Mutational spectrum of DNA damage and mismatch repair genes in prostate cancer.

Over the past few years, a number of studies have revealed that a significant number of men with prostate cancer had genetic defects in the DNA damage repair gene response and mismatch repair genes. Certain of these modifications, notably gene alterations known as homologous recombination (HRR) genes; PALB2, CHEK2 BRCA1, BRCA2, ATM, and genes for DNA mismatch repair (MMR); MLH1, MSH2, MSH6, and PMS2 are connected to a higher risk of prostate cancer and more severe types of the disease. The DNA damage repair (DDR) is essential for constructing and diversifying the antigen receptor genes required for T and B cell development. But this DDR imbalance results in stress on DNA replication and transcription, accumulation of mutations, and even cell death, which compromises tissue homeostasis. Due to these impacts of DDR anomalies, tumor immunity may be impacted, which may encourage the growth of tumors, the release of inflammatory cytokines, and aberrant immune reactions. In a similar vein, people who have altered MMR gene may benefit greatly from immunotherapy. Therefore, for these treatments, mutational genetic testing is indicated. Mismatch repair gene (MMR) defects are also more prevalent than previously thought, especially in patients with metastatic disease, high Gleason scores, and diverse histologies. This review summarizes the current information on the mutation spectrum and clinical significance of DDR mechanisms, such as HRR and MMR abnormalities in prostate cancer, and explains how patient management is evolving as a result of this understanding.

Pioneering Informed Consent for Return of Research Results to Breast Cancer Patients Facing Barriers to Implementation of Genomic Medicine: The Kenyan BRCA1/2 Testing Experience Using Whole Exome Sequencing.

INTRODUCTION: Obtaining informed consent from study participants and disseminating the findings responsibly is a key principle required for ethically conducted clinical and genetic research. Reports from African researchers providing feedback on insights gained during the return of whole exome sequencing (WES) results to breast cancer patients treated in resource-limited settings is lacking. AIM: The empirical process used to fill this gap in relation to BRCA1/2 variant detection using WES provided unique insights incorporated into a pathology-supported genetic testing algorithm for return of research results to Kenyan breast cancer patients. METHODS: The Informed consent form approved by the Moi Teaching and Referral Hospital in Kenya was adopted from a translational research study conducted in South Africa. Initially, the informed consent process was piloted in 16 Kenyan female patients referred for breast surgery, following a community-based awareness campaign. A total of 95 female and two male breast cancer patients were enrolled in the study from 2013 to 2016. Immunohistochemistry (IHC) results of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status were obtained from hospital records. DNA of patients with a family history of cancer was extracted from saliva and screened for pathogenic variants in the BRCA1/2 genes as the first step using WES. RESULTS: Ten patients approached for participation in this study declined to sign the informed consent form. Data on IHC used as a proxy for molecular subtype were available in 8 of 13 breast cancer patients (62%) with a family history of cancer. Five BRCA1/2 variants of uncertain clinical significance were detected, as well as a pathogenic BRCA2 variant (c.5159C > A; S1720∗) in a female patient eligible for return of WES results. CONCLUSION: Experience gained during the qualitative pilot phase was essential to overcome challenges associated with the translation of sophisticated genetic terms into native African languages. Detection of a pathogenic BRCA2 variant in a patient with familial breast cancer, frequently associated with hormone receptor-positive breast carcinoma as reported in this case, led to a high level of confidence on which to base risk management in future. Implementation of new technologies alongside standard pathology provides a practical approach to the application of genomic medicine in Africa.

Epstein barr virus IgG and EBER-1 in Burkitt's lymphoma children at a referral hospital in western Kenya.

Burkitt's lymphoma (BL) is a frequent childhood B cell non-Hodgkin's lymphoma (NHL) in equatorial Africa associated with infections. Chronic Epstein Barr virus (EBV) infections can lead to host immune stimulation that may trigger genetic translocation(s), neoplastic transformation and proliferation of B cells. We determined EBV immunoglobulin G (IgG) in sera from participants and EBER-1 in tumour sections in confirmed BL cases at Moi Teaching and Referral Hospital (MTRH). A cross sectional study of children with clinical and histology diagnosis of NHL from whom BL status were confirmed by immunohistochemistry (IHC) was carried out. Epstein Barr virus IgG in sera was determine using Enzyme-linked immunosorbant assay, IHC for EBER-1 and MYC protein in tumour sections. Demographic and clinical information were obtained from questionnaires and hospital files respectively. Ninety three percent of sera were EBV IgG positive of which 31.7% were confirmed as BL. All jaw BL tumours and 86.7% of BL tumours carried EBER-1 antigen. Odds ratio of EBER-1 positive was 1.39, 95% CI: 0.16-12.19 in BL tumours regardless of age or gender. EBV infection among the study participants may be associated with BL, however, EBER-1 and MYC negative in BL tumours suggest alternative BL pathogenesis or variant.

Cytokines associated with Burkitt's lymphoma in western Kenya.

OBJECTIVE: Burkitt's lymphoma (BL) is a common aggressive non-Hodgkin's lymphoma in East and Central Africa among children. Persistent infections with Epstein Barr virus or Plasmodium falciparum are associated with immune hyperstimulation. It is hypothesised that inadvertent cytokine responses to infections indirectly or directly influence B cell neoplastic transformation through c-myelocytomatosis (c-myc) gene translocation. We sought to describe cytokines in children and adolescents with BL. Participants were recruited from western Kenya with parental consent, diagnosis confirmed using histology and consensus panel of immunohistochemistry antibodies. T helper1/2/17A and transforming growth factor-ß1 (TGF-ß1) cytokines were estimated using cytometric bead array in plasma. Complete blood counts (CBC) were determined by Beckman Coulter®. RESULTS: Out of 104 enrolled participants, 32% were confirmed BL and 68% grouped as non-BL. Mean (pg/ml) levels of cytokines in BL and non-BL were: interleukin (IL)-6 100.3 and 39.4 p = 0.152; IL-10 11.5 and 12.5 p = 0.363; IL-17A 17.8 and 64.9 p = 0.094 respectively. Expressions of interferon-γ, IL-2 and tumour necrosis factor-α were low and TGF-ß1 undetectable in both groups. Mean CBC differed between the two groups before and after chemotherapy, WBC being significantly so. Interleukin-6, IL-17A and IL-10 responses to infections in the study area may be associated with pathogenesis and be potential therapeutic targets.

Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya.

BACKGROUND: Tumors commonly are infiltrated by leukocytes, or tumor infiltrating leukocytes (TILs). It remains unclear, however, if the density and type of individual TILs has a direct or simply correlative role in promoting poor prognosis in breast cancer patients. Breast cancer in Kenyan women is aggressive with presentation at a young age, with advanced grade (grade III), large tumor size (>2.0 cm), and poor prognosis. We previously observed that the tumors were predominantly estrogen receptor positive (ER+) but also included both a high percentage of triple negative tumors and also increased immune cell infiltration within the tumors. We used breast tumor tissues from each patient to make tissue microarrays that were then stained for leukocyte and myeloid markers including CD4, CD8, CD20, CD25, CD68, and CD163 using immunohistochemical techniques. The immune cell infiltration into the cancer tissue included increased numbers of macrophages (CD68+), helper T cells (CD4+), and CD25+ lymphocytes compared to benign tissue. RESULTS: This study characterized the grade, molecular subtypes, and proliferation index of these tumors and determined if TIL density was enriched across any of these factors. We analyzed 49 malignant patient tissue samples for this study. The patient population had a mean age of 51.9 years. The tumors analyzed were heterogeneous by grade: grade I (6%), grade II (47%), and grade III (39%). Most patients presented with large tumors (>2.0 cm) (69%). We classified the tumors into molecular subtypes based on clinical marker expression. Based on this analysis, the molecular subtype distribution was heterogeneous with luminal B (41%), basal/triple negative (TN) (37%), luminal A (14%) and HER2 (8%) breast cancer subtypes. While the basal/TN subtype had a much higher proliferative index (Ki-67+) than did the other molecular subtypes, we did not see a significant correlation between TIL density and either subtype or tumor grade. Therefore, TIL density is independent of molecular subtype and grade. CONCLUSION: This study identified a Kenyan patient cohort that develops large, high-grade tumors primarily of the luminal B and basal molecular subtypes. After analyzing the TILs within these tumors, we found that immune cell infiltration of these tumors correlated with increased proliferation but not grade or molecular subtype. Future research is required to determine if the aberrant recruitment of TILs to tumors contributes to cancer progression and response to cancer treatments.

Hepatitis B infection is highly prevalent among patients presenting with jaundice in Kenya.

BACKGROUND: Viral hepatitis is a major concern worldwide, with hepatitis A (HAV) and E (HEV) viruses showing sporadic outbreaks while hepatitis B (HBV) and C (HCV) viruses are associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study determined the proportion, geographic distribution and molecular characterization of hepatitis viruses among patients seeking medical services at hospitals throughout Kenya. METHODS: Patients presenting with jaundice at four selected hospitals were recruited (n = 389). Sera were tested for the presence of antibody to hepatitis viruses A through E, and HBV surface antigen (HBsAg). Nucleic acid from anti-HAV IgM antibody and HBsAg positive samples was extracted, amplified and sequenced. RESULTS: Chronic HBV infection was the leading cause of morbidity among patients with symptoms of liver disease seeking medical help. Incident HCV, HEV and HDV infection were not detected among the patients in this study, while the proportion of acute HAV was low; HAV IgM positivity was observed in 6.3 % of patients and sequencing revealed that all cases belonged to genotype 1B. HCV seropositivity upon initial screening was 3.9 % but none were confirmed positive by a supplementary immunoblot assay. There was no serological evidence of HDV and acute HEV infection (anti-HEV IgM). HBsAg was found in 50.6 % of the patients and 2.3 % were positive for IgM antibody to the core protein, indicating probable acute infection. HBV genotype A was predominant (90.3 %) followed by D (9.7 %) among HBV DNA positive specimens. Full genome analysis showed HBV/D isolates having similarity to both D4 and D6 subgenotypes and D/E recombinant reference sequences. Two recombinant sequences demonstrated > 4 % nucleotide divergence from other previously known D/E recombinants. CONCLUSIONS: HBV is highly prevalent among patients seeking care for symptoms consistent with hepatitis, compared to the general population. Molecular characterization of HBV isolates indicated recombinant strains that may give rise to new circulating variants. There is a need to document the prevalence, clinical manifestation and distribution of the variants observed. HAV genotype 1B, prevalent in Africa, was observed; however, the absence of HCV, HDV and acute HEV in this study does not rule out their presence in Kenya.

Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration.

BACKGROUND: Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. METHODS: We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. RESULTS: Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59% were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30% were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. CONCLUSIONS: We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells.

Career choices and global health engagement: 24-year follow-up of U.S. participants in the Indiana University-Moi University elective.

BACKGROUND: Global health experiences evoke a profound awareness of cultural differences, inspire learners to prioritize professional values, and provide a lens for addressing global health care challenges. This study compares the long-term career and practice choices of participants in a 2-month Indiana University-Moi University, Kenya elective from 1989-2013 with those of a control group. METHODS: Global health elective (GHE) participants and a random sample of alumni without GHE experience were surveyed on their clinical practice, public health and global health activities. RESULTS: Responses from 176 former participants were compared with a control group of 177 alumni. GHE participants were more likely than similar controls to provide care to underserved U.S. populations (p=0.037), spend time in global health, public health, and public policy activities (p=0.005) and be involved in global health advocacy (p=0.001). Using multivariable analysis, GHE participants were more likely to be generalists (p<0.05), report that healthcare costs influenced medical decision-making (p<0.05), and provide healthcare outside the U.S. for ≥1 week/year (p<0.001). CONCLUSIONS: Many years out of training, GHE participants were more likely to be generalists working with underserved populations, to be cost-conscious in their healthcare decision-making, and to be involved in global health, public health or public policy. IMPLICATIONS: With the primary care provider shortage and need for greater awareness among providers of healthcare costs, our study shows that that global health experiences may yield broader benefits to the U.S. medical system.

High Prevalence of HIV Low Abundance Drug-Resistant Variants in a Treatment-Naive Population in North Rift Kenya.

The advent of antiretroviral treatment (ART) has resulted in a dramatic reduction in AIDS-related morbidity and mortality. However, the emergence and spread of antiretroviral drug resistance (DR) threaten to negatively impact treatment regimens and compromise efforts to control the epidemic. It is recommended that surveillance of drug resistance occur in conjunction with scale-up efforts to ensure that appropriate first-line therapy is offered relative to the resistance that exists. However, standard resistance testing methods used in Sub-Saharan Africa rely on techniques that do not include low abundance DR variants (LADRVs) that have been documented to contribute to treatment failure. The use of next generation sequencing (NGS) has been shown to be more sensitive to LADRVS. We have carried out a preliminary investigation using NGS to determine the prevalence of LDRVS among a drug-naive population in North Rift Kenya. Antiretroviral-naive patients attending a care clinic in North Rift Kenya were requested to provide and with consent provided blood samples for DR analysis. DNA was extracted and amplified and nested PCR was conducted on the pol RT region using primers tagged with multiplex identifiers (MID). Resulting PCR amplicons were purified, quantified, and pyrosequenced using a GS FLX Titanium PicoTiterPlate (Roche). Valid pyrosequencing reads were aligned with HXB-2 and the frequency and distribution of nucleotide and amino acid changes were determined using an in-house Perl script. DR mutations were identified using the IAS-USA HIV DR mutation database. Sixty samples were successfully sequenced of which 26 were subtype A, 9 were subtype D, 2 were subtype C, and the remaining were recombinants. Forty-six (76.6%) had at least one drug resistance mutation, with 25 (41.6%) indicated as major and the remaining 21 (35%) indicated as minor. The most prevalent mutation was NRTI position K219Q/R (11/46, 24%) followed by NRTI M184V (5/46, 11%) and NNRTI K103N (4/46, 9%). Our use of NGS technology revealed a high prevalence of LADRVs among drug-naive populations in Kenya, a region with predominantly non-B subtypes. The impact of these mutations on the clinical outcome of ART can be ascertained only through long-term follow-up.

Level of CD8 T Lymphocytes Activation in HIV-Infected Pregnant Women: In the Context of CD38 and HLA-DR Activation Markers.

Background. To date the effect of pregnancy on the immune activation of CD8 T cells that may affect HIV disease progression has not been well studied and remains unclear. Objective. To determine the effect of pregnancy on CD8 T lymphocyte activation and its relationship with CD4 count in HIV infected pregnant women. Study Design. Case control. Study Site. AMPATH and MTRH in Eldoret, Kenya. Study Subjects. Newly diagnosed asymptomatic HIV positive pregnant and nonpregnant women with no prior receipt of antiretroviral medications. Study Methods. Blood samples were collected from the study participants and levels of activated CD8 T lymphocytes (CD38 and HLA-DR) were determined using flow cytometer and correlated with CD4 counts of the study participants. The descriptive data focusing on frequencies, correlation, and cross-tabulations was statistically determined. Significance of the results was set at P < 0.05. Results. HIV positive pregnant women had lower activated CD8 T lymphocyte counts than nonpregnant HIV positive women. Activated CD8 T lymphocyte counts were also noted to decrease in the second and third trimesters of pregnancy. Conclusion. Pregnancy has a significant suppression on CD8+ T lymphocyte immune activation during HIV infections. Follow-up studies with more control arms could confirm the present study results.

Esophageal Cancer, the Topmost Cancer at MTRH in the Rift Valley, Kenya, and Its Potential Risk Factors.

Esophageal cancer at Moi Teaching and Referral Hospital (MTRH) is the leading cancer in men with a poor prognosis. A case control study (n = 159) aimed at the histology type, gender, and risk indicators was carried out at MTRH. Mantel Haenszel chi-square and logistic regression were employed for analysis. Squamous-cell carcinoma was the common histological type occurring in the middle third portion of the oesophagus. The occurrence of the cancer in males was 1.4 times that of females. The mean age was 56.1 yrs. Low socioeconomic, smoking, snuff use, alcohol, tooth loss, cooking with charcoal and firewood, hot beverage, and use of mursik were independently associated with esophageal cancer (P < 0.05). Using logistic regression adjusted for various factors, alcohol consumption was associated with the increased risk of esophageal cancer. AHR was 0.45 and 95% CI: 0.205-0.985, P = 0.046. A societal component of low socioeconomic conditions, a lifestyle component with specific practices such as the consumption of mursik, chang'aa, busaa, snuff, smoking, hot tea, poor oral hygiene, and an environmental component with potential exposure to high levels of nitrosamines, passive smoking, and cooking with coal, could be involved. The increase in experts at MTRH capable of diagnosing could be responsible for the increase in reporting this neoplasm.

TP53 mutations, human papilloma virus DNA and inflammation markers in esophageal squamous cell carcinoma from the Rift Valley, a high-incidence area in Kenya.

BACKGROUND: Squamous Cell Carcinoma of Esophagus is one of the most common malignancies in both men and women in eastern and south-eastern Africa. In Kenya, clinical observations suggest that this cancer is frequent in the Rift Valley area. However, so far, there has been no report on the molecular characteristics of esophageal squamous cell carcinoma (ESCC) in this area. RESULTS: We have analyzed TP53 mutations, the presence of human papilloma virus (HPV) DNA and expression of inflammation markers Cyclooxygenase 2 (Cox-2) and Nitrotyrosine (NTyR) in 28 cases (13 males and 15 females) of archived ESCC tissues collected at the Moi Teaching and Referral Hospital in Eldoret, Kenya. Eleven mutations were detected in TP53 exons 5 to 8 (39%). All ESCC samples were negative for HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Immunohistochemical analysis of Cox-2 and NTyR showed a low proportion of positive cases (17.4% and 39.1%, respectively). No association between the above markers and suspected risk factors (alcohol or tobacco use, hot tea drinking, use of charcoal for cooking) was found. CONCLUSION: Our findings suggest that mechanisms of esophageal carcinogenesis in eastern Africa might be different from other parts of the world. Low prevalence of TP53 mutation compared with other intermediate or high incidence areas of the world highlights this hypothesis. Our data did not support a possible ole of HPV in this series of cases. Further studies are needed to assess and compare the molecular patterns of ESCC from Kenya with those of high-incidence areas such as China or Central Asia.

Cerebral malaria in children: serum and cerebrospinal fluid TNF-alpha and TGF-beta levels and their relationship to clinical outcome.

This was a prospective study conducted at the Moi Teaching and Referral Hospital, Eldoret, Kenya. Twenty-three children admitted to the hospital with cerebral (CM) and 10 children with non-cerebral malaria (NCM) were studied. The aim of the study was to establish and compare levels of tumour necrosis factor (TNF-alpha) and transforming growth factor (TGF-beta1) in these children. Serum and cerebrospinal fluid (CSF) cytokine levels were assayed using ELISA kits. In serum, TGF-beta1 and TNF-alpha decreased over 5 days after admission to the hospital in both groups of patients with CM and NCM. In the CSF of cerebral cases the levels of TNF-alpha and TGF-beta1 were low and inversely related. Children in deeper coma had lower levels in serum of TGF-beta and higher levels of TNF-alpha than those in lighter levels of coma. The serum TNF-alpha levels in CM children were the same irrespective of the duration of illness before admission, but children with NCM who had been sick for a shorter duration before admission tended to have higher serum levels of TNF-alpha and higher levels of TGF-beta than those with a longer duration of illness before admission. In conclusion, this study shows that TNF-alpha and TGF-beta1 may not be useful in predicting the outcome for CM. They may, however, be useful in detecting children at risk of developing deep coma. TNF-alpha and TGF-beta levels were inversely related both in serum and CSF.