MOLECULAR CHARACTERIZATION OF BREAST CANCER AMONG  HIV  POSITIVE  AND HIV   NEGATIVE   FEMALES USING IMMUNOHISTOCHEMICAL MARKERS IN AHMADU  BELLO   UNIVERSITY   TEACHING   HOSPITAL    ZARIA, NIGERIA

PRINCIPAL INVESTIGATOR: DR ALIYU, USMAN BAPPA

DEPARTMENT OF PATHOLOGY AHMADU BELLO UNIVERSITY ZARIA

DR ALIYU USMAN BAPPA

MOLECULAR CHARACTERIZATION OF BREAST CANCER AMONG HIV

ABSTRACT

Introduction:  Invasive  breast  cancer  (IBC)  is  a  public  health  problem  particularly   in developing countries. It is the most common cancer  of  women  and  a  leading  cause of mortality in Nigeria. Majority of  our  patients  who  are  incidentally  in  the  peak  age  of  economic  productivity  present  to  the  hospital  in  late  stage  when  cure cannot be attained.

The treatment of  IBC  is  determined  by  its  morphologic,  immunohistochemistry  (IHC)  and  molecular  characteristics,  and  these,  along  with  hematologic  parameters, have significant prognostic and predictive values in  determining  the  clinical course and response to therapy.  The  successes  recorded  in  the  management of human immunodeficiency virus  (HIV)  infection  over  the  past  decades have led to more patients living longer however more  vulnerable  to  developing IBC. The synergy between  IBC  and  HIV  infection  suppresses  the immune system thereby conferring  worse  prognosis.  In  fact,  in  HIV  infected  patients, IBC affects women  at  relatively  younger  age,  tends  to  be  more  aggressive and has high mortality.

Aim:  To  determine  the  molecular  subtypes  of  IBC  and  the   associated  hematologic  parameters  in  HIV  seropositive  and  negative  patients  in   ABUTH  Zaria

Methodology: This will be a cross-sectional  study  that  will  be  carried  out  among  sixty (60) consenting patients with IBC in  ABUTH  Zaria,  over  a  period  of  ten  months,  from  the  day  the  study  commences.  The  patients  will  be  enrolled  into HIV  positive  and  HIV  negative   groups  with  each  group  having  thirty  patients   who  will  be  selected  consecutively  as  they  present  to  the  Breast  cancer  clinic and  consent  to  the  study.  Structured,  self-administered  questionnaire  will  be  issued to consenting patients so as to  get  their  Biodata,  risk  factors  for  IBC  and  HIV as well  as  other  co-morbid  conditions.  Breast  tissue  biopsy  from  the  lesion  will  be  taken  and  auto-processed  for  morphologic  features  (using  hematoxylin   and eosin)  and  molecular  subtyping  using  IHC  (ER,  PR,  Her2,  ki-67  and  p53)  with  Autotissue  processor,  Leica  TP1020;  Austria  and  Autostainer  Link  48,   DAKO; United Kingdom. The biopsies  that  reveal  2+  positivity  (++)  for  Her2  antibody  will  have  reflex  Fluorescent   In  Situ  Hybridization  (Reflex  FISH)   done   on them. An EDTA anticoagulant containing bottle will be used to sample 5ml  of  venous  blood  for  complete  blood  count  using   hematology   analyzer   (Swelab   Alfa, Boule Medical Lab, Sweden; December 2014) and HIV status of all the

participants will be determined by standard  ELIZA  technique  (pre-  and  post- screening counseling  will  be  offered).  The  CD4  T-cell  count  and  HIV  viral  load  will be determined for patients that  are  HIV  positive  using  Cyflow  counter  and  Cobas ampliprep/ Cobas Taqman 96 respectively.

The data obtained will be analyzed  using  SPSS  version  20.0  where  frequency  tables, coefficients  of  correlation  and  association  as  well  as  chi  square  test  will  be  calculated  and  p  value  </=  0.5  will  be  considered  significant.  The  result  of  the research will  be  used  in  guiding  therapy.  At  the  end  of  the  study,  a  report  will be generated and the outcome will be disseminated via  conferences  and  publishing articles in peer reviewed journals.

NIH – FORM BIOSKETCH

BIOGRAPHICAL SKETCH—Pilot Format (To Be Used for Specific FOAs only)

Provide the following information for the Senior/key personnel and other significant contributors.

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EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

NOTE: The Biographical Sketch may not exceed five pages. Follow the formats and instructions below.

1. Personal Statement

I have the necessary motivation and zeal as a junior faculty to carry out this research work. My area of interest in Pathology is Breast and Gynecologic Pathology. I attended conferences and seminars on Breast Pathology organized from within Nigeria including but not limited to those organized by West Africa Division of International Academy of Pathology (WADIAP) and Pathologists friends of Africa in Diaspora.

2. Positions and Honors

2006 - present: Lecturer, Department of Pathology, Ahmadu Bello University, Zaria May 2016 - present: Fellow in Pathology, Department of Pathology, ABU Zaria

3. Contributions to Science

NIL

4. Research Support

NIL

BIOGRAPHICAL SKETCH—Pilot Format (To Be Used for Specific FOAs only)

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

NOTE: The Biographical Sketch may not exceed five pages. Follow the formats and instructions below.

1. Personal Statement

I have the training, expertise and necessary motivation to successfully carry out the proposed research project. I also have a broad background in anatomic pathology with expertise in cancer diagnosis, immunohistochemistry for definitive diagnosis of specific cancers and data analysis in the cancer registry. My research includes Cancer pattern in Zaria and skin tumours in general. In addition, I have collaborated with other researchers, and produced several peer-reviewed publications. I am also a member of the hospital multidisciplinary team in cancer management with commensurate publication in cancer distribution, diagnosis and management. As a result of my previous experiences in research, I am aware of the importance of frequent communication among project members and of constructing a realistic research plan, timeline, and budget. The current application builds logically on my prior work.

2. Positions and Honors

2004 - present: Lecturer, Department of Pathology, Ahmadu Bello University, Zaria 2005 - present: Consultant Pathologist, Department of Pathology, ABU Zaria

2011- present: Consultant in Charge/Director- Zaria Cancer Registry, ABUTH 2012- present: Consultant &Facilitator- Immunohistochemistry training

2014: Professor of Pathology- Ahmadu Bello University, Zaria

3. Contributions to Science

Collaborator  in  grant  application  “Center  for  Research  Excellence  in  Cancer  and  Nutrition  Epidemiology  in   Nigeria   (CRECNEN)”   which   among   other things is designed to  support  cancer  registration  and  cancer  epidemiology  in Nigeria.  This  will  articulates  a  well-conceived  framework  that  builds  on  the Nigerian  National  System  of  Cancer  Registries   (NSCR)   that   was   developed   with  support  from  the  University  of  Maryland  Greenebaum  Cancer  Center  and   the NIH/NCI funded Capacity Development for Research into AIDS Associated Malignancies (CADRE) grant

Cancer registration in Nigeria has improved  in  recent  years  as  evidenced  by  Nigerian cancer registry data being included  in  the  International  Agency  for  Research  on  Cancer’s  (IARC’s)  GLOBOCAN  2012   database   and   several scientific publications based on Nigerian cancer registry data in international peer-reviewed journals.

Our registry is hospital based and also  captures  cancer  data  from  other  governmental and private hospitals within Zaria metropolis.

I have updated the mechanism of our data collection and  analysis  as  well  as  coverage  of  relevant  clinics  and  departments  that  treat  cancer  patients  within    and outside the hospital.

I published our cancer data over a 5year period detailing frequencies  and  morphological  cancer  types  and  this  publication  is  available  as  a  reference  booklet for cancer study with emphasis on the Zaria experience.

I have over 50 peer reviewed publication in reputable journals and also tutor Postgraduate students of Clinical Laboratory management Research Methods.

BUDGET

BUDGET JUSTIFICATION

Estrogen  receptor  (ER)  antibody:  1  unit  of  1ml/vial  will  be  required  to  determine the ER status of each patient

Progesterone (PR) receptor antibody: 1 unit of 1ml/vial  will  be  required  to determine the PR status of each patient

Human epidermal growth receptor (Her2) antibody: 1 unit of 1ml/vial will  be  required to determine the Her2 receptor status of each patient

Ki-67 antibody: 1 unit of 1ml/vial will  be  required  to  determine  the  proliferation  index of the tumor in each patient

P53 antibody: 1 unit of 1ml/vial will  be  required  to  determine  the  p53  gene  mutation status of each patient

Antibody diluents: 1 vial of 250ml  will  be  needed  to  dilute  the  concentrated  primary antibodies  to  desired  dilution/mix  (as  will  be  specified  by  the  manufacturer) to attain the desired titer

Antigen retrieval buffer: 10 units of 500ml each of proteolytic enzymes  will  be  needed  to  treat  the  formalin  fixed  paraffin  embedded  tissues  so  as  to  expose   the  antigen  binding  sites  for   the   requisite   antigen-antibody   reaction   and complex formation

Dual Rabbit Mouse horseradish peroxidase (HRP) combined with diaminobenzidine  (DAB):  1  unit  of  100ml  will  be  needed.  The  HRP  is  an enzyme label that forms colored substance  which  marks  the  site  of  antibody-  antigen complex while DAB  is  the  chromogen  that  absorbs  light  and  produces  color that will then be viewed as positive reaction on microscopy

IHC glass slides: 5 units will be required to mount  the  processed  tissues  and  covered for viewing under the microscope

Reflex  FISH:  All  the  patients  whose  IHC  for  Her2  shows  positivity  of  ++  will have reflex FISH done  to  establish  whether  they  may  benefit  from  anti-Her2  therapy (estimated to be about 20% of all patients)

Complete blood count: This will be performed of all patients  at  the  time  of  diagnosis, before the commencement of therapy and after treatment

Viral load: All the patients that test  positive  for  HIV  infection  will  have  their  viral load estimated before and after treatment

Research Assistant: An Assistant  will  be  needed  to  help  with  basic  tasks  over  the period of the study, ie 12 months.

PROJECT NARRATIVE

This  project  sets  to  determine  the  molecular  characteristics   and  prognosis  of   IBC in Zaria. It will define,  by  reflex  FISH,  borderline  positive  (++)  Her2  patients  that may benefit from anti-Her2 therapy using  Trastuzumab  and  so  predict  the  clinical course and response to treatment. The main goals of the study  are  to  determine  the  molecular  characteristics  of  IBC   among   HIV   positive   and  Negative  females,  establish  baseline  hematologic  parameters,  determine  HIV  status and viral load as well as build  the  capacity  of  the  PI  with  hands  –on  skills that will guide further studies.

RESEARCH PLAN

Specific aims

1. To determine the molecular subtypes of IBC in HIV infected and non infected  patients
2. To establish the baseline hematologic parameters in the study groups.
3. To establish the relationship between molecular subtypes and hematologic parameters.

BACKGROUND AND SIGNIFICANCE

Invasive breast cancer is the most common  cancer  of  women  in  developing  countries including Nigeria. Majority of the patients in Zaria which  is  the  national  center  of  Excellence  in  Radiotherapy  and  Oncology,  present  in  their  fourth  to   fifth decades of life with late stage disease when cure cannot be attained.¹ The molecular  classifications  of  this  disease   determine   treatment,   response   to therapy and prognosis as well as predict the clinical course of the disease.² Derangements  in  hematologic  parameters,  HIV  infection  and  Her2  negative   tumors  have  been  associated  with  poor   prognosis.^2-9  The   successes   recorded   in the treatment of HIV infection has led to more patients living longer  and  so  becoming more vulnerable  to  developing  IBC.  There  are  few  studies  in  the literature about patients with HIV infection who have IBC.  Still  fewer  are  studies  linking  molecular  classification  of  IBC  with  hematologic  parameters  and  HIV  status. This study is significant because it will  determine  the  molecular  characterization of IBC, establish the baseline hematologic parameters and any derangement during and  after  treatment  and  establish  the  viral  load  in  patients  with HIV infection. Furthermore, it  will  build  the  capacity  of  the  PI  and  set  the  stage for future studies.

PRELIMINARY DATA

There is no preliminary data.

EXPERIMENTAL DESIGN AND METHODS

Sixty consecutive and  consenting  IBC  patients  that  will  present  to  the  Surgical  and/  or  Pathology  departments  of  ABUTH  between   the   periods   covering   January to October 2017 will be enrolled to participate in the study. They will  be grouped into HIV Positive and  HIV  Negative  categories  with  each  group  having  thirty patients. Details  of  the  risk  and  the  benefits  of  the  study  will  be  explained  to  them  in  the  language  they  choose.  Structured,   self-administered   questionnaires will  be  issued  to  consenting  patients  so  as  to  get  their  Biodata,  risk factors for IBC and HIV as  well  as  other  co-morbid  conditions  and  for  those  that cannot read and write,  the  questionnaires  will  be  filled  on  their  behalf  by  the PI and/ or the research assistant after which they will be made to thumb print it.

Tissue biopsy using wide bore  needle  or  knife  will  be  taken  from  the  patient  by  the surgeon for histologic diagnosis. In  addition  5ml  of  venous  blood  will  be  sampled  for  complete  blood  count  and  determination  of  HIV  status,   the   latter  will  involve  both  pre  and  post  screening   counseling.   Using   auto-tissue   processor and auto-stainer, H/E and IHC stained  slides  will  be  made  from  the  biopsy while hematology analyzer will be used in  conducting  the  CBC.  The  HIV  status will be determined by  standard  ELISA  while  the  viral  load  of  HIV  seropositive  patients   will   be   determined   using   Cobas   ampliprep/   Cobas Taqman  respectively.  The  diagnosis  of  IBC  will  be   made   using   H/E   stained slide  on  light  microscopy  and  the  receptor  status  of  the  tumor  will   be   determined using the IHC slides. These  will  be  corroborated  with  the  clinical  features and the stage of the disease at presentation. All the patients that  have  positivity of ++  on  Her2  stained  slides  will  he  reflex  FISH  done  in  private laboratory in Kaduna. In the case of IHC IBC specimen that tested  positive  for  hormone receptors will be used as  control.  The  results  of  the  CBC  will  be  compared  between  the  two  groups  (HIV  Positive   and   HIV   Negative   groups). The HIV status will be documented and all the patients that test positive for  HIV infection will have their CD4 count done and viral load determined.

The  results  of  the  above  stated  parameters  will  be  processed  and  analyzed   using statistical package for social sciences (SPSS) version 20.0. Frequency  distribution  tables  will  be  made  so  also  cross  tabulations  to   examine   relationships  between  the  variables.  Tests   for   associations   using   chi   square  and  Pearson’s  correlation  coefficient  as  well  as  analysis  of  variance   will   be done. The p value of  /=  0.05  will  be  considered  significant.  At  the  end  of  the  work,  report  will  be  written,  presentations  may  be  made  at  conference  and  articles will be published in peer reviewed journals.

FUTURE DIRECTIONS

In ABUTH,  IBC  patients  with  Her2  positivity  of  ++  go  for  treatment  using  anti-  Her  2  drugs.  This  cost  more  money  and  yield  no  establish   advantage.   The result of this study  will  help  in  ascertaining  the  percentage  of  Her2  ++  patients  that will need not waste time  and  money  on  medications  that  may  not  be  of  optimal importance  to  them.  The  result  of  this  study  will  help  in  adding  to  the calls for  the  establishment  of  a  national  breast  cancer  screening  program.  More the  hands  on  experience  that  will  be  obtained  by  the  PI  will  assist  in  establishing Reflex FISH as a routine investigation in Pathology  department  of  ABUTH.

PLAN FOR THE PROTECTION OF HUMAN SUBECTS

The  benefits  for  the  study  participants  include  establishing  the  need  for   anti- Her2 in Reflex FISH positive patients and vice versa as well  as  determining  beforehand the possibility of blood transfusion thereby priming the minds  of  the  patients and their relatives as it pertains cost which is an established cause  of  treatment failure and default especially  that  most  of  the  patients  are  poor  and  health  insurance  does  not  cover  cancer  treatment  in  Nigeria.  Informed  consent  will be obtained  after  a  detailed  interaction  and  enlightenment  of  the  patients  about the benefits and possible risks of the study. They  will  be  made  to  sign  a  written consent and non literate patients will be talked to in the language they understand  the  best.  Strict  confidentiality  in  accordance  with   the   Physicians’   oath will be ensured.

REFERENCES

1. Samaila MOA, Ayeni EI and Ahmed SA. Cancer Trend in Zaria: A Five  Year  Hospital Based Analysis (2009  –  2013).  The  Zaria  Cancer  Registry  Report;  2014:33. ABU Press Zaria Nigeria.
2. Juan  Rosai.  Rosai  and  Ackerman’s  Surgical  Pathology  10^th  Ed.   Vol   2 Elsevier Philadelphia; 2011
3. Sayed S, Moloo Z, Wasike R, Bird P, Oigara R, Govender D et  al.  Is  Breast  Cancer  from  Subsaharan  Africa  Truly   Receptor   Poor?   Prevalence   of ER/PR/Her2 in Breast Cancer from Kenya. The Breast 2014;23(5):591-596
4. McCormeck  V, Joeffe M, van der Berg  E, Broeze N, Silva IS, Romieu I, et al.   Breast  Cancer  Receptor  Status  and  Stage  at  Diagnosis  in  Over  1200  Consecutive Public Hospital Patients  in  Soweto,  South  Africa:  A  case  Series.  Breast Cancer Res 2013;15(5):84
5. Ufelle SA, Ukaejiofo  EO,  Neboh  EE,  Achukwu  PU,  Ikekpeazu  EJ,  Maduka  IC  et al. Some Hematologic  Parameters  in  Pre-  and  PostSurgeryBreast  Cancer  Patients in Enugu Nigeria. Int J Cur Bio Med Sci 2012;2:188-190
6. Ali LO. Study Effect of Breast Cancer on Some Hematologic and Biochemical Parameters in Babylon Province, Iraq. IOSR-JPBS 2014;9:20-24
7. Akinbami  A,  Popoola  A,  Adediran  A,  Dosunmu  A,  Oshanaike  O,  Adebola  P,  et al. Full Blood Count Pattern of Pre- Chemotherapy Breast  Cancer  Patients  in  Lagos, Nigeria. Caspian J Int Med 2013; 4:574-579
8. Preeti C, Ritu Y, Kaushal V and Preeti B.Prognostic  Significance  of  Complete  Blood Count in Breast  Cancer  Patients.  Indian  J  Med  R  Pharm  Sci,  2016;  3:52-  57
9. Rama APS, Kaur M, Zonunsanga B, Puri A and  Kukah  AS.  Preoperative  Peripheral Blood Count  in  Breast  cancer:  Predictors  of  Prognosis  or  a  Routine  Test

PROJECT MENTOR

PROFESSOR MODUPEOLA OMOTARA ALIYU-SAMAILA

Professor of Pathology/ Director, Zaria Cancer Registry

Ahmadu Bello University/Ahmadu Bello University Teaching Hospital Shika-Zaria

Kaduna State

IRB APPROVAL

An    approval    has    been     obtained     from    the    Health     Research    and         Ethics Committee (HREC) of ABUTH for the study.

STAMINA MENTORED RESEARCH SUMMARY REPORT

APPLICATION #: 33

1. Significance

Additional literature search revealed that there may not be difference between the IHC staining for ER, PR, Ki-67, p53 and Her-2 between those with and without HIV. Hence this rationale has been stepped down.

2. Innovation

3. Approach

STUDY DESIGN: This will be a cross-sectional study that will be carried out among sixty

(60) consenting patients with IBC in ABUTH Zaria, over a period of ten months, from the day the study commences. The patients will be grouped into HIV positive and HIV negative groups with each group having thirty patients who will be selected consecutively as they present to the Breast cancer clinic and consent to the study.

FOLLOW UP: The women will not be followed to evaluate for outcomes of breast cancer

SAMPLE SIZE: An average of nine new cases per month of breast cancer is seen in ABUTH Zaria. Over the period of ten months an estimated ninety cases may be recorded. However due to the possibility of industrial action and refusal to give permission for the study by some patients, two-thirds of the number (60) is estimated.

GROUPING PATIENTS: The patients will be enrolled into HIV positive and HIV negative groups with each group having thirty patients who will be selected consecutively as they present to the Breast cancer clinic and/ Pathology Department and consent to the study.

CLARIFICATION ON CBC: The results of the CBC will be compared between the two groups (HIV Positive and HIV Negative groups).

PURPOSE OF QUESTIONNAIRE: Structured, self-administered questionnaire will be issued to consenting patients so as to obtain their Biodata, risk factors for IBC and HIV as well as other co-morbid conditions.

Tissue biopsy: Breast tissue Biopsy from the lesion will be taken at the time of presentation to the hospital.

Title  modification:  Molecular  Characterization  of  Breast  Cancer  among  HIV  Positive and  HIV  Negative  Females  using  Immunohistochemical  Markers  in  Ahmadu Bello University Teaching Hospital Zaria, Nigeria

DEPENDENT VARIABLE: The dependent variable is Hematologic parameter

STATISTICS:    The    interventions    to    be    administered    are    Chemotherapy                          and Radiotherapy with/ without Surgery. This will be a cross-sectional study of 60