Prevention of chemotherapy toxicity by agents that neutralize or degrade cell-free chromatin.

BACKGROUND: Toxicity associated with chemotherapy is a major therapeutic challenge and is caused by chemotherapy-induced DNA damage and inflammation. We have recently reported that cell-free chromatin (cfCh) fragments released from dying cells can readily enter into healthy cells of the body to integrate into their genomes and induce DNA double-strand breaks, apoptosis and inflammation in them. We hypothesized that much of the toxicity of chemotherapy might be due to release of large quantities of cfCh from dying cells that could trigger an exaggerated DNA damage, apoptotic and inflammatory response in healthy cells over and above that caused by the drugs themselves. METHODS: We tested this hypothesis by administering cfCh neutralizing/degrading agents namely, anti-histone antibody complexed nanoparticles, DNase I and a novel DNA degrading agent-Resveratrol-Cu concurrently with five different chemotherapeutic agents to examine if chemotherapy-induced toxicity could be minimized. RESULTS: We observed (i) significant reduction in chemotherapy-induced surge of cfCh in blood; (ii) significant reduction in chemotherapy-induced surge of inflammatory cytokines CRP, IL-6, IFNγ and TNFα in blood; (iii) abolition of chemotherapy-induced tissue DNA damage (γH2AX), apoptosis (active caspase-3) and inflammation (NFκB and IL-6) in multiple organs and peripheral blood mononuclear cells; (iv) prevention of prolonged neutropenia following a single injection of adriamycin and (v) significant reduction in death following a lethal dose of adriamycin. CONCLUSION: Our results suggest that toxicity of chemotherapy is caused to a large extent by cfCh released from dying cells and can be prevented by concurrent treatment with cfCh neutralizing/degrading agents.

Sentinel node biopsy versus low axillary sampling in women with clinically node negative operable breast cancer.

BACKGROUND: Sentinel node biopsy (SNB) was initially conceived as excision of the first station axillary lymph node(s) (LN) identified by radioactive and/or blue dye uptake. The definition was subsequently enlarged to also include palpable lymph nodes in the vicinity of sentinel node(s) (SN). We reasoned that the excision of this combination of nodes might be best achieved by sampling the lower axilla. METHODS: Each patient underwent low axillary sampling (LAS) and identification of SN in the excised specimen followed by complete axillary lymph node dissection (ALND). LAS was defined as excision of all fibrofatty tissue overlying the second digitation of serratus anterior below the intercostobrachial nerve and was carried out following a pre-operative injection of radioactive colloid and an intra-operative injection of blue dye. Blue and/or hot nodes (B&/HN) in the dissected tissue and remaining axilla, along with any palpable nodes within the sampled tissue, were defined as SN. The primary endpoint of the study was to compare false negative rates (FNR) of SN with that of LAS in predicting axillary LN status (NCT00128362). FINDINGS: The study was performed between March 2004 and December 2011 in 478 women with clinically node negative axilla. On histopathological evaluation the median tumor size was 2.5 cm and axillary nodal metastases were found in 34.1% of patients. The FNR of SNB (12.7%, 95% CI 8.1-19.4) and LAS (10.5%, 95% CI 6.6-16.2) were not significantly different (p = 0.56). The FNR of B&/HN alone, without palpable nodes, (29.0%, 95% CI 22.5-36.6) was significantly inferior to those of SNB (p = 0.0007) and LAS (p = 0.0003). INTERPRETATION: LAS is as accurate as SNB in predicting axillary LN status in women with clinically node negative operable breast cancer. Confining SNB procedure to excision of B&/HN, significantly increases the risk of leaving behind metastatic lymph nodes in the axilla. LAS is an effective and low cost procedure that minimizes axillary surgery and can be implemented widely. Registry Name: Clinicaltrials.gov. REGISTRATION NUMBER: NCT00128362.

Validation of EORTC quality-of-life questionnaire in Indian women with operable breast cancer.

BACKGROUND: The European Organization for Research and Treatment of Cancer (EORTC) module QLQ-C30 and the breast cancer-specific module BR-23 have been validated worldwide to assess the quality of life (QOL) in women with breast cancer. No such study has been published on Indian women using EORTC questionnaires. METHODS: QOL was assessed in relation to surgery, adjuvant chemotherapy, radiation therapy and hormone therapy in 299 Indian women with operable breast cancer (OBC) at the Breast Unit of Tata Memorial Hospital (TMH), Mumbai, from October 1998 to September 2001. The QLQ-C30 module was used to assess physical health, emotional, cognitive and social functioning, and the BR-23 module to assess breast cancer treatment-related symptoms. Assessment was done at 3 visits: visit 1 (after surgery); visit 2 (during adjuvant therapy) and visit 3 (on completion of adjuvant therapy). RESULTS: Of the 299 women at first visit, 274 (91.6%) completed the visit 2 questionnaire and 239 (80%) completed the visit 3 questionnaire. Only those women who filled the questionnaires at all 3 visits were included as 'valid visits' for analysis (193 of 299; 64.5%). The reliability and validity of the English and translated versions of the questionnaires were tested by Cronbach alpha (0.61-0.96) and item-scale correlation (0.63-0.93). Women with breast conservation treatment had a superior body image as compared to those with mastectomy (p <0.001). Physical, emotional and cognitive functions were not related to the type of surgery. Global QOL, physical, sexual and role functioning were found to deteriorate with chemotherapy (p < or = 0.01). Radiotherapy had only local adverse effects (p < 0.001 ), while hormone therapy had no adverse impact on QOL. CONCLUSION: QLQ-C30 and BR-23 questionnaires can be used reliably to assess QOL in Indian patients. The translated versions were found to be valid for further use in clinical trials on Indian women with breast cancer.

Breast screening in the emerging world: high prevalence of breast cancer in Cairo.

The Cairo Breast Screening Trial (CBST) was designed to evaluate the role of clinical breast examination as a primary screening modality in the context of primary care, as in Egypt breast cancer is usually diagnosed at an advanced stage. A specialised medical centre in Cairo (the Italian Hospital) was selected as the headquarters of the study. The initial target group was women age 35-64 living in a geographically defined area around the Italian Hospital, 4116 being contacted by social workers and invited to attend a Primary Health Centre for clinical breast examination. High rates of breast cancer were observed; 8 per 1000 at the first examination and approximately 2 per thousand among those who attended for re-screening. The initial prevalence suggests that many women in the community with early but palpable breast cancer fail to seek medical attention until their cancer is advanced. The detection rate on re-screening, and after follow-up of those who only received one or no screens, ( approximately 3/1000) is similar to expectation.

Analysis of BRCA1 involvement in breast cancer in Indian women.

The involvement of the familial breast-ovarian cancer gene (BRCA1) in the molecular pathogenesis of breast cancer among Indian women is unknown. We have used a set of microsatellite polymorphisms to examine the frequency of allele loss at the BRCA1 region on chromosome 17q21, in a panel of 80 human breast tumours. Tumour and blood leukocyte/normal tissue DNA from a series of 80 patients with primary breast cancer was screened by PCR-amplified microsatellite length polymorphisms to detect deletions at three polymorphic BRCA1 loci. PCR-allelotype was valuable in examining allele losses from archival and small tumour samples. Loss of alleles at BRCA1 in the patient set, confirmed a noteworthy role of this gene in the molecular patho-genesis of breast cancer and was in accordance with its well-documented tumour suppressive function.

Timing of surgery during the menstrual cycle and prognosis of breast cancer.

There are conflicting reports on the differential effect of surgery performed during the two phases of the menstrual cycle, namely, follicular and luteal, and prognosis of operable breast cancer. A statistical meta-analysis of the published evidence suggests a modest survival benefit of 15+/-4% when the operation is performed during the luteal phase. Further research in this area might provide a novel avenue to understand the natural history of breast cancer. A spin off from these studies might be the understanding of the importance of events that occur at the time of surgery in determining long term prognosis.

Timing of surgery with regard to the menstrual cycle in women with primary breast cancer.

There is sufficient evidence to support both the hormonal influence on the outcome of breast cancer surgery and the SDA hypothesis. The SDA model produces a paradigm shift in the understanding of the natural history of breast cancer. It offers opportunities to try modifying a tumor's biological potential for metastasis (e.g., by tamoxifen, progesterone, antiprotease, or angiostatin) in the neoadjuvant setting. It continues to support the beneficial effects of detection and surgery early in the natural history of disease. It would be worthwhile to plan a trial comparing standard practice (unplanned surgery as the patient enrolls) with surgery during the luteal phase of the menstrual cycle in premenopausal women. Another possibility, based on studies of circulating progesterone, would be to compare primary progesterone treatment (for 4 to 10 days before surgery) with standard practice. Such a trial of primary progesterone is already under way, conducted by the Indian Breast Group. More than 200 patients have enrolled so far. The details of the trial are available from Clinical Research Secretariat, Tata Memorial Centre, Parel, Mumbai, India (e mail: tmho3@bom2.vsnl.in).

Oestrogen increases S-phase fraction and oestrogen and progesterone receptors in human cervical cancer in vivo.

Although cancer of the cervix is traditionally considered not to be responsive to steroid hormones, an in vitro study has reported that the addition of oestrogen increased cellular proliferation in a cervix cancer cell line that was inhibited by progesterone. We investigated whether the reported in vitro effects of oestrogen and progesterone on cellular proliferation can be replicated in locally advanced cervical cancer in vivo and whether these effects, if any, are related to oestrogen and progesterone receptor (ER and PgR) content of the tumour. One hundred post-menopausal patients with locally advanced cervical cancer were systematically allocated by rotation to the four treatment groups: (1) control group receiving no treatment; (2) ethinyl oestradiol 50 micrograms: (3) norethisterone 5 mg: (4) a combination of ethinyl oestradiol and norethisterone. Hormone treatment (five doses) was given orally every 12 h. Tissue biopsies were taken before and 12 h after the last hormone treatment. S-phase fraction (SpF) was measured by flow cytometry, and ER and PgR were measured by enzyme immunoassay. Results were analysed using two-factor analysis of variance, the factors being oestrogen-absent or present- and progesterone-absent or present. The main effects of oestrogen were increases in SpF, ER and PgR, which were statistically significant (P = 0.0056, 0.0009 and 0.01 respectively), indicating that there is much greater change in these three parameters in the presence of oestrogen (mean changes 7.808%, 6.258 fmol mg-1 and 12.716 fmol mg-1 for SpF, ER and PgR respectively) than in its absence (mean change -1.986%,-3.041 fmol mg-1 and 1.736 fmol mg-1 respectively). The progestogen main effect and the oestrogen-progestogen interaction were not significant. The rise in SpF, ER and PgR in the presence of oestrogen had a correlation coefficient with the initial ER values of -0.0565, -0.2863 and -0.1230 respectively, none being statistically significant, suggesting that the oestrogen actions were not strictly related to baseline ER concentrations. The combined median baseline ER and PgR values of the four groups were 1.48 fmol mg-1 and 0.80 fmol mg-1 respectively. Our results show that oestrogen is capable of increasing SpF in locally advanced cervical cancer in vivo and may help to revive interest in the use of oestrogen as a radiosensitizing agent in the treatment of this disease.

A comparative study of detection of p53 mutations in human breast cancer by flow cytometry, single-strand conformation polymorphism and genomic sequencing.

The accuracy of immunodetection by dual parameter flow cytometry (FCM), polymerase chain reaction-mediated single strand conformation polymorphism (PCR-SSCP) and genomic sequencing to detect p53 mutations were compared. Analysis by the last two techniques was restricted to exons 5-8. Initially, 110 breast tumours were screened for p53 expression by FCM. Seventy (64%) of tumours were immunopositive. Fifteen highly immunopositive and 15 completely immunonegative tumours were selected for further analysis by PCR-SSCP and genomic sequencing. Eleven out of 15 immunopositive tumours were found to have mutation by PCR-SSCP. Genomic sequencing confirmed the presence of mutation in 10 of these 11 immunopositive tumours. Therefore, four immunopositive tumours failed to show mutation by SSCP and five by genomic sequencing. Of the 15 immunonegative tumours, one showed mutation by both PCR-SSCP and genomic sequencing and one tumour has undergone deletion of the p53 gene. Overall, immunoreactivity correlated with both PCR-SSCP and genomic sequencing in 80% of cases (24/30), and there was 96.5% (28/29) concordance between PCR-SSCP and genomic sequencing. We conclude that there is good concordance between mutations detected by PCR-SSCP and genomic sequencing, but immunochemical detection of p53 overexpression is not an absolute indicator of p53 gene mutation.

Multicentricity of breast cancer: whole-organ analysis and clinical implications.

We studied the spatial relationship within the breast between multicentric foci (MCF) and the primary tumour in 30 modified radical mastectomy specimens using Egan's correlated pathological-radiological method using 5 mm slices of the whole breast. The relative positions within the breast of the primary tumour and MCF were used to calculate the relative distribution of primary tumour and MCF in the four quadrants of the breast and the per cent breast volume that would be required to be excised to include all MCF. Nineteen (63%) breast harboured MCF. The relative distribution of primary tumour and MCF in the four breast quadrants was significantly different (P = 0.034). MCF were present beyond the index quadrant (25% of breast volume including the tumour) in as many as 79% (15/19) of breasts that harboured MCF; and in half the cases (15/30) when all breast were considered. This is in variance with the suggestion put forward previously that MCF are contained within the index quadrant in 90% of cases. Although the number of patients in the present series is small, the probability of our finding being due to play of chance is 1 in 1500. In a large series of breast conservation studies > 90% of early breast recurrences have been found to occur in the index quadrant. Our finding, that in half the patients (15/30) MCF are present in quadrants other than the index quadrant, suggests that MCF do not give rise to early breast recurrence.