsive tumors.

Helicobacter pylori infection affects the expression of PCNA, p53, c-erbB-2 and Bcl-2 in the human gastric mucosa.

OBJECTIVE: Helicobacter pylori infection has been related to gastric carcinogenesis. This association is based on epidemiological data, pathological changes observed in the gastric mucosa, and chemical products from bacteria that may induce damage of DNA. In the present study we examined gastric endoscopic biopsies from patients with chronic gastritis, with and without H. pylori infection, and surgical biopsies from gastric cancer patients to evaluate whether this bacteria may induce changes in the expression of molecular markers associated with carcinogenesis. PATIENTS AND METHODS: the study involved 57 biopsies from the antral region of the stomach of patients with chronic gastritis and gastric cancer that were analyzed by immunohistochemistry. Molecular markers examined were: PCNA (Proliferating Cell Nuclear Antigen), p53, c-erbB-2, Bcl-2, and p21 H-ras. RESULTS: PCNA content of epithelial cells was significantly higher in H. pylori infected biopsies. Treatment aimed to eradicate H. pylori decreased the level of PCNA-positive cells in the group of patients that became H. pylori-negative as well as in H. pylori-positive patients. Nuclear p53 expression (used here as a surrogate marker for p53 mutation/inactivation) and c-erbB-2 expression were observed only in the group of patients that remained with the bacteria after treatment. A higher bcl-2 expression in lymphoid cells was observed in H. pylori-positive biopsies, and treatment did not change the expression of this protein. No significant expression of p21 H-ras was observed in the studied biopsies. CONCLUSION: this study suggests that H. pylori is involved in the induction of molecular changes that might predispose human gastric mucosa cells to pre-neoplastic and neoplastic events.

La infección por Helicobacter pylori afecta la expresión de PCNA, p53, c-erbB-2 y Bcl-2 en la mucosa gástrica humana / Helicobacter pylori infection affects the expresion of PCNA, p53, c-erbB-2 and Bcl-2 in the human gastric mucosa

Objetivo: la infección por Helicobacter pylori ha sido relacionada con la carcinogénesis gástrica. Esta asociación se basa en datos epidemiológicos y en cambios patológicos observados en la mucosa gástrica y, además, en los productos químicos elaborados por esta bacteria que dañan al ADN. En el presente estudio hemos examinado biopsias endoscópicas de estómago de pacientes con gastritis crónica, con y sin infección por H. pylori, y biopsias quirúrgicas de pacientes con cáncer gástrico para evaluar si esta bacteria induce cambios en la expresión de ciertos marcadores moleculares asociados con la carcinogénesis. Pacientes y métodos: el estudio involucró el análisis inmunohistoquímico de 57 biopsias provenientes de la región antral del estómago de pacientes que padecían de gastritis crónica y cáncer gástrico. Los marcadores moleculares evaluados fueron: el antígeno nuclear de proliferación celular (o Proliferating Cell Nuclear Antigen - PCNA), p53, c-erbB-2, Bcl-2 y p21H-ras.Resultados: el contenido de PCNA en las células epiteliales fue significativamente más alto en las biopsias que provenían de las muestras de los pacientes infectados por el H. pylori. El tratamiento dirigido a erradicar al H. pylori disminuyó la cantidad de células PCNA positivas en el grupo de pacientes que se volvieron H. pylori-negativos así como en aquellos pacientes en donde permaneció la bacteria. La acumulación nuclear de p53 (una indicación de p53 mutado/inactivado) y la expresión de c-erbB-2 fueron observadas solamente en el grupo de pacientes en los cuales el H. pylori n o pudo ser erradicado después del tratamiento. Se observó una mayor expresión de Bcl-2 en las células linfoides de las biopsias provenientes de los pacientes H. pylori positivos, y el tratamiento no indujo cambios en la expresión de esta proteína. En todas las biopsias estudiadas no se observó una expresión significativa de p21H - r a s. Conclusión: este estudio sugiere que el H. pylori está involucrado en la inducción de cambios moleculares que podrían predisponer a las células de la mucosa gástrica a eventos pre-neoplásicos o neoplásicos (AU)

Effect of neonatal chronic stress on expression of Hsp70 and oestrogen receptor alpha in the rat oviduct during development and the oestrous cycle.

A chronic unpredictable stress model used to produce depressive disorders in adult rats was applied to neonatal rats to investigate whether this type of stress can induce changes in the expression of Hsp70 and oestrogen receptor alpha in the oviduct, as detected by immunohistochemistry. Rats stressed during neonatal development showed changes in the expression pattern of Hsp70. In neonatal control rats, Hsp70-positive cells observed in the isthmus did not show any changes. Moreover, rats exposed to this stress model that reached adulthood had higher expression of Hsp70 in the isthmus (P<0.01) but not in the ampulla during oestrus than did the control rats. In contrast, during dioestrus, no significant changes were noted in adult rats that were stressed during neonatal development or in rats that were stressed in adulthood. These findings indicate that the isthmus is very sensitive to stressful stimuli and that repeated pre-weaning stress can change the expression of heat shock proteins in early and adult life. These subtle changes of expression in the oviduct did not affect the fertility of the rats that reached adulthood or that were mated under unstressed conditions. However, the control animals stressed during adulthood showed a disruption of the oestrous cycle: this finding is not observed in rats stressed during neonatal development that show an attenuated oestrous cycle disruption induced by chronic stress in adulthood. Moreover, there was dissociation between the expression of oestrogen receptor alpha and Hsp70. The amount of oestrogen receptor alpha remained constant in the epithelium of the oviduct in the control and in the stressed rats. Expression of oestrogen receptor alpha was noted in the stroma of the oviduct without the concomitant expression of Hsp70. It is possible that in certain cells and tissues Hsp70 is not necessary for oestrogen receptor alpha to be functional or Hsp70 might be present at very low amounts but is sufficient for the receptor to function.

Immunohistochemical analysis of Ki-67, p21waf1/cip1 and apoptosis in marker lesions from patients with superficial bladder tumours treated with vinorelbine intravesical therapy in a preliminary phase I trial.

OBJECTIVE: To investigate Ki-67 and p21Waf1/Cip1 expression and apoptosis, before and after treatment, in tumour biopsies obtained from patients with superficial bladder cancer who underwent vinorelbine intravesical therapy. PATIENTS AND METHODS: Twenty patients with high-risk superficial bladder cancer (including one or more of the following parameters: tumour diameter > 3 cm, histological grade 3, or multicentric tumours) were treated 1-6 times (weekly) with intravesical vinorelbine (50 mg/mL) instillations. Transurethral tumour marker biopsies were obtained one week before the first instillation of the drug and one week after the last. The biopsies were immunostained for Ki-67 and p21Waf1/Cip1 with monoclonal antibodies, on tissue sections derived from paraffin-embedded samples obtained before and after vinorelbine treatments. In addition, apoptosis was determined using a terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labelling (TUNEL) technique. RESULTS: There were no significant differences in the cell proliferation marker Ki-67 in biopsies taken before or after treatment. However, p21Waf1/Cip1 showed significantly higher expression in biopsies obtained after vinorelbine treatment, with median (range) values of 40 (20-90)% before and 70 (50-80)% after (P < 0.001, paired nonparametric Wilcoxon test). The apoptotic index was significantly higher after vinorelbine therapy, with median (range) values of 0.89 (0.06-3.8)% before and 2.25 (0.17-18.7)% after treatment (P < 0.001, paired nonparametric Wilcoxon test). Despite the brief treatment and few patients there was a clinical response in nine patients, together with low toxicity in all. CONCLUSION: The intravesical treatment of tumours with vinorelbine affects p21Waf1/Cip1 expression without blocking cell proliferation, although increasing apoptosis. The preliminary results suggest that vinorelbine may be useful for treating superficial bladder tumours, and thus a phase II study is warranted.

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)

Marcadores moleculares en pacientes con cáncer de cuello uterino tratadas con quimioterapia neoadyuvante y radioterapia / Molecular markers in patients with cervical cancer treated with induction chemotherapy and radiation therapy

Objetivos: En este estudio prospectivo de determinaron las modificaciones en la expresión y el valor predictivo de p53, p21 wafi/sdII/cipi, PCNA, hMLH1, hMSH2, Bcl'2 y TUNEL en pacientes con cáncer de cervix localmente avanzado tratadas con quimioterapia de inducción y radioterapia. Pacientes y métodos: Se obtuvieron muestras de 24 pacientes (IB'bulky/IIIB, 95 por ciento carcinomas escamosos) antes de la quimioterapia y a los 30 días del tratamiento. Trece pacientes recibieron un esquema de drogas basado en cisplatino y como la respuesta a esta terapia no fue buena, a las otras 11 pacientes se les administró vinorelbine e ifosfamida. Luego de la quimioterapia todas las pacientes recibieron radioterapia. La expresión de los marcadores moleculares en las biopsias pre- y post quimioterapia se estudió por inmunohistoquímica y la apoptosis fue evaluada por la técnica del TUNEL mejorada recientemente. Para comparar los cambios en la expresión de los marcadores moleculares y para correlacionarlos con la evaluación clínica (media de seguimiento: 31 meses para las pacientes que recibieron cisplatino y 19 para las que recibieron vinorelbine e ifosfamida) se realizaron análisis estadísticos. Resultados y conclusiones: La quimioterapia de inducción no aumentó la sobrevida de las pacientes, el 50 por ciento tuvo enfermedad progresiva (EP) o falleció (F). La expresión de p21waf1/sdII/cip1, hMLF1, hMSH2, y Bcl-2 no mostró cambios significativos después de la quimioterapia y no correlacionó con la evaluación clínica. La expresión de p53 no se modificó luego de la quimioterapia, las pacientes con tumores p53 positivos mostraron una tendencia a tener una sobrevida menor. Las pacientes con EP o que fallecieron mostraron niveles altos de PCNA, a diferencia de aquellas que estuvieron libres de enfermedad (LE) o con enfermedad estable (EE) (50 por ciento versus 17 por ciento, respectivamente, p<0.004). La sobrevida de las pacientes con bajos índices de TUNEL (igual o menor al valor medio entre las biopsias pre y post-quimioterapia de 1.5) fue significativamente más corta que las pacientes que presentaron índices de TUNEL altos (p<0.009). Nuestros resultados muestran que la quimioterapia de inducción (los dos tratamientos aplicados en este estudio) no mejoró la sobrevida de pacientes con cáncer de cervix...

Marcadores moleculares en pacientes con cáncer de cuello uterino tratadas con quimioterapia neoadyuvante y radioterapia / Molecular markers in patients with cervical cancer treated with induction chemotherapy and radiation therapy

Objetivos: En este estudio prospectivo de determinaron las modificaciones en la expresión y el valor predictivo de p53, p21 wafi/sdII/cipi, PCNA, hMLH1, hMSH2, Bcl2 y TUNEL en pacientes con cáncer de cervix localmente avanzado tratadas con quimioterapia de inducción y radioterapia. Pacientes y métodos: Se obtuvieron muestras de 24 pacientes (IBbulky/IIIB, 95 por ciento carcinomas escamosos) antes de la quimioterapia y a los 30 días del tratamiento. Trece pacientes recibieron un esquema de drogas basado en cisplatino y como la respuesta a esta terapia no fue buena, a las otras 11 pacientes se les administró vinorelbine e ifosfamida. Luego de la quimioterapia todas las pacientes recibieron radioterapia. La expresión de los marcadores moleculares en las biopsias pre- y post quimioterapia se estudió por inmunohistoquímica y la apoptosis fue evaluada por la técnica del TUNEL mejorada recientemente. Para comparar los cambios en la expresión de los marcadores moleculares y para correlacionarlos con la evaluación clínica (media de seguimiento: 31 meses para las pacientes que recibieron cisplatino y 19 para las que recibieron vinorelbine e ifosfamida) se realizaron análisis estadísticos. Resultados y conclusiones: La quimioterapia de inducción no aumentó la sobrevida de las pacientes, el 50 por ciento tuvo enfermedad progresiva (EP) o falleció (F). La expresión de p21waf1/sdII/cip1, hMLF1, hMSH2, y Bcl-2 no mostró cambios significativos después de la quimioterapia y no correlacionó con la evaluación clínica. La expresión de p53 no se modificó luego de la quimioterapia, las pacientes con tumores p53 positivos mostraron una tendencia a tener una sobrevida menor. Las pacientes con EP o que fallecieron mostraron niveles altos de PCNA, a diferencia de aquellas que estuvieron libres de enfermedad (LE) o con enfermedad estable (EE) (50 por ciento versus 17 por ciento, respectivamente, p<0.004). La sobrevida de las pacientes con bajos índices de TUNEL (igual o menor al valor medio entre las biopsias pre y post-quimioterapia de 1.5) fue significativamente más corta que las pacientes que presentaron índices de TUNEL altos (p<0.009). Nuestros resultados muestran que la quimioterapia de inducción (los dos tratamientos aplicados en este estudio) no mejoró la sobrevida de pacientes con cáncer de cervix... (AU)

Differential anthracycline sensitivity in two related human colon carcinoma cell lines expressing similar levels of P-glycoprotein.

Chemosensitivity of the human colon carcinoma HCT-15 cell line to 4'-epidoxorubicin proved to be 100-fold higher than that of its variant HCT-15 EDR. Confocal scanning microscopy showed significant less drug accumulation in HCT-15 EDR. A 2-fold increase in hsp27 expression was found in HCT-15 EDR, with no alteration in hsp70. The expression of the drug exporter Pgp was similar in both cell lines, despite the lower drug accumulation shown by HCT-15 EDR in respect to HCT-15. Other molecules implicated in the acquisition of enhanced chemoresistance or a more active Pgp variant present in HCT-15 EDR, could explain the phenomenon.

Constitutive expression of heat shock proteins hsp25 and hsp70 in the rat oviduct during neonatal development, the oestrous cycle and early pregnancy.

Certain heat shock proteins are regulated by steroid hormones and are associated with oestrogen receptor function in reproductive tissues, indicating that these proteins have a role during implantation, decidualization and placentation. In the present study, the expression of hsp25, hsp70 and oestrogen receptor alpha were examined by immunohistochemistry in oviducts from rats during neonatal development, the oestrous cycle and during early pregnancy. Oestrogen receptor alpha was the first protein observed in the neonatal oviduct, and its expression preceded that of hsp70 and hsp25. Although these heat shock proteins have been associated with the oestrogen receptor, this study showed that during early development of the oviduct, the receptor protein was not associated with the concomitant expression of hsp25 and hsp70. However, these heat shock proteins were expressed when oviductal cells became differentiated. In the adult oviduct, hsp70 was more abundant than hsp25, moreover, there were no significant modifications in expression of hsp25 during the oestrous cycle. In contrast, the expression of hsp70 was significantly higher in epithelial cells during dioestrus, when the maximum amount of oestrogen receptor alpha was also observed. Therefore, the present study shows that hsp70, but not hsp25, is an oviductal protein modulated by the oestrous cycle and that it is a protein marker for specific phases of the oestrous cycle. In addition, hsp70 was more responsive to the hormonal changes in the infundibulum and ampullar regions of the oviduct. During early pregnancy, hsp25 expression was downregulated (unlike in the endometrium), whereas hsp70 was relatively abundant in the oviduct. hsp70 was observed in all functional segments of the oviduct during pregnancy, indicating that in the oviduct, this protein is modulated by oestrogens and progesterone and possibly by other pregnancy-related hormones.

Molecular markers for predicting response to tamoxifen in breast cancer patients.

Tamoxifen is one of the most effective treatments for breast cancer. Standard practice is to select patients who are likely to respond to this therapy through the evaluation of estrogen receptor (ER) and progesterone receptor (PR) in the primary tumor tissue. Over the past 25 yr that physicians have been using ER determination to guide tamoxifen use, numerous studies have demonstrated that this molecular marker is useful in predicting benefit from tamoxifen. ER has been analyzed for many years using ligand-binding assays. However, current practice involves the use of immunohistochemical-based assays to detect ERalpha Immunohistochemistry (IHC) has several advantages. For example, IHC evaluates tumor cell heterogeneity, can be used to study small samples, is less expensive, and allows direct correlation with multiple histopathological tumor features and other molecular markers. PR, an estrogen-responsive protein, can also be useful in predicting response to tamoxifen in specific clinical situations. In recent years, several other markers of tamoxifen response have been examined, including: pS2 (another estrogen-regulated protein), heat-shock proteins 27 and 70, bcl-2 protein, c-erbB-2 (HER-2/neu) oncoprotein, and mutated p53 tumor suppressor protein. In this article, we present an analysis of the data on these new molecular markers. Overall, from numerous studies, the data indicate that in addition to ERalpha bcl-2 is a potential candidate to help further improve our ability to predict response to tamoxifen. ER and bcl-2 are the most useful molecular markers to better identify breast cancer patients who will respond to tamoxifen and who will have prolonged survival.

Improved detection of apoptotic cells using a modified in situ TUNEL technique.

We are in the process of assessing the response of cancer tissues to chemotherapy, evaluating, among other points, the proportion of cancer cells undergoing apoptosis. However, the apoptotic index obtained with the original TUNEL technique was lower than that obtained by evaluation of apoptosis on H&E-stained sections. Here we describe a small modification of the TUNEL technique that significantly increases the sensitivity of the assay. In the nonmodified TUNEL technique, a digoxigenin-labeled probe is detected using a direct peroxidase-conjugated system, whereas here we report the advantage of using a streptavidin-biotin-immunoperoxidase system. This, in conjunction with pretreatment of tissue sections with proteinase K and microwave irradiation, improved the detection of apoptotic cells.

c-erbB-2 (HER-2/neu) protein and drug resistance in breast cancer patients treated with induction chemotherapy.

Expression of c-erbB-2 protein has been associated with poor prognosis and poor response to chemotherapy in breast cancer patients. In the present prospective study, we have analyzed whether c-erbB-2, p53 and P170 proteins may be determinants of tumor resistance in locally advanced breast cancer patients treated with induction chemotherapy. Biopsies (n = 60) were examined by immuno-histochemistry; in 62% of cases core or incisional biopsies were taken before drug administration, allowing comparison in paired biopsies of the cytological and molecular changes induced by treatment Sixty percent of the patients received relatively high doses of FAC or FEC (5-fluorouracil, doxorubicin or epirubicin and cyclophosphamide), and 40% received relatively high doses of doxorubicin or epirubicin alone. No significant changes were observed in the molecular markers studied following chemotherapy; in the few biopsies where changes appeared, the changes did not exhibit any significant or similar trend. For 30 of the patients who received FAC/FEC treatment, follow-up reached a median of 34 months. In these cases, neither the clinical (reduction in tumor size) nor the histological (evaluated after neoadjuvant chemotherapy) responses showed statistically significant differences between the patients who developed distant metastases and the disease-free patients. c-erbB-2 was over-expressed in 50% of patients who developed distant metastases vs. 7% of the disease-free patients. Disease free survival (DFS) curves between c-erbB-2-positive and c-erbB-2-negative patients were statistically significant. No correlation between p53 or P170 expression with DFS was found. Our results suggest that c-erbB-2 protein expression is associated with development of distant metastases in breast cancer patients treated with relatively high doses of anthracyclines in induction chemotherapy.

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy.

Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.