Molecular Genetics of Lobular Breast Cancer in a South African cohort and effect of HIV infection.

Breast cancer has become the most common malignancy among women in sub-Saharan Africa, and cause of cancer-related death. Changes in reproductive behaviours and westernized lifestyle factors are leading to increased breast cancer rates. Sub-Saharan Africa has nearly 70% of the world's cases of human immunodeficiency virus (HIV) and HIV incidence rates are rising faster among women than among men. There is evidence both from USA and Africa that HIV-infected cancer patients have poorer cancer-specific survival than HIV-uninfected patients, even after accounting for cancer stage and treatment. Data about prognosis in HIV-positive breast cancer patients are sparse.

Preliminary data from the South African Breast Cancer and HIV-Outcomes cohort study (SABCHO) reveals that breast cancer in HIV positive women occurs at younger age, has a higher mortality rate but has a similar subtype distribution to breast cancer in HIV cancer negative patients, with estrogen receptor positive (ER+) cancers being most common.

In this project we will use samples from the SABCHO study to investigate a subtype of breast cancer, known as lobular breast cancer that has distinct etiological, clinical and biological characteristics compared with the more common invasive ductal (IDC). There is some evidence that they are less chemo-sensitive than IDC and that the 10-year survival rate of women with ILC is lower than that of ER+ IDCs. ILC accounts for 10-15% of all invasive breast cancer in Western countries and is less frequent in lower and middle income countries presumably due to lifestyle factors and lack of hormone replacement therapy use. However, there is evidence from the USA that African Americans have more aggressive ILC and worse outcomes. It is also likely that with changes in reproductive behaviors and westernized lifestyle factors there will be an increase in lobular breast cancer in sub-Saharan Africa.

From the SABCHO study we have identified a series of 67 cases of ILC that occurred in black women of whom 13 (19%) are HIV positive with tissue blocks and blood samples available for analysis. The mean age at diagnosis of HIV positive patients was 45 years compared to 60 years for the HIV negative cases. The HIV positive cases were also more likely to present with stage 3-4 disease (77% vs 46%). Clinico-pathological features (age, nodal status, size, receptor status, grade and lymphocytic infiltrate) will be assessed in these samples and compared to a comparable UK cohort. We will also assess whether HIV infection has an effect on the composition of the lymphocytic infiltrate. Molecular differences will be assessed using targeted sequencing and copy number analysis.

As ILCs are more chemoresistant than IDCs the mainstay of treatment is Tamoxifen in premenopasual women. Tamoxifen is activated by liver enzymes to the therapeutic metabolite endoxifen, this activation can be affected by drugs such anti-retrovirals used in the treatment of HIV infection and by genetic variants of the liver enzymes, ~30% of African populations have such a genetic variant, CYP2D6*17. Patients for this study will be eligible if they have ILC or ER+ HER2-negative IDC and have been receiving tamoxifen treatment for at least 3 months. In this pilot phase 50 women who are on anti-retroviral therapy for HIV and 50 HIV-uninfected patients will have blood taken and analysed for tamoxifen and its metabolites and genotyped for genetic variants. This will allow us to study the effects of anti-retroviral therapy and the African-specific CYP2D6*17 polymorphism on tamoxifen metabolism.

We will also perform a small pilot to explore the utility of analysing levels of tumour mutations in plasma DNA from patients before and after therapy for metastatic ILC. This will be a small prospective study on ten patients (5 HIV+, 5 HIV-) collecting plasma before and after treatment.

Objectives 1 & 2: Characterize clinco-pathological features and molecular genetics of ILC in African women
Somatic copy number aberrations will be assessed using the Illumina HumanCytoSNP-12 BeadChip Kit (300,000 SNPs). Raw SNP array data will be processed with the GenomeStudio 2.0 software (Illumina). Segmentation will be done using SNP Rank segmentation algorithm on the Nexus 8.0 software (BioDiscovery) and absolute copy number determined using the Tumour Aberration Prediction Suite 2.0. Targeted sequencing will be performed using the SureSelect XT low input Target Enrichment System (Agilent). Captured libraries will be run on the HiSeq2500 and analysed using our standard pipeline.
Objective 3: Does anti retroviral therapy interferes with the efficacy of tamoxifen therapy?
Tamoxifen and its metabolite concentrations will be analyzed on a triple quadruple mass spectrometer using a reverse phase column and an isocratic mobile phase system composed of acetonitrile and formic acid solution. Population pharmacokinetic simulation tools in NONMEM will be used to estimate the AUC (a measure of total drug exposure) and Cmax (maximum drug exposure). Multiplex genotyping will be performed on the open-array QuantStudio 12K Flex Real-Time PCR System. The microarray panel will simultaneously detect genetic variants of CYP2C9, 2C19, 2D6, 3A4, 3A5.
Objective 4: Assess whether ctDNA can be used to detect metastatic ILC
Blood samples will be collected in Cell Preserver tubes (Nonacus.com), centrifuged and plasma transferred into sterile microfuge tubes and stored at -200C before shipping on dry ice to KCL. ctDNA will be extracted using the Cell Xtract protocol (Nonacus) and quantified by Qubit analysis. The protocol for set up of fluorescent assays for wildtype and mutant alleles, droplet generation and PCR is as previously described (Gao F et al, J Thorac Oncol 2015;10: 212-217). Results are read using the QX100 optical reader and processed using QuantaSoft (BioRad) software

This research will contribute to development of evidence-based guidelines for the management of ER+ breast cancer in HIV-positive women and improve outcomes for all women in limited resource settings.

Specifically it will:

1. Explore the molecular mechanisms underlying the aggressive behavior of ER+ breast cancer in HIV-positive women which will help us tailor our treatments for this subgroup of women.

2. Understand how anti-retroviral therapy and /or the CYP2D6*17 polymorphism affect endoxifen levels and whether these women require dose adjustment of tamoxifen to improve their survival or would be better treated with complete estrogen blockade.

3. Explore whether circulating tumour DNA can be used to monitor response to treatment in the metastatic setting in ER+ breast cancer in low and middle income countries and replace expensive cross-sectional imaging.


Though this pilot proof of concept research will focus on a small proportion of hormone responsive breast cancers, namely the invasive lobular group, it will serve to establish a long- term research collaboration on the complete group of hormone responsive breast cancers between clinicians and scientists from King's College London and from the University of the Witwatersrand, Johannesburg South Africa. Future projects focused on the optimum management and improved survival of ER+ breast cancer patients will build on findings from this pilot programme. For example, once proven, the circulating tumour DNA technology may be applicable to regular interval blood samples to detect early signs of resistance to tamoxifen in patients on long-term treatments to enable timeous switch to different anti-estrogen therapies. An immediate opportunity for regular blood sampling for tamoxifen and metabolite concentration measurements would be to verify patient compliance to long-term tamoxifen treatments and early interventions to improve compliance in patients not adhering to treatments. If we find that ART or the CYP2D6*17 polymorphism affects endoxifen levels, we will apply for funding to follow our study participants to assess the associations of endotoxin levels with medication adherence and survival. We would also assess the prevalence of the CYP2D6*17 polymorphism in a large cohort to assess future tamoxifen treatment guidelines, and may also seek funding for a trial of tamoxifen dose adjustment among our HIV-infected patients. Such applications would address the challenge of pilot and pump-priming studies, which are necessarily limited in sample size.

This research will assist the development of much needed cancer research capacity in South Africa. Mid-career academics and postgraduate students will work on the current and future research projects and develop their research expertise and publication track records.

Finally, ER+ breast cancers constitute the majority of breast cancers among all racial groups. They are less responsive to expensive and resource intensive chemotherapy treatments. Because endocrine treatment (ET) is far less toxic, inexpensive and much easier to administer than chemotherapy, it is an important treatment option for hormone responsive breast cancers in low-resource settings. In the longer term, we envisage that our research collaboration will expand the effective use of hormonal treatments to patients in low- and middle- income countries where chemotherapy treatments are largely unaffordable and specialist oncologist resources are few.