Breakthroughs in Health Education and Healthcare

in the 4th Industrial Revolution

Samuel D. Bernal
MD, PhD, JD, LLM, MBA
Professor of Medicine Emeritus, University of California, Los Angeles, USA
Physician, Hematology & Oncology, Cedars-Sinai Medical Center, Beverly Hills CA, USA
Chairman and Founder, GlobeTekPro International, USA, Czech Republic, Philippines
Scientific , Institute of Personalized Molecular Medicine, The Medical City, Philippines
Adjunct Professor of Business Innovation, Asian Institute of Management, Philippines
President, International Institute of Law and Technology, USA, Czech Republic, Philippines
Visiting Professor in Law, Science and Medicine - Russia, Georgia, Czech Republic, Poland
 Statement of the Problem:
The Current Health Education and Healthcare System – Do they work well?
A. Don’t we already have many drugs approved by FDA in countries around the world
that can be conveniently bought in pharmacies with standard dosing schedules?
B. Weren’t drug treatments proven to have efficacy by the use of RCT – randomized
clinical trials in the framework of EBM – Evidence-Based Medicine?
C. Aren’t there accepted standards for drug therapy that are spelled out in general
guidelines for treatments according to disease classifications?
D. Haven’t we developed effective strict safety guidelines that allow us to protect
patients from adverse drug events and serious drug interactions?
E. Don’t the disease classifications we use lead to effective management strategies by
the associations with efficacy of therapies using population-based statistics?
F. Don’t we have very effective educational systems and specialty training programs
based upon organ systems (neurology, cardiology, nephrology) that create efficient and
well-organized methods for delivery of care?
G. Don’t our healthcare programs and professionals already emphasize preventive
medicine, nutritional management, social, cultural and spiritual support systems?
Paradigms of Health Education and Healthcare in the 21th Century DC Whitcomb 2012

Randomized protocols, general guidelines - Personalized Molecular & Cellular Therapy

- RCT – “Evidence”-Based Medicine - Personalized Molecular Profiling-Based

 Statement of the Problem:
Updating Health Education and Healthcare System – What changes are needed?
A. Multidisciplinary approaches – bringing together the different skill sets of physicians, nurses,
medical technologists, counselors, nutritionists, dieticians, patient advocates, other health practitioners
in coordinated teams – preventing the automatic prescription ‘drug’ trap.
B. Interdisciplinary approaches – bringing together the different skill sets of chemists, biochemists,
molecular biologists, genetic engineers, biophysicists, computer and data scientists, media and
communication practitioners, economists, financial planners, lawyers, bioethicists – integrating the skill
sets in working teams, avoiding the ‘specialist’ shell.
C. The Patient as the focus and center of health planning, health maintenance and decision making. The
family and close associates as support providers. Health practitioners as guides and resources for
information, insight and empathy, not as the decision-maker for the patient.
D. Vigilance of drug adverse effects - highlight potential for drug-drug, drug-supplement, drug-diet
interactions, influence of age, sex, genetic characteristics – pharmacogenomics.
E. Educational emphasis on molecular and cellular mechanisms of how the body maintains wellness
and heals itself, and the how disruptions in these mechanisms lead to disease – should be the basis for
therapeutic interventions.
F. Systems approaches in health education and training programs – emphasizing the interrelatedness of
organism, organ, tissue, cell and molecular pathways – not organ-oriented compartments.
G. Strengthen education in preventive medicine, nutritional management, social, cultural and spiritual
support systems – judicious use of prescription drugs. Increase awareness of economic realities in
interventions.
Molecular - Cellular Therapy for Chronic Diseases
 Cardiac cell regeneration –
post myocardial infarction,
cardiomyopathies
 Diabetes, metabolic
syndromes
 Neuro-Degenerative
Diseases
 Kidney – renal failure
 Liver regeneration –
cirrhosis, hepatitis
 Bone and connective tissue
regeneration – post trauma,
fractures, osteoporosis,
osteoarthritis
 Auto-immune diseases
Vasculitis, Lupus, Rheumatoid
Arthritis
 Lung – lung fibrosis, COPD
 Eye – cornea, retina
 Skin – burns, aesthetics
 Molecular-Cellular Treatments for Cancer in Philippines
(2004-2019 – 650 Patients)
• Lung • Head / Neck •Non-Hodgkin’s Lymphoma
• Breast • Prostate •Acute Lymphocytic
• Ovary Leukemia
• Kidney
• Uterus •Acute Myelogenous
• Bladder Leukemia
• Liver • Melanoma •Chronic Lymphocytic
• Pancreas • Skin Squamous Leukemia
• Colon • Sarcoma •Hodgkin’s Lymphoma
• Esophagus • Mesothelioma •Multiple Myeloma
 Referrral of Patients for Molecular and Cellular
Treatments for Cancer in Philippines
• United States • Russia • India
• Canada • Czech Rep. • Pakistan
• Mexico • Slovakia • Sri Lanka
• Norway • Italy • Hongkong
• Denmark • France • China
• Latvia • Spain • Singapore
• Poland • Australia • Malaysia
• England • New Zealand • Thailand
 Chemical Drug Efficacy and Toxicity
A. Efficacy
• Most of the drugs approved by the US FDA fall in the range of 50–75% response rates (Abbott Pharmaceutical
Research - Spear et al, 2001).
• Many of the drugs were used for lifestyle diseases that could have been managed or prevented by non-
pharmaceutical methods – depression, anxiety, asthma, diabetes, obesity, arthritis, chronic pain syndromes.
• For chemical drugs, the lowest overall response is 25% for cancer chemotherapy.
• Every day, more than 130 people in the United States die after overdosing on opioids. The misuse of
and addiction to opioids—including prescription pain relievers, heroin, and synthetic opioids such as
fentanyl—is a serious national crisis that affects public health as well as social and economic welfare. The total
"economic burden" of prescription opioid misuse alone in the United States is $78.5 billion a year, including
the costs of healthcare, lost productivity, addiction treatment, and criminal justice involvement. (National
Institute on Drug Abuse, Jan 2019)

B. Toxicity
• Despite the intensive efforts of pharmaceutical companies to develop safer drugs and of the regulatory
agencies to maintain strict safety guidelines many drugs lead to serious toxicities. This is particularly true of
cancer chemotherapy where some patients die because of the treatment, not because of the disease.
• Of the 1232 chemical entities approved as drugs in the USA, 193 (16%) - associated with adverse events
severe enough to require a ‘black box’ warning on the product label (Merck Index, 54th Ed.). In
• A published analysis (Lazarou J et al, JAMA 1998) reported 1.8 million people were hospitalized for adverse
drug events in the USA in 1994, with over 100,000 deaths.
 Consumer Reports, 2017
“The amount of harm stemming from inappropriate prescription medication is staggering. Almost 1.3 million people went to U.S.
emergency rooms due to adverse drug effects in 2014, and about 124,000 died from those events. That’s according to estimates
based on data from the Centers for Disease Control and Prevention and the Food and Drug Administration. Other research
suggests that up to half of those events were preventable.”
“All of that bad medicine is costly, too. An estimated $200 billion per year is spent in the U.S. on the unnecessary and improper
use of medication, for the drugs themselves and related medical costs, according to the market research firm IMS Institute for
Healthcare Informatics.”
BB. Spear et al. Clinical Trends in Molecular Medicine, Volume 7, Issue 5, 1 May 2001, pages 201 - 204.
BB. Spear et al. Clinical Trends in Molecular Medicine, Volume 7, Issue 5, 1 May 2001, pages 201 - 204.
 - effective, less toxic therapies for individual patients

BB. Spear et al. Clinical Trends in Molecular Medicine, Volume 7, Issue 5, 1 May 2001, pages 201 - 204.
The impact of personalised medicine on delivering better treatments for patients
 Impact of Personalized Medicine in Diverse Cancers
A. Personalized Approach showed superior outcomes versus NonPersonalized Approach
• The personalized approach, compared with a nonpersonalized approach, consistently and
independently correlated with higher median RR (31% v 10.5%, respectively; P < .001) and
prolonged median PFS (5.9 v 2.7 months, respectively; P < .001) and OS (13.7 v 8.9 months,
respectively; P < .001).
• Nonpersonalized targeted arms had poorer outcomes compared with either personalized targeted
therapy or cytotoxics, with median RR of 4%, 30%, and 11.9%, respectively; median PFS of 2.6, 6.9,
and 3.3 months, respectively (all P < .001); and median OS of 8.7, 15.9, and 9.4 months, respectively
(all P < .05).
• Personalized arms using a genomic biomarker had higher median RR and prolonged median PFS and
OS (all P < .05) compared with personalized arms using a protein biomarker.
B. Personalized strategy was associated with a lower treatment-related death rate than a
nonpersonalized strategy
• Treatment related death median, 1.5% in Personalized v 2.3% in NonPersonalized, P < .001).
C. Conclusion
• Comprehensive analysis of phase II, single-agent arms revealed that, across malignancies, a
Personalized strategy was an independent predictor of better outcomes and fewer toxic deaths.
• Nonpersonalized targeted therapies were associated with significantly poorer outcomes than
cytotoxic agents, which in turn were worse than personalized targeted therapy.
 Genetic Analysis and Personalized Molecular and Cellular Medicine – Applications in Cancer
1. Personalized Molecular-Genetic Diagnostics
1. Genomics – genetic mapping, DNA sequencing of sets of genes, interactions of genes with each other
2. Transcriptomics – array of mRNA transcripts produced in a particular cell or tissue type
3. Proteomics – complete set of proteins expressed by an organism, tissue, or cell, the study of changes in
protein synthesis, modification and degradation
4. Metabolomics – chemical processes involving metabolites, the small molecule intermediates and
products of metabolism
5. Epigenetics - modification of gene expression rather than alteration of the genetic code itself
6. Live Cell Functional Analytics – functions and interactions of cells in the living state
2. Personalized Diagnostics to Personalized Clinical Therapeutics
1. Classification of cancers – beyond morphologic criteria
2. Prognostication – risk of relapse, rapid progression
3. Identification of drug resistance – gene expression associated with resistance to chemical drugs
4. Patient selection for aggressive treatment – e.g. high dose chemotherapy with stem cell
transplant
5. Choice of chemotherapeutic drugs – classical chemical treatment
6. Selection of Targeted agents – e.g. tyrosine kinase inhibitors
7. Immunotherapies – passive immune treatments, monoclonal antibodies
8. Selection of Targets for Adoptive cellular therapies – Dendritic Cells, Cytokine Induced Killer
Cells, CAR-T cells (Chimeric Antigen Receptor T Cells)
Sanger Sequencing:
• Single genes NGS Sequencing:
• 1-100 amplicons • >100 genes at a
at lowest cost time cost
• Up to 96 samples effectively
at a time without • Finding novel
barcoding variants,
• Microbial expanding no. of
identification targets per run
• Fragment • Samples with low
analysis, high amounts of
throughput starting material
genotyping • Microbial
• Microsatellite or genomes for
STR analysis pathogen
• NGS confirmation subtyping

Bunnik & Le Roch, 2013

RNA-Seq: Bisulfite-Seq
determining mRNA profiling the genome-
levels to study gene wide locations of
expression and its methylated cytosines
regulation

SNP-Seq:
complete genome or
Faire-Seq, Maine-Seq: exome (all coding
investigating various regions of the
aspects of chromatin genome) to identify
structure and single-nucleotide
regulation of gene polymorphisms (SNP-
expression by Seq)
determining or other DNA
nucleosome positioning mutations

ChIPSeq:
histone modifications
or transcription factor
binding
Bunnik & Le Roch, 2013
Proteomics:
Analysis of the protein content
of cells can be
performed by two-
dimensional gel
electrophoresis,
where proteins are separated
first by size, and then by
charge, followed by MS.
Multidimensional
protein identification
technology (MudPIT) starts
with protein digestion into
peptides, followed by 2D gel
and MS.
Metabolomics:
tools most widely used are
nuclear magnetic resonance
(NMR)
and liquid chromatography
coupled to Mass Spectroscopy.
Leads to understanding
of complex cellular metabolism,
energy metabolism, Live Cell
Analytics, Electrical Energetics,
characterize new metabolic
pathways, and identify new
targets for therapeutic
intervention in cancer.
Interactome:
Protein-protein interactions by
Two-hybrid method, to analyze
interactions of two proteins -
one protein
of interest is fused to a DNA
binding domain,
another protein of interest is
fused to an activation
domain. Both fusion proteins are
then expressed in the same cell.
If the proteins interact, a
reporter gene is transcriptionally
activated, which will change the
phenotype of the cell and allow
for an easy readout.
Affinity purification methods can
be used to study the organization
of proteins into
complexes. A tag is fused to a
protein of interest, which is
subsequently used to
isolate this protein together with
all proteins that are bound. The
bound proteins are then analyzed
by MS.
Bunnik & Le Roch, 2013
1997 Publication, Switzerland
Drug resistance develops quickly in
tumors treated with chemotherapy alone.
Specific genes, related to stem cell
differentiation, greatly influence
resistance to chemotherapy and
biotherapy.
To overcome drug resistance, multiple
modalities, such as membrane molecular-
targeted therapies, antibodies, and
immune stem cells can be used along
with chemotherapy.
1992 Publication, Switzerland

Cancer cells display maturation

characteristics that are similar to
normal stem cells.
The one-size-fits-all approach to the
treatment of cancer does not work.
Each cancer has to be profiled at the
molecular level, in order to
customize treatment.
Mitochondrial fluorescent
markers related to the
voltage level of
mitochondrial inner
membrane electrical
potential.

Confocal microscopy of
living cells in real time
shows that mitochondria
are filamentous, moving
along tracks of
microtubules, increasing or
decreasing electrical
voltages relative to
metabolic needs of
organelles within the cell,
eg. nucleus, endoplasmic
reticulum, calcium fluxes.
 Live Cell Confocal 3D Microscope
with CO2 temperature controlled incubator
Rhodamine 123 efflux for studies of ATP Binding Cassette Transporters
related to chemotherapy drug resistance
2D Gel
MembraneProteins
of Human Small
Cell Lung
Carcinoma

Bernal et al, 1982

Molecular Mechanisms of Drug Resistance in Human Cancers

Bernal et al, 1984

 LEUKEMIA & MYELODYSPLASTIC SYNDROME Panel
BCR-ABL1 CBL EP300 GNAS KRAS NRAS PTEN STAG2

ASXL1 CBLB ERBB2 IDH1 MEF2B NSD1 PTPN11 STAT3

ATM CD79A ETV6 IDH2 MPL NUP98 RAD50 STAT5B

ATRX CD79B EZH2 IKZF1 MYC PAX5 RECQL4 TET2

BCL2 CDKN2A FAS JAK2 MYD88 PDGFRA RHOH TNFAIP3

BCL6 CEBPA FLT3-ITD KDM2B NF1 PHF6 RUNX1 TNFRSF14

BCOR CREBBP FOXO1 KDM6A NLRP1 PIGA SETBP1 TP53

BCORL1 CSF3R GATA1 KIT NOTCH1 PIM1 SF3B1 U2AF1

BRAF CXCR4 GATA2 KMT2A NOTCH2 PLCG2 SGK1 WT1

BTK DNMT3A GNA13 KMT2D NPM1 PRDM1 SRSF2 ZRSR2

CALR
 LYMPHOMA Panel
ARID1A CD79B GNAI2 MYD88 SYK
ATM CDKN2A IKZF1 NOTCH1 TET2
B2M CDKN2B IKZF3 NOTCH2 TNFAIP3
BCL2 CIITA IRF4 PIK3CD TP53
BCL6 CREBBP IRF8 PIM1 XPO1
BCL10 CSFR2B ITPBK PLCG2
BRAF DDX3X KMT2C PPRDM1
BTK EP300 KMT2D PRKDC
CARD11 EZH2 KRAS PTEN
CCND1 FAS MFHAS1 SGK1
CCND3 FOXO1 MTOR STAT3
CD79A GNA13 MYC STAT6
 MULTIPLE MYELOMA Panel
AKT1 CDK4 GRB2 JAK2 PIK3R2 STAT3

AKT2 CDK7 IDH1 KDM6A PIM1 TGFBR2

ATM CDKN2A IDH2 KRAS PIM2 TLR4

B2M CRBN IDH3A MYC PIM3 TP53

BIRC2 CUL4A IFNGR2 MYD88 PSMA1 TRAF3

BIRC3 CXCR4 IGF1R NR3C1 PSMB5 WHSC1

BRAF DIS3 IKZF1 NRAS PSMD1 XBP1

CCND1 EGFR IKZF3 PIK3CA PSMG2

CD38 FAM46C IL6 PIK3CG PTPN11

CDK1NB FGFR3 IRF4 PIK3R1 RB1

 Drug Resistance Gene Expression Panels
cDNA RT-PCR (partial list)

• Multi-Drug Resistance: ABCB1 (PGY1, MDR1), ABCC1 (MRP1), ABCC2 (cMOAT, MRP2),
ABCC3 (MOAT-D, MRP3), ABCC5 (MRP5), ABCC6 (MRP6, CFTR), ABCG2 (BCRP), BAX,
BCL2, BCL2L1 (bcl-x), MVP (LRP), RB1, TOP1, TOP2A, TOP2B, TP53 (p53).
• Drug Metabolism: ARNT, BLMH, CLPTM1L (CRR9/DKFZP761M2324), CYP1A1, CYP1A2,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, DHFR, EPHX1,
GSK3A, GSTP1, NAT2, SOD1, SULT1E1 (STE), TPMT.
• DNA Repair: APC, ATM, BRCA1, BRCA2, ERCC1, ERCC3, MSH2, XPA, XPC.
• Cell Cycle: CCND1 (cyclin D1), CCNE1 (cyclin E1), CDK2, CDK4, CDKN1A (p21Waf1,
p21Cip1), CDKN1B (p27Kip1), CDKN2A (p16Ink4), CDKN2D (p19Ink4d).
• Growth Factor Receptors and Oncogenes: EGFR (ERBB1), ERBB2 (Her-2), ERBB3,
ERBB4, FGF2, IGF1R, IGF2R, MET, Kras, Alk.
• Hormone Receptors: AR, ESR1 (ER-alpha), ESR2 (ER-beta-cx), PPARA, PPARD, PPARG,
RARA, RARB, RARG, RXRA, RXRB.
• Transcription Factors: AHR, AP1S1, ELK1, FOS (c-fos), HIF1A, MYC (c-myc), NFKB1,
NFKB2, NFKBIB (TRIP9), NFKBIE, RELB (l-rel), TNFRSF11A (Rank).
 Analysis of Drugs Commonly Used in Hematologic Malignancies
Antitumor antibiotics Interact directly with DNA in the nucleus of cells, interfering with cell survival • Bleomycin
sulfate (Blenoxane®) • Daunorubicin (Cerubidine®) • Idarubicin (Idamycin®) • Doxorubicin (Adriamycin®) •
Mitoxantrone (Novantrone®)

Antimetabolites Block cells’ ability to form RNA or DNA, preventing cell growth and accelerating cell death •
Cladribine (Leustatin®, 2-CdA) • Cytarabine (cytosine arabinoside, ARA-C, Cytosar-U®) • Fludarabine (Fludara®)
• Hydroxyurea (Hydrea®, Droxia®) • 6-Mercaptopurine (Purinethol®) • Methotrexate • 6-Thioguanine
(Thioguanine®, Tabloid®) • Azacitidine (Vidaza®) • Decitabine (Dacogen®) • Clofarabine (Clolar®

Plant alkaloids Act on certain proteins (enzymes) in the cell nucleus that normally repair injury to DNA (DNA-
repair enzyme inhibitors) • Etoposide (VP-16, VePesid®, Etopophos®, Toposar®) • Teniposide (VM-26, Vumon®)
• Topotecan (Hycamtin®) Impair structures in the cell that are required for cells to divide into two daughter
cells (block mitosis) • Vinblastine (Velban®) • Vincristine (Oncovin®) • Paclitaxel (Taxol®)

Alkylating agents Alter DNA and enhance cell death • Bendamustine (Treanda®) • Busulfan (Myleran®,
Busulfex®) • Carboplatin (Paraplatin®) Carmustine (BCNU, BiCNU®) • Chlorambucil (Leukeran®) • Cisplatin
(Platinol®) • Cyclophosphamide (Cytoxan®, Neosar®) • Dacarbazine (DTIC-Dome®) • Ifosfamide (Ifex®) •
Lomustine (CCNU®, CeeNU®) • Mechlorethamine (nitrogen mustard, Mustargen®) • Melphalan (Alkeran®) •
Procarbazine (Matulane®)
 Genetic Mutations in Hematologic Malignancies
Diagnostic Tools Used in Hematologic Malignancies:
• Morphology
• Flow cytometry
• Fluorescence in situ hybridization (FISH)
• Polymerase chain reaction (PCR)
• Molecular genetics
• Cytogenetics, karyotyping
• Chromosomal microarray – molecular karyotype
FISH panel can detect the presence of core binding factor inv(16) or t(8;21), both of which are associated with a
better prognosis.
Cytogenetics and FISH should be used to diagnose the acute promyelocytic leukemia subtype of AML, which
is characterized by t(15;17). All-trans retinoic acid (ATRA) to treat patients with suspected acute promyelocytic leukemia, even
prior to the diagnosis being confirmed - rare example in oncology in which early treatment is essential, because the disease can
lead to death in just a few days. Treatment with ATRA is so benign that there is no reason not to implement it
immediately if the clinician has even the smallest suspicion of acute promyelocytic leukemia.

Chromosomal microarray (CMA) improves the diagnostic yield to identify genetic changes that are not detected by conventional
chromosome analysis or FISH studies. Although many chromosomal abnormalities are large enough to be detected with
conventional chromosome analysis, many others are below its limits of resolution, and conventional chromosome analysis does
not detect copy-neutral loss of heterozygosity. CMA utilizes >1.9 million copy number probes and approximately 750,000 single
nucleotide polymorphism probes to detect copy number changes and regions of copy-neutral loss of heterozygosity (LOH).
Dawson et al. 2011
 Summary 1 – Bernal et al, 1992
Determinants of Cancer Response to Treatment:
• Histological type – tissue of origin, but also differentiation level
• Gene Expression - molecular characteristics of cancer cells, individual ”pharmacogenomics”
• Treatment history – intrinsic resistance, acquired resistance
Each cancer is different at the molecular level:
• The one-size-fits-all approach to cancer treatment does not work.
• Advances in analysis of abnormal genes and gene products of a patient’s tumor biopsy have made it
feasible and cost-effective to apply individualized approaches to cancer treatment.
• Molecular analysis increasingly being used to select the most effective cytotoxic and biotherapic drugs
for a particular patient’s tumor.
Chemotherapy pharmacogenomics for initial selection of treatment - BUT, even if a tumor is initially
sensitive to the chosen drugs, resistance develops quickly – need to address ways to avoid or reverse
drug resistance at the beginning.
• After chemotherapy, emergence of multi-drug resistance including resistance to drugs that the cells
were never exposed to.
• Need to:
• Identify genes that contribute to drug resistance
• Consider approaches that can interfere with function of drug resistance molecules.
• Therapeutic approaches that have mechanisms of action different from commonly used drugs,
potentially synergistic and no overlapping toxicities.
 Summary 2 (Bernal et al, 1997)
Targeted Biotherapies, such as tyrosine kinase inhibitors, can interfere
with action of some drug resistance molecules.
Potential Targets: Surface membrane drug resistance molecules (eg.
ATP binding cassette transporters) - antibodies and antibody
fragments; Intracellular drug resistance pathways - small molecules
Immunotherapy using dendritic cells, cytokine induced killer ,
chimeric antigen receptor T (CARTcells have been developed that can
target even the cancer cells that are resistant to drugs.
Personalized molecular profiling of a patient’s cancer can also be used
to create specific targeting to specific antigens of the tumor.
- Future treatments for advanced cancers - may involve the application
of molecular profiling to design personalized combinations of
chemotherapy, biotherapy and molecular-targeted immune
therapies.
 Summary 3 (Bernal et al, 1992)
Intertumor Heterogeneity - Tumors even within the same diagnostic
category are not the same at the molecular levels, and particularly in
their genetic expression – influencing the response of each tumor to
chemotherapy, biotherapies or immune therapies
Intratumor Heterogeneity – within each tumor, there is a great deal of
cellular heterogeneity at the molecular level, so the there are
subpopulations that differ in growth kinetics and response to
treatment.
Cancer Stem Cells - present in each patient’s cancer tend to be resistant to
chemotherapy and radiation. Even if the non-stem cell populations within a
cancer responds to chemotherapy or radiation, the cancer stem cells are
usually not eradicated and will be responsible for recurrence of the cancer,
which becomes more resistance and aggressive after chemotherapy and
radiation. Cancer stem cells are be identified by their molecular signatures
and can be controlled by molecular-targeted immune therapies.
 Major Technological Development in Health – Personalized Molecular-Cellular Medicine

1. Personalized Cellular Therapeutics

1. Live Immune Cell Therapies for Personalized Treatment of Cancer – Dendritic
Cells, Cytokine Induced Killer Cells, Chimeric Antigen Receptor T Cells
2. Live Cell Modulators of Immune Function – Mesenchymal Stem Cells, T-
Regulatory Cells
3. Modulators of Immune Cell Function - Immune Checkpoint Inhibitors
4. Replacement and Regenerative Live Cell Therapeutics – Adult and Umbilical Cord
Stem Cells (versus Embryonic, Aborted Fetal, Induced Pluripotent Stem Cells,
Animal Cells)
1. Neurological & Spinal 8. Pancreas
2. Eye 9. Kidney
3. Cardiac 10.Bladder
4. Vascular 11.Bones
5. Pulmonary 12.Joints & Connective Tissue
6. Gastro-Intestinal 13.Skin
7. Liver 14.Hair Follicle
Credence Research
 Personalized Molecular-Cellular Medicine
1. “Personalized Molecular-Cellular Medicine” - the use of molecular and cellular tools to individualize treatment, predict
appropriate therapies and prevent adverse health outcomes. This approach has made significant impact especially in the
field of oncology, leading to individualized treatments for each cancer patient.
2. Personalized medicine is a new framework for medical care that involves modelling and simulation of a disease on the
basis of underlying mechanisms. This strategy must replace the 20th century paradigm of defining disease by pathology
or associated signs and symptoms, conducting outcomes research that is based on the presence or absence of the
disease syndrome, and old tenets of randomized clinical trials in “evidence-based medicine” that population-based
statistical averages. Instead, we need to realize that the “one size fits all approach” does not work in medicine and new
technologies, molecular and cellular technologies including next-generation sequencing, the ‘omics’, molecular-cellular
biology and powerful computers provide basic information about the uniqueness of each individual, in their
predisposition to disease, the course of their disease, and response to treatments.
3. The framework from which medicine operated in the 20th century was useful for infectious disease and disorders with a
single etiology. Medical care for complex inflammatory disorders, functional disorders and cancers has not improved to
the same degree, and additions of powerful new technologies have not appreciably improved patient outcomes. A new
framework that is effective in individual patients is needed.
4. The personalized medicine approach not only provides better care than other approaches, it probably saves money.
Savings are made by minimizing diagnostic costs, avoiding high-risk diagnostic procedures, limiting the use of therapies
to patients who are most likely to benefit, and addressing the etiology to minimize the rate of disease progression, and
thereby avoiding the high cost, suffering and disability associated with advanced disease.
5. In contrast to current practice, health care should now not focus on disease, but on health; not on disease status but on
trajectory; not on treatment based on pathology but on avoiding pathology; not on treatment trial and error but on
selection of the best treatment with continuous optimization.
DC Whitcomb 2012
 Personalized Molecular Medicine
1. Providers of personalized medicine for cancer identified four domains which still
cause legal, ethical and social concerns:
1. informed consent for cancer genomic testing,
2. privacy, confidentiality, and disclosure of genomic test results,
3. access to genomic testing and targeted therapies in oncology,
4. costs of scaling up pharmaco-genomic testing and targeted cancer therapies.
2. In addition, there are issues of liability for practitioners and institutions that expose
patients to toxic drugs not guided by molecular testing or not referring patients to
practitioners and institutions that carry out and interpret molecular testing.
3. These specific concerns are not unique to oncology, or even genomics. However,
those most invested in the success of personalized medicine view oncologists’
responses to these challenges as precedent-setting because oncology is farther along
the path of clinical integration of genomic technologies than other fields of medicine.
4. The rapid emergence of personalized medicine approaches in clinical oncology
provides an important opportunity for identifying and managing potential frictions
and pitfalls that emerge as health care paradigms shift in these directions.
M McGowan et al 2014
 Personalized Molecular Medicine

1. Next-generation sequencing—NGS - rapidly evolving technology that uses massive parallel sequencing with
computer reconstruction of DNA sequence fragments to generate a partial or full genomic sequence. It is a major
advance over genome-wide association study (GWAS), because it reveals disease-causing genetic variants rather
than nearby markers and is faster and costs less than traditional Sanger sequencing of a single large gene. Clinical
application of next-generation sequencing reveals a tremendous number of genetic variations among individuals,
with many new mutations and multiple susceptibly risk factors seen in each patient. It is nearly impossible to
resolve the complexity of a patient’s genome using epidemiology, statistics and patient subclassification strategies
within the framework of 20th century medicine. However, application of next-generation sequencing into new
paradigms will be the foundation of medicine in the 21st century.
2. Using expensive technology to diagnose patients on the basis of the old framework is inefficient and ineffective,
because the complex data sets cannot be adequately interpreted. What is needed is a new framework so that new
types of data relevant to individual patients can be integrated into a treatment plan that targets the true aetiology
precisely.
3. In contrast to current practice, health care should now not focus on disease, but on health; not on disease status
but on trajectory; not on treatment based on pathology but on avoiding pathology; not on treatment trial and
error but on selection of the best treatment with continuous optimization .The process of developing disease
models is challenging but can be accomplished using reverse engineering, an approach used to understand other
complex systems. The system is broken into its component parts, the function of each component is modelled, the
modelled components are reintegrated, and the effects of the integrated components on overall processes are
simulated under multiple conditions. Such modelling and simulation is that it can be done on a patient-by-patient
basis, incorporating the unique variables that each patient possesses.
DC Whitcomb 2012
 How is Personalized Molecular & Cellular Medicine Applied to
the Field of BioRegenerative Medicine (BRM)?
BioRegenerative Medicine (BRM) - Innovative medical therapies that involve
the engineering of living cells, tissues and organs for the purpose of preserving
and enhancing organ function to prevent disease, maintain wellness and to
restore or replace organ function lost or impaired due to disease, injury or aging
with the purpose of improving the quality of life.
Its scope goes beyond stem cell transplantation and includes genomics and
molecular tests to identify the underlying factors of each patient’s disease or
risk of disease, tissue / biomaterials engineering, growth and differentiation
factors and energy metabolism.
It makes use of cutting-edge techniques and technologies.
• It takes advantage of the body’s inherent mechanism of healing itself.
• Using one’s own cells avoids the problem of an unwanted or adverse immune reaction
occurring.
It falls under innovative practice, uses the tools of Personalized Molecular
Medicine and Value-Based Medicine.
 9
Personalized Molecular Medicine – Tomorrow’s Medicine Applied Today
• “Personalized Medicine” - Treatments are directed to individual patients based upon their
molecular profile
• Difference from another term used “Precision Medicine” – precision based upon
identification of catagories of molecular markers but not necessarily individualized.
• It is less prone to unwanted and unintended side effects.
• Requires extensive bioinformatics analysis to allow prediction and simulation of the
underlying basis of each patient’s condition and the response to treatment or combinations
of treatments.
• Does not rely on population-based statistical averages, guidelines or standards of care that
are based on trials from randomized groups of patients diagnosed and grouped by traditional
diagnostic techniques.
• Requires the expertise of multidisciplinary teams of scientists – in molecular biology,
molecular biology, molecular genetic, pharmacogenetics, genomic bioinformatics – working
alongside multidisciplinary teams of physicians and health care providers.
43
 Personalized Molecular Medicine:
Scientific Areas
Molecular Genetics and Embryology
Cell Biology and Biochemistry
Cellular and Tissue Electrophysiology
Bio-Engineering and Bio-Manufacturing
Organic Chemistry
Biophysics and Quantum Mechanics
Biocomputation and Computer Modeling
Artificial Intelligence and Robotics
 Specimen cDNA Conversion
Collection
http://www.made-in-china.com/showroom/fukangmedical/product-
detailLqvEjiSuCTRK/China-Yellow-Top-Vacuum-Vascular.html

http://www.newworldencyclopedia.org/entry/Bone_marrow

RNA Extraction
http://www.dnassequencing.com/2011/01/08/dna-synthesis-5/

Cancer
http://theolderman.com/2010/11/a-twist-of-fate/
Molecular cDNA MicroArray

Profiling
http://www.ovca.org/ovarian-cancer-treatment/chemotheraphy

Selection of Appropriate Chemotherapeutic

and Biological Agents
Procedure

Cancer Molecular Profile vs

Published Clinical Studies Data Analysis
 Drug Resistance gene expression panels of patient cancers –
cDNA RT-PCR (partial list)

• Drug Resistance: ABCB1 (PGY1, MDR1), ABCC1 (MRP1), ABCC2 (cMOAT, MRP2),
ABCC3 (MOAT-D, MRP3), ABCC5 (MRP5), ABCC6 (MRP6, CFTR), ABCG2 (BCRP), BAX,
BCL2, BCL2L1 (bcl-x), MVP (LRP), RB1, TOP1, TOP2A, TOP2B, TP53 (p53).
• Drug Metabolism: ARNT, BLMH, CLPTM1L (CRR9/DKFZP761M2324), CYP1A1, CYP1A2,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, DHFR, EPHX1,
GSK3A, GSTP1, NAT2, SOD1, SULT1E1 (STE), TPMT.
• DNA Repair: APC, ATM, BRCA1, BRCA2, ERCC1, ERCC3, MSH2, XPA, XPC.
• Cell Cycle: CCND1 (cyclin D1), CCNE1 (cyclin E1), CDK2, CDK4, CDKN1A (p21Waf1,
p21Cip1), CDKN1B (p27Kip1), CDKN2A (p16Ink4), CDKN2D (p19Ink4d).
• Growth Factor Receptors and Oncogenes: EGFR (ERBB1), ERBB2 (Her-2), ERBB3,
ERBB4, FGF2, IGF1R, IGF2R, MET, Kras, Alk.
• Hormone Receptors: AR, ESR1 (ER-alpha), ESR2 (ER-beta-cx), PPARA, PPARD, PPARG,
RARA, RARB, RARG, RXRA, RXRB.
• Transcription Factors: AHR, AP1S1, ELK1, FOS (c-fos), HIF1A, MYC (c-myc), NFKB1,
NFKB2, NFKBIB (TRIP9), NFKBIE, RELB (l-rel), TNFRSF11A (Rank).
 Program in Molecular Profiling for Personalized Cancer Treatments

• Pre-Clinical: Cell lines, fixed tissues and human tumors implanted in immune-incompetent mice
• Histologic types, differentiaion markers of human cancers compared with human fetal and adult normal tissues
• Series of drug sensitive and drug resistant cancer cells
• 2D gel electropheresis, isolation of membrane antigens, monoclonal and polyclonal antibody development,
immunoblot, immunohistochemistry of fixed tumor samples, DNA probe development, gene cloning, Fluorescence in
situ hybirdization
• Live cell analysis – flow cytometry, cell sorting, live cell confocal microscopy, mitochondrial membrane potential
Rh123, JC1; calcium fluxes, apoptosis
•
• Clinical Programs I: IRB clearance, Ethics Review, patient informed consent
• Initial Diagnosis – collection of fresh and fixed tumor tissue
• Fresh isolates of cancer cells –
• flow cytometry – analysis of cancer antigens, cancer stem cell markers
• cell sorting – collection of live cancer cells
• Live cell confocal fluorescence microscopy – temperature controlled, media infused chambers for live single cell
analysis of mitochondrial potential, calcium fluxes, before and during exposure to treatments with chemotherapy,
biotherapy and immune therapies
 Program in Molecular Profiling for Personalized Cancer Treatments
• Clinical Programs II:
• Frozen and fixed tumor tissue
• Isolation of mRNA, cDNA synthesis, cDNA microarray, RT-PCR for genes related to treatment
resistance, PCR mutational analysis EGFR (ex 19 response to erlotinib, gefitinib), kRas (mut less resp
cetuximab), EML4-Alk trans (resp to crizotinib); fluorescence in situ hybirdization, her2neu, Bcr-Abl
• Isolation of membrane antigens, construction of liposomal constructs of antigens
• Collection of hematopoietic stem cells - leukapheresis
• Induction of stem cell differentiation to dendritic cells, sensitization with personalized cancer
antigens, injection intradermally
• Preparation and expansion of cytokine induced killer cells, sensitized with personalized dendritic
cells in vitro, infused intravenouisly..
• Monitoring of specific anti-cancer immune response in vivo – monthly collection of live immune cells
– B cells – patient antibody immunoblot to cancer antigens, T Cells – interferon gamma release after
cancer antigen challenge, target cancer cell cytotoxicity.
• Monitoring of clinical response – CAT scans, Fluoro-Deoxyglucose Positron Emission Tomography for
changes in viable cancer signals even when no change in CAT scan measurements, patient symptoms,
quality of life measures, disease free interval, overall survival compare with” standard therapy.”
• If no response, mixed response, progression of tumor, rebiopsy tumor when feasible, repeat
analysis, modify treatment strategy.
 ERCC-1 and CDDP Resistance in Ovarian Cancer
• ERCC-1 is a protein encoded by the excision repair cross-complementation group 1 gene. The ERCC1 protein belongs to the nucleotide
excision repair (NER) pathway and is activated when DNA is damaged by platinum-based chemotherapeutic agents such as cisplatin and
carboplatin.
• Although platinum is largely used in ovarian cancer, approximately 20% of patients do not respond to treatment, and early responders
present risk for disease progression. Genetic alterations of the cellular repair system modify the patient response to chemotherapy.
• Increased NER activity and a high expression of ercc1 mRNA have been found to play a crucial role in cisplatin-resistant ovarian cancer
cell lines . An elevated expression of ERCC-1 in ovarian cancer has also been correlated with the development of resistance to platinum-
based therapy .
• An SNP in codon 118 of the ercc1 gene is responsible for a C to T transition that can be used as a marker of ERCC-1 expression. T
substitution in ovarian cancer cells is associated with lower levels of ercc1 mRNA and consequently a reduced DNA repair capability.
• In ovarian cancer patients has stressed the association between the C/C genotype and increased resistance to platinum-based
chemotherapy, consistent with a higher transcription activity of the AAC codon.
• Ercc1 SNP codon 118 influences ovarian cancer treatment and survival. Consistent with previous studies regarding the C/C genotype
and its relationship with poor treatment response (38), these authors found that ovarian cancer patients harboring C/C genotypes were
less responsive to chemotherapy and maintained a higher risk for disease progression and death in contrast to those harboring C/T or T/T
genotypes.
• Ovarian cancer patients with a high ERCC-1 expression or the C/C genotype at codon 118 may benefit from a platinum/paclitaxel
combination, whereas patients with a low ERCC-1 expression or the C/T or T/T genotype may respond well to platinum without
paclitaxel.
• In vitro, paclitaxel blocks ERCC-1 activity, inhibits the DNA repair system and prevents cellular resistance to drug-induced DNA
damage.
• If ERCC-1 expression in ovarian cells is arrested by paclitaxel, then paclitaxel treatment may offset the chemoresistance to platinum
therapy in ovarian cancer patients.
ERCC1 Polymorphism and Survival of Ovarian CA Patients on Carbo-Taxol
Kim et al, 09
ERCC1 mRNA Expression and Survival of Ovarian CA Patients on
Platinum (A,C) v Platinum + Paclitaxel (B,D) Smith et al, 07
 SPARC Expression in Cancer
The matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) - is an extracellular Ca2-binding matricellular glycoprotein:
• associates with cell populations undergoing migration, morphogenesis, and differentiation.
• its principal functions in vitro are counteradhesion and antiproliferation
• plays major roles in regulation of cell adhesion and proliferation
• influences tumorigenesis and metastasis (Yiu et al, 01).

Overexpression of SPARC is associated with increased tumor invasion and metastasis, leading to poor prognosis in multiple tumor types:
• including breast, prostate, esophagus, gastric, colorectal, liver, lung, kidney, melanoma, bladder, head and neck, thyroid, and brain cancers.

But in ovarian cancer, SPARC also abrogates carcinoma cell adhesion, a key step in peritoneal implantation.
• SPARC inhibits integrin-mediated ovarian cancer cell adhesion to extracellular matrix proteins, as well as to peritoneal mesothelial cells mediated
in part by - attenuation of cell surface expression and clustering of v-integrin subunit, v3- and v5-heterodimers in ovarian cancer cells.
• SPARC suppressed both anchorage-dependent and -independent activation of AKT and mitogen-activated protein kinase survival signaling
pathways in ovarian cancer cells (Said et al, 07).

The effects of tumor SPARC as a negative regulator of ovarian cancer are mediated through decreased recruitment of macrophages and
downregulation of the associated inflammation (Said et al, 08).
• reduces macrophage chemoattractant protein-1 production and its macrophage chemotactic effect
• attenuates the response of ovarian cancer cells to the mitogenic and proinvasive effects of macrophage chemoattractant protein-1 and decreased
macrophage-induced cancer cell invasiveness. Overexpression of SPARC in
• Decreases macrophage- and mesothelial cell–induced production and activity of interleukin-6, prostanoids (prostaglandins E2 and 8-isoprostanes)
as well as matrix metalloproteinases and urokinase plasminogen activator.
• effects of SPARC overexpression in ovarian cancer cells were mediated, in part, through inhibition of nuclear factor-κB promoter activation.

SPARC was first identified as a glycosylated 43-kDa secreted protein with high binding affinity to albumin, prompting investigations for targeted
therapy.
 SPARC and Response to Chemotherapy in Cancer

• Response to nab-paclitaxel was higher for SPARC-positive head and neck cancer patients
(10/12, 83%) than SPARC-negative patients (1/4, 25%). (Desai et al, 09)
• SPARC-negative patients exhibited significantly lower response than the overall response
rate among all 60 patients (1/4, 25% vs 45/60, 75%).
• Because of SPARC-albumin interaction, tumoral SPARC facilitates the accumulation of
albumin in the tumor and increases the effectiveness of nanoparticle albumin-bound
paclitaxel (nab-paclitaxel).
• Nanoparticle albumin-bound paclitaxel (nab-paclitaxel), Abraxane, is a Cremophor-free,
albumin-bound 130-nm particle form of paclitaxel.
• It was approved by the U.S. FDA in January 2005 for the treatment of breast cancer after
failure of combination chemotherapy for metastatic disease or relapse within 6 months of
adjuvant chemotherapy.
• Compared with conventional paclitaxel formulation known as Taxol nab-paclitaxel does
not require the use of Cremophor-EL, thus avoids the severe toxicities associated with this
vehicle.
Higher SPARC – greater response to nab paclitaxel v docetaxel
Improved effectiveness of nanoparticle
albumin-bound (nab) paclitaxel versus
polysorbate-based docetaxel in multiple
xenografts as a function of HER2 and
SPARC status.
Desai, Neil; Trieu, Vuong; Hwang, Larn;
Wu, Rujin; Soon-Shiong, Patrick;
Gradishar, William

Anti-Cancer Drugs. 19(9):899-909,

October 2008.
DOI: 10.1097/CAD.0b013e32830f9046

Fig. 6 Relative efficacy of nanoparticle

albumin-bound (nab)-paclitaxel compared
with polysorbate-based docetaxel in tumor
xenografts. The relative antitumor efficacy
(polysorbate-based docetaxel tumor
volume/nab-paclitaxel tumor volume) in
different tumor xenografts was calculated
using data from 120 mg/kg nab-paclitaxel
group (15 mg/kg for MX-1) and 15 mg/kg
polysorbate-based docetaxel group on day
32 posttreatment. *Relative efficacy could
not be calculated for MDA-MB-231 tumors
as nab-paclitaxel treatment resulted in
complete response in all 10 mice at day 31
posttreatment. HER2, human epidermal
growth factor receptor-2; SPARC, secreted
protein, acidic and rich in cysteine.

© 2008 Lippincott Williams & Wilkins, Inc. Published by Lippincott Williams & Wilkins, Inc. 8
U.S. regulators have approved a first-of-a-kind test that looks for mutations in hundreds of cancer genes
at once, giving a more complete picture of what’s driving a patient’s tumor and aiding efforts to match
treatments to those flaws. The U.S. Food and Drug Administration approved Foundation Medicine’s test
for patients with advanced or widely spread cancers, and the Centers for Medicare and Medicaid Services
proposed covering it. The dual decisions, announced late Thursday, will make tumor-gene profiling
available to far more cancer patients than the few who get it now, and lead more insurers to cover it.
“It’s essentially individualized, precision medicine,” said Dr. Kate Goodrich, chief medical officer for the
Medicare oversight agency. Currently, patients may get tested for individual genes if a drug is available to
target those mutations. It’s a hit-and-miss approach that sometimes means multiple biopsies and wasted
time. In lung cancer alone, for example, about half a dozen genes can be checked with individual tests to
see if a particular drug is a good match. The new FoundationOne CDx test can be used for any solid tumor
such as prostate, breast or colon cancer, and surveys 324 genes plus other features that can help predict
success with treatments that enlist the immune system. “Instead of one or two, you have many” tests at
once from a single tissue sample, said the FDA’s Dr. Jeffrey Shuren. The tests give better and more
information to guide treatment and can help more patients find and enroll in studies of novel therapies,
he said.
“This will be a sea change” for patients, said Dr. Richard Schilsky, chief
medical officer of the American Society of Clinical Oncology, the
association of doctors who treat the disease.
Foundation Medicine, based in Cambridge, Massachusetts, and others
have sold tumor profiling tests for several years under more lax rules
governing lab-developed tests. But insurers have balked at paying for the
tests, which cost around $6,000. Now, the FDA’s approval gives
assurance of quality, Shuren said, and the government’s proposed
coverage for Medicare and other public insurance programs means
private insurers will more likely follow.
Coverage is proposed for patients with recurrent, widely spread or
advanced cancers, in people who have decided with their doctors to
seek further treatment and who have not previously had a gene
sequencing test. “A lot of these folks have run out of treatment options,”
The impact is expected to be greatest on lung cancer, since so many of
those tumors are found at an advanced stage and multiple gene-
targeting drugs are available to treat it. In mid-November, the FDA also
approved a gene-profiling test developed by Memorial Sloan Kettering
Cancer Center, but it’s used almost exclusively on patients at that cancer
center and is not envisioned to be a widely available commercial test.
The federal decisions will make gene sequencing a more routine
component of cancer care, “just like we normally look with a
microscope” to classify the stage of a patient’s disease, said Dr. David
Klimstra, pathology chief at the cancer center.
Another leader in this field, Caris Life Sciences, says it also intends to
pursue FDA approval for its widely used tumor profiling test, sold now
through lab certifications. It’s also working on a newer tool to profile
tumor genes from a blood sample.
 Personalized Molecular-Cellular Medicine:
Molecular-Cellular Analytics = Basis for Molecular-Cellular Therapeutics
• Stem cells – the origin of dividing and maturing cells
• Normal vital processes – self renewal and specialized
functions in the natural and continuous process of self –
healing
• Hype and misconceptions
• Benefits vs Dangers
• Conflicting commercial interests, monopolies & control
• Ethical, moral, legal issues – conflict of laws
Embryonic Stem cells
Induced Pluripotent Stem cells
 Legal Aspects of Personalized Molecular and Cellular Medicine
1. Providers of personalized medicine for cancer identified four domains which still
cause legal, ethical and social concerns:
1. informed consent for cancer genomic testing,
2. privacy, confidentiality, and disclosure of genomic test results,
3. access to genomic testing and targeted therapies in oncology,
4. costs of scaling up pharmaco-genomic testing and targeted cancer therapies.
2. In addition, there are issues of liability for practitioners and institutions that expose
patients to toxic drugs not guided by molecular testing or not referring patients to
practitioners and institutions that carry out and interpret molecular testing.
3. These specific concerns are not unique to oncology, or even genomics. However,
those most invested in the success of personalized medicine view oncologists’
responses to these challenges as precedent-setting because oncology is farther along
the path of clinical integration of genomic technologies than other fields of medicine.
4. The rapid emergence of personalized medicine approaches in clinical oncology
provides an important opportunity for identifying and managing potential frictions
and pitfalls that emerge as health care paradigms shift in these directions.
M McGowan et al 2014
 Conflict of Laws and Regulations in Stem Cells
SD Bernal, Int. Jour. of Law and Medicine, 2014

• As stem cell therapies expand to an increasing number of applications, the

market is expected to increase toUS$8.8 billion by 2016, although some
estimates expected it to reach more than US $10 billion by 2023
• In contrast to the well-established bone marrow and umbilical cord
(BM/UC) stem cell treatments, the emergence of other types of stem cells,
such as embryonic stem cells (ES), aborted fetal (FS), genetically altered
(GA), and induced pluripotent (IPS) stem cells, led to major controversies
and wide differences in opinion, leading to conflicting laws and regulations
on stem cells worldwide.
• In the Philippines, autologous and allogeneic adult and umbilical cord stem
cells are permitted, genetically altered and IPS stem cells are restricted,
and embryonic and aborted fetal stem cells are prohibited.
 Points of Emphasis
• Distinguish:
• Type 1 – human adult and umbilical cord stem cells
• Type 2 - embryonic, aborted fetus, genetically altered and xenobiotic – animal and
plant stem cells.
• Laws and Regulations – have to distinguish these two general types of stem
cells.
• Serious safety issues for Type 2 – Mandate strict clinical trials
• Stem Cell Procedures versus Drugs
• Global Competition
• Conflict of Laws
• Regulations on stem cell practices – weed out unsafe and unethical
practices, focus on:
• Safe practices of stem cells
• Multidisciplinary clinical specialists and scientific experts
• Data gathering on safety and efficacy, sharing information and training.
 PESTLE - Global Factors in Stem Cells – Part 1
• Political Factors
• Politics of Stem Cells, agenda of a few “international associations,” U.S. FDA, patients ignoring going abroad, now
pressuring other countries to “regulate”, but advanced countries broadening patents. Business competition and
scare tactics used in the U.S. and E.U. on “medical tourism” and “stem cell tourism”
• Religious organizations (eg. Vatican) , direct influence on individual attitudes beyond state legislation
• Economic Factors
• The business of umbilical cord blood (UCB) banking – UCB v Umb Cord SC, directed v non-directed banking, Phil v
foreign banks, banking v patient treatment
• Funding of SC research, Funding of clinical trials in the U.S. and E.U. – effective “barrier to entry”
• Standard of care v Innovative practice and patients paying for treatments abroad
• Business competition and its effect on reporting of “scientific data,” journal articles and press releases – Korean
fraud, misinformation by some U.S. doctors, influencing intl. associations, ISSCR
• Social Factors
• Impact on professionals in health, science, engineering, service industries, tourism; sustainablility provides
justification for public support of infrastructure.
• Misinformation by individuals and groups with vested interests in state and private money –Contrary to their
proclamations, increasing number scientists recognize versatility of adult and umbilical cord SC
• Demands by Patients and Patient Rights Groups
• Rapidly fatal illnesses with no effective treatments or very toxic treatments (cancer, neurodegenerative diseases
eg. XDP, ALD, ALS, immune disorders)
• Chronic diseases that progress despite pharmaceutical treatments (diabetes, heart disease, COPD)
• Aesthetics, wellness and prevention
• Technological Factors PESTLE - Global Factors in Stem Cells – Part 2
• Embryonic SC v. Adult SC v. Umbilical Cord SC v.
• Control of cell growth, molecular factors v Control of cell differentiation, molecular markers
• Established, safe CT practices worldwide– The “Good”
• adult SC (autologous – patient’s own and allogeneic – matched donor, related or unrelated)
• umbilical cord SC (related or unrelated)
• Problems with safety – the “Bad”
• Embryonic SC
• Aborted Fetal SC
• Problems with manipulations that have potential to cause mutations - the “Ugly”
• induced pluripotent SC – manipulations that make cells behave like embryonic cells
• Genetically modified SC – complications with mutations, harm from vectors
• Legal issues
• Legislation worldwide – banned, restricted, allowed, encouraged
• FDA regulations, EU directives, pressure to “harmonize” laws, hidden agendas
• Conflict of laws, US Federal v State, US State v State, EU member states, Russia, China, India, ASEAN
• Intellectual property law and patent litigation
• Insurance coverage and pricing of treatments
• Environmental Factors
• Biohazards from genetic manipulations
• Sources of materials from natural sources eg. plants
• Dangers of sources of cellular materials from animal sources, eg. sheep
 Problematic Stem Cells: Embryonic SC
• Embryonic stem cells make tumors containing multiple different cell types.
Formation of tumors is the "gold standard" test of whether a stem cell is, in fact,
an embryonic stem cell that is capable of producing multiple mature tissues.
Tumor formation is, therefore, not merely an incidental problem with ESCs - it is a
fundamental aspect of the biology of ESC.
• Embryonic stem cells, just like any other cells or organs from another person,
would be rejected by the immune system of the patient, unless a very close
immune match, or human cloning is performed. Yet unlike conventional organ
transplant, injected stem cells spread throughout the body and they cannot be
removed when the patient's body rejects them. The patient may then develop a
fatal immune rejection.
• Ethical objections – religious groups contend that – “the extraction of stem cells
from embryos, which involves the willful taking of human life — the embryo is
human life and not just a ‘clump of cells’ — is considered morally and ethically
wrong in every instance.”
 Problematic Stem Cells: Aborted Fetal SC
• Similar to ESC, fetal SC have problems of uncontrolled
proliferation and carcinogenic capacity.
• In fact, patients injected with fetal SC for neurological
diseases have already been reported to develop tumors.
• Similar to ESC, immune rejection is also a problem.
• Ethical objections – the use of materials from aborted
fetuses- whether spontaneous or induced is considered
to be morally objectionable.
PLoS Med 6(2): e1000029. doi:10.1371/journal. pmed.1000029
Issues on Safety: Genetically altered (Induced Pluripotent
Stem Cells
• Similar to ESC, IPS have problems of uncontrolled
proliferation and carcinogenic capacity.
• Developed initially from adult skin cells transfected
with viral vectors containing oncogenes (Yamanaka).
• More recently, IPS developed without viral vectors but
still transfected with cancer genes.
• Gold standard of confirmation of IPS - teratoma
tumor formation.
• Interesting for studies on the basic biology of cell
differentiation (and cancer causing genes) but not for
injecting into patients.
 Issues on Safety: Animal “fresh cells”
Fetal sheep (Niehans) , calf cells – banned in the U.S. since 1985; serious immunological reactions,
fatal complications, fatal gas gangrene, polyradiculitis, leukoencephalitis, Guillian-Barré syndrome,
immune complex vasculitis, encephalopathy
• Gelband H and others. Cellular treatment. In Gelband H and others. Unconventional cancer
treatments. Washington, DC, U.S. Government Printing Office, 1990. pp 97-98.
• Kuettner H. Verimpfung an stelle der transplantation hochwertiger organe. Zentralblatt fur
Chirugie 1:390-397, 1912., as cited in Popov IM and others. Journal of the International
Academy of Preventive Medicine 40:74-82, 1977.
• Unproven methods of cancer management: Fresh cell therapy. CA -- A Cancer Journal for
Clinicians 41:126-128, 1991.
• Lindeman B, Cubbison C: Cellular therapy: A shabby clinic offered rejuvenation but delivered
death. Today's Health 53:36-41, 1975.
• de Ridder M and others. Two cases of death following cell therapy. Deutsche Medizinische
Wochenshrift 112:1006-1009, 1987.
• Goebel HH and others. Fresh cell therapy followed by fatal coma. Journal of Neurology 233:242-
247, 1986.
• Cell therapy. FDA Talk Paper T84-78. Rockville, MD: Food and Drug Administration, Nov 5, 1984.
• Last PM. Cell therapy: A cruel and dangerous deception. A drama in three acts. Journal of
Paediatrics and Child Health 26:197-199, 1990.
• Stephen Barrett, Cellular Therapy
http://www.quackwatch.org/01QuackeryRelatedTopics/Cancer/cellular.html
 Effective and Safe: Adult and Umbilical Cord Stem Cells
•

•
 Legitimate Uses of Stem Cells
• Hospital Based Programs for Stem Cell Transplantation:
The Medical City, Lung Center, National Kidney & Transplantation Institute
• Specialized Clinical Facilities
• Multidisciplinary Clinical Teams
• Subspecialty Board Certified Physicians
• Sterile laboratory facilities
• Advanced cell and molecular biology
• Participation of Academic Departments – Ateneo School of Medicine and
Public Health, Ateneo School of Science and Engineering, UP College of
Medicine, UP Molecular Biology and Biotechnology
• Accreditation by Department of Health, Food and Drug Administration,
Joint Commission International (TMC)
 Innovative Practice and Clinical Trials
Helsinki Convention
World Medical Association
Ethical Principles for Medical Research Involving Human
Subjects
Ethical uses of Innovative Treatment:
• Article 35: “In the treatment of a patient, where proven interventions
do not exist or have been ineffective, the physician, after seeking
expert advice, with informed consent from the patient or a legally
authorized representative, may use an unproven intervention if in the
physician’s judgement it offers hope of saving life, re-establishing
health or alleviating suffering.”
 Innovative Practice and Clinical Trials
Helsinki Convention
World Medical Association
Ethical Principles for Medical Research Involving Human Subjects
Ethical uses of Innovative Treatment:
• Article 35: Whenever possible, the innovative treatment should also be
made the object of research, designed to evaluate its safety and efficacy.
• However, unlike formal clinical trials which are based on gathering data
rather than benefiting an individual patient, the principle of innovative
therapy is not solely aimed at producing generalizable knowledge but
rather to improve an individual patient’s condition.
• Patrick L. Taylor, “Overseeing Innovative Therapy Without Mistaking it for Research: A Function-Based Model Based on Old Truths, New Capacities,
and Lessons From Stem Cells”, Journal of Law Medicine and Ethics 2010, p.287.
• Insoo Hyun, “Allowing Innovative Stem Cell-Based Therapies Outside of Clinical Trials: Ethical and Policy Challenges,” Journal of Law, Medicine and
Ethics, Symposium on Law, Science and Innovation, Summer 2010, p. 279.
 Stem Cells only small part of Personalized Molecular Medicine

Stem cells are not enough for regeneration.

Stem cells by themselves do not work.
Many other components are needed for tissue and organ
regeneration – growth and maturation factors, extra-cellular and
intracellular matrices, electrical signals and energy regulation.
Molecular medicine into account takes all factors needed to
restore tissue and organ structure and function.
Molecular Medicine leads to Personalized Medicine – at the
molecular level, no two individuals, no two diseases are the same.
Regeneration has to be directed to the unique molecular defects in
each disease state of each individual.
Requires specialized facilities, specialized scientific and clinical
expertise, cannot be done in a general hospital, and definitely not
in a clinic, hotel, or mall.
 Cellular and Molecular Components of Regenerative Therapy

1. Living Cells

a. Stem cell collection – adult, cord blood (not embryonic)
b. Cell Culture – expansion, induction, activation, sensitization
c. Epigenetic and genetic modifications of living cells
2. 3D Scaffolding or architecture
Extracellular – matrix, anchoring, intercellular
Intracellular – intermediate filaments, microfilaments, microtubules
3. Soluble Circulating Factors
Protein growth factors, protein differentiation factors
4. Electrical energy and signaling
Extracellular, intracellular, mitochondrial electrical potential
 Opportunities for Developing Countries
Through the recent advances in molecular biology, we are now able to replace,
repair, or enhancing organ function by bio-engineering living cells in culture
applied to BioRegenerative Medicine (BRM) with potential applications to
human medicine.
The technology goes beyond stem cells, involves integrative medicine and
basic sciences.
 Personalized Medicine becomes a reality with BRM – focusing on the
uniqueness of each individual at the personal and molecular levels.
 BRM is ETHICAL and SAFE, when done properly. Need major medical center
and university with basic science departments in chemistry, molecular biology,
physics, engineering.
 BRM –Competitive advantage of developing countries – BRM requires global
technologies and is labor-intensive. Opens opportunities for partnerships.
 Molecular Profiling
of Solid Tumors

Institute of Personalized Molecular Medicine

The Medical City, Philippines (not only a stem cell center)
 Accreditation
Joint Commission International
Department of Health, Food and Drug Administration
Philippines

TMC Laboratory of Regenerative Medicine

• Biosafety laboratory infrastructure
• Sterile procedures, Infection control
• Sampling handling, identity checks
• TMC Clinical Section – Institute of Personalized
Molecular Medicine
• Bioethics, Institutional Review, Management
• Informed consent, Patient protection
• Data collection, biostatistics
Collection of Adult Stem Cells - Leukapheresis
Collection of Adult Stem Cells – Bone Marrow Aspiration

commons.wikimedia.org
Stem Cell Maturation

commons.wikimedia.org
Stem Cell Culture in Incubators

Christopher Reeve Foundation

Stem Cell Expansion

Healthline
Stem Cell Maturation and Separation

HealthCentral
Regenerative Medicine Laboratory
The Medical City – positive pressure Operating Room type rooms
Cryogenic Liquid N2 Storage for Adult and Umbilical Cord Stem Cells
Gene Probes- Fluorescent In-Situ Hybridization (FISH)
Sterile Laminar Flow Hoods: Human Cell Culture for Transplantation
Real Time Polymerase Chain Reaction, Gene Isolation and DNA Sequencing
Aria III - Live Cell Sorter
Cell Analysis and Sorting Room
Aria III Live Cell Sorter – 5 lasers, 16 wavelengths
the first in Asia
 Fluidics Subsystem

• Hydrodynamic focusing guides particles in a single-file

stream through the cuvette
• Positive air pressure forces sample through an optically
gel-coupled cuvette flow cell
Sorting
• Droplet sorting is an efficient
method for distributing
populations of particles or
cells into collection containers
Preparative Cell Sorting of
Adult Stem Cells:

Aria III fractionation by

leukapheresis collected
stem cells of a 55 y/o male
with metastatic lung cancer,
TMC
CD45 is expressed by all
leucocytes and a plot of SS
versus CD45 generally gives
a good estimate of the most
important leucocyte sub-
types.

B cell: CD5, CD10, CD19, CD20, CD45,

Kappa, Lambda;

T cell: CD2, CD3, CD4, CD5, CD7, CD8,

CD45, CD56;

Myelomonocytic: CD7, CD11b, CD13,

CD14, CD15, CD16, CD33, CD34, CD45,
CD56, CD117, HLA-DR;

Plasma cell: CD19, CD38, CD45,

CD56, CD138.
Preparative Cell
Sorting of Umbilical
Cord Blood Stem
Cells:

Aria III fractionation of

UCB collected at TMC
Stem Cell and Immune Cell Fractionation and Live Cell Sorting: CD3, CD45, CD4, CD8
Live Cell Confocal 3D Microscope with CO2 temperature controlled
incubator – for live cell analysis of metabolic processes in real time
 Conflict of Laws and Regulations in Stem Cells
SD Bernal, Int. Jour. of Law and Medicine, 2014

• As stem cell therapies expand to an increasing number of applications, the

market is expected to increase toUS$8.8 billion by 2016, although some
estimates expected it to reach more than US $10 billion by 2023
• In contrast to the well-established bone marrow and umbilical cord
(BM/UC) stem cell treatments, the emergence of other types of stem cells,
such as embryonic stem cells (ES), aborted fetal (FS), genetically altered
(GA), and induced pluripotent (IPS) stem cells, led to major controversies
and wide differences in opinion, leading to conflicting laws and regulations
on stem cells worldwide.
• In the Philippines, autologous and allogeneic adult and umbilical cord stem
cells are permitted, genetically altered and IPS stem cells are restricted,
and embryonic and aborted fetal stem cells are prohibited.
35 y/o Doctor Presented with Carcinoma of Unknown Primary Increasing
on Chemotherapy
Mar 2004
 Ovarian Cancer – Chemotherapy is not enough - search for more effective treatments
with lasting remissions
• Ovarian cancer (OVCA) remains the leading cause of mortality for patients
with gynecologic malignancies, despite recent advances in post-surgical
chemotherapy.
• Numerous patients with OVCA are diagnosed at an advanced stage of the
disease, but may be treated with surgery followed by chemotherapy.
• Initially, OVCA may be chemotherapy-sensitive and the majority of patients
achieve clinical remission following primary surgical debulking and adjuvant
platinum-based therapy.
• However, the majority of patients who respond to such therapy ultimately
succumb due to relapse from recurrent and drug-resistant disease.
• Following initial treatment, 70-90% of patients relapse.
• With additional second-line chemotherapy, some patients return to partial or
complete remission. However, further relapse is likely to occur
 Immunotherapy for Metastatic Ovarian Cancer.
Abstract Bernal et al. 2012
A 35 year old woman with metastatic carcinoma who presented with large pelvic
masses and large metastatic lesions in the liver and lungs was initially diagnosed
with adenocarcinoma of unknown primary, later found to have metastatic ovarian
cancer. Her initial treatment consisted of chemotherapy with carboplatinum,
taxol and VP16, leading to a partial response but had she had persistent large
masses in the pelvis, and residual lesions in the liver and lungs. After 10 courses
of chemotherapy, she had increased pelvic pain with enlargement of a pelvic
mass. She then underwent resection of the pelvic mass, total abdominal
hysterectomy and bilateral salpingo-oopherectomy. A large cystic mass was found
in the left ovary which in histologic sections showed endometrioid
adenocarcinoma consistent with an ovarian primary. She was then treated with
dendritic cells (DC) fused with autologous ovarian carcinoma cells and liposomal
construct of ovarian carcinoma antigens. Following DC treatment, all her
metastatic lesions markedly decreased in size. Two years after initial diagnosis
she has shown no progression of tumor. This report describes a successful
treatment metastatic ovarian cancer with dendritic cells following chemotherapy.
Resolution of Lung Metastases
Mar 2004 Jan 2006
 Resolution of Liver Metastases
Mar 2004 Jan 2006
Enlargement then Resolution of Pelvic Masses
Mar 2004 Dec 2004

Jan 2006
Response of Ovarian Cancer to Immune Cell Rx
•Chemotherapy achieved initial mixed response but later
increased size in some lesions.
•Surgical resection (TAH-BSO) of increasing pelvic mass
because of increased pain, showed ovarian mass.
•Stem cell (dendritic cell vaccine) had minimal side
effects, led to progressive decreased size of all lesions.
CA125 normal, patient asymptomatic
•Patient unable to tolerate more chemotherapy because
of neuropathy, thrombocytopenia
Immune therapy: Dendritic cells and cytokine
induced killer cells, Chimeric Antigen Receptor T
(CART-T) Cells targeted to the antigens of the
patients tumor.

Monitoring of immune response in vivo:

B Cell antibody – immunoblot
T Cell Interferon-gamma release and cytotoxicity
Zhang et al,
2016
 Generations of CARs and Armored CAR-T cells for Improved Antitumor Therapy
(A) Chimeric Antigen Receptors and Cytokines Zhang et al,
2016
• 1. First-generation CARs, including activating receptors, such as CD3z;
• 2. second-generation CARs combine activating and costimulatory signals, such as
CD28;
• 3. third-generation CARs combined two costimulatory and activating signals, such as 4-
1BB, etc.;
• 4. fourth-generation CAR-T cells, also called “TRUCK” cells, are engineered with
additional inducible cytokines, which can secrete cytokines upon the activation of
CARs.
(B) Modified CAR-T cells - recognize tumor cells by their tumor-associated antigen in a
non-MHC restrictive manner. CAR signaling activates T cells, and the T cells
• then secrete cytokines, which kill tumor cells and induce them to attack other tumor
cells.
(C) The fourth-generation CAR-T cells with Dendritic Cells and Effector Cells - have the
additional advantage of activating the innate immune system, which recruits innate
immune cells (macrophages or DCs) to attack tumor cells and regulate the tumor
microenvironment.
CAR-T Preparation and Processing (Wang et al, 2016)
 Advantages of CAR-T Cells in Immunotherapy of Tumors
Han et al, 2017

• 1. CAR-T cells have ability to recognize antigens on any human

leukocyte antigen background - whereas natural T cell receptors
(TCRs) expressed on T cell surface need to be matched to the
patients' haplotype.
• 2. HLA expression may be downregulated on some tumor cells, and
these tumor cells may escape the TCRs-mediated immune response.
In contrast, CAR-T cells are still capable of eradicating these escaping
tumor cells.
• 3. CAR-T cells have a much wider range of potential targets on the
tumor cells in that not only traditional proteins but also
carbohydrates, glycoproteins and glycolipids could form the specific
targets for CAR-T cells.
Comparisons of Traditional T Cell Receptor with Chimeric Antigen Receptor Figueroa et al,
2015
Martin Fries, a 62-year-old
pharmacist from
Kissimmee, Fla., received CAR-T
cell therapy at Moffitt Cancer
Center in Tampa.
By Jonathan D. Rockoff
April 26, 2018 7:00 a.m. ET
The emergence of genetics-based medicines is pushing the cost of treating certain diseases to new
levels, forcing hospitals and health insurers to reckon with how to cover total costs per patient
approaching a million dollars. The therapies deliver new genes or genetically altered cells to tackle
some of the hardest-to-treat diseases, including in children. They come at a high
price: Novartis AG listed its newly approved cell therapy for cancer at $475,000, while Gilead
Sciences Inc. priced its rival drug at $373,000.
But the price of the drugs is just the beginning, hospitals and insurers say. Administering these therapies
can add hundreds of thousands of dollars to the tab, including lengthy hospital stays and use of other
services and medicines. It isn’t clear how much hospitals will get paid for these new treatments.
Current payment systems generally cover infusions of drugs, or episodes of hospital care, but aren’t set
up to deal with treatments that combine both. What is clear is that the total cost will be far more than
the list price of the drugs themselves.
“It is about systems that don’t work well and aren’t set up for cutting-edge new technologies” says Gary
Goldstein, a business manager at Stanford University’s health system, which is offering the new
treatments. “And if we don’t get this one right, what about the next one?”

Million-Dollar Treatment
A new wave of gene-based therapies for cancer and other diseases threatens to bring the cost of
treatment to a million dollars, because both the drug and related care are expensive.
One of the first genetics-based treatments was Gilead’s lymphoma drug Yescarta, approved last
October for use in patients who have failed other drugs. Yescarta is a form of cell therapy
known as CAR-T, for chimeric antigen receptor T-cells. It uses a patient’s own immune cells,
which are extracted, modified in a lab and then put back into the patient where they hunt
down and attack cancer. Martin Fries’s recent treatment with Yescarta included a 13-day
hospital stay, use of several other drugs and a variety of procedures that he anticipates will cost
between $750,000 and $1 million.

The 62-year-old pharmacist from Kissimmee, Fla., first spent a half-day at Moffitt Cancer Center
in Tampa hooked up to a so-called apheresis machine. It harvested his immune cells, which
were shipped to Gilead to be turned into the drug. Mr. Fries then received two chemotherapies
over three days to prepare his body for the altered cells. He was admitted to the hospital in
December, where the engineered cells were infused and he was monitored for side effects.
Monitoring usually costs a few thousand dollars a day, hospitals say. Mr. Fries says he was
treated for fever of 104.8 degrees and received a blood-plasma drug and a steroid to combat
neurological side effects.
Traditionally, hospitals get a lump sum for care they give in the hospital, including
drugs, or a payment for outpatient infusing of a drug that covers extra costs. But
hospitals say neither of those pay enough to cover their tab for drugs like CAR-T
therapies.
“What is the incentive then for hospitals to provide these therapies, which are
complicated and require a large investment of time and resources, if there is not a way
to at least recoup costs?” says Aaron Chrisman, director of Stem Cell Transplant and
Cellular Therapy Administration at the University of Chicago Medicine. Insurers don’t
question the benefits of the treatments but say they are affordable only if the cost is
spread over time. There is currently no payment mechanism to do so. “We either
need to accept we are going to see a bump-up in premiums—which I don’t think we
want, honestly–or we have to figure out how to pay for them over time,” says Michael
Sherman, chief medical officer of Harvard Pilgrim Health Care Inc. The federal
government’s Centers for Medicare and Medicaid Services hasn’t said whether it will
cover the drugs, according to hospitals. The decision is left to contractors around the
country that process Medicare claims, only some of which have said Medicare will pay.
Hospitals have been lobbying for a reimbursement pathway.
 Immune Cell Treatments for Cancer in Philippines
(2004-2019 – 650 Patients)
• Lung • Head / Neck •Non-Hodgkin’s Lymphoma
• Breast • Prostate •Acute Lymphocytic
• Ovary Leukemia
• Kidney
• Uterus •Acute Myelogenous
• Bladder Leukemia
• Liver • Melanoma •Chronic Lymphocytic
• Pancreas • Skin Squamous Leukemia
• Colon • Sarcoma •Hodgkin’s Lymphoma
• Esophagus • Mesothelioma •Multiple Myeloma
 Referrral of Patients for Molecular and Cellular
Treatments for Cancer in Philippines
• United States • Russia • India
• Canada • Czech Rep. • Pakistan
• Mexico • Slovakia • Sri Lanka
• Norway • Italy • Hongkong
• Denmark • France • China
• Latvia • Spain • Singapore
• Poland • Australia • Malaysia
• England • New Zealand • Thailand
Immune therapy: Dendritic cells and cytokine
induced killer cells, Chimeric Antigen Receptor T
(CART-T) Cells targeted to the antigens of the
patients tumor.

Monitoring of immune response in vivo:

B Cell antibody – immunoblot
T Cell Interferon-gamma release and cytotoxicity
Trends in Immunotherapy (2018) Volume 2
doi:10.24294/ti.v2i3.1064

CAR-T Therapy for Solid Tumors: Development of

New Strategies
Marvin de los Santos , Samuel D. Bernal
1 1,2*

1 GlobeTekPro, Los Angeles, California 91367, USA, Globetek Science Foundation, Manila, 1227,
Philippines,
midelossantos1215@gmail.com
2 Cedars-Sinai Medical Center, Section of Hematology & Oncology, 8700 Beverly Blvd, Los Angeles,

California 90048,
USA , sberne@ucla.edu.
ABSTRACT
The recent approval of two CAR-T therapies by US Food and Drug Administration (FDA) marks a very
significant
development in cell-based cancer immunotherapy. This milestone was demonstrated by the effectiveness of
eradicating
hematologic cancers using CD19-specific CARs. The success spurred development of immune cell
therapies for other
cancers, especially solid tumors. The generation of novel CAR constructs for these cancer types represents
Zhang et al, 2016
 Generations of CARs and Armored CAR-T cells for Improved Antitumor Therapy
(A) Chimeric Antigen Receptors and Cytokines
• 1. First-generation CARs, including activating receptors, such as CD3;
• 2. second-generation CARs combine activating and costimulatory signals, such as
CD28;
• 3. third-generation CARs combined two costimulatory and activating signals, such as 4-
1BB, etc.;
• 4. fourth-generation CAR-T cells, also called “TRUCK” cells, are engineered with
additional inducible cytokines, which can secrete cytokines upon the activation of
CARs.
(B) Modified CAR-T cells - recognize tumor cells by their tumor-associated antigen in a
non-MHC restrictive manner. CAR signaling activates T cells, and the T cells
• then secrete cytokines, which kill tumor cells and induce them to attack other tumor
cells.
(C) The fourth-generation CAR-T cells with Dendritic Cells and Effector Cells - have the
additional advantage of activating the innate immune system, which recruits innate
immune cells (macrophages or DCs) to attack tumor cells and regulate the tumor
microenvironment.
CAR-T Preparation and Processing (Wang et al, 2016)
 Advantages of CAR-T Cells in Immunotherapy of Tumors
Han et al, 2017

• 1. CAR-T cells have ability to recognize antigens on any human

leukocyte antigen background - whereas natural T cell receptors
(TCRs) expressed on T cell surface need to be matched to the
patients' haplotype.
• 2. HLA expression may be downregulated on some tumor cells, and
these tumor cells may escape the TCRs-mediated immune response.
In contrast, CAR-T cells are still capable of eradicating these escaping
tumor cells.
• 3. CAR-T cells have a much wider range of potential targets on the
tumor cells in that not only traditional proteins but also
carbohydrates, glycoproteins and glycolipids could form the specific
targets for CAR-T cells.
Comparisons of Traditional T Cell Receptor with Chimeric Antigen Receptor Figueroa et al,
2015
De Los Santos & Bernal 2019 Strategies for Improvement of CAR-T for Solid Tumors
 Strategies for Improvement of CAR-T for Solid Tumors
A. CAR-T cell trafficking.
B. Homing and extravasation of CAR-T cells to tumor site.
C. CAR-T cells infiltrating the Tumor MicroEnvironment (TME).
1. Tumor growth-inducing cells.
2. Immune cells and associated immune-suppressing cells
3. Non-cellular components of TME
D. Potentiating tumor targeting by fine-tuning scFv affinity of CAR.
E. Neutralizing CAR-T toxicity.
F. Targeting heterogeneous population of cancer cells in the tumor site.
G. Evading complexity of cancer genomic instability.
H. Bypassing immune checkpoint inhibition.
I. Circumventing down-regulation of antigen targets.
J. Molecular profiling of tumors to identify known and neo-antigens
 WT1-Expressing Malignancies Oka et al,
2007
• Hematopoietic malignant disorders
• Acute myeloid leukemia (AML)
• Acute lymphocytic leukemia (ALL)
• Chronic myelogenous leukemia (CML)
• Myelodysplastic syndromes (MDS)
• Non-Hodgkin lymphoma (NHL)
• Multiple myeloma (MM)
• Solid cancers
• Lung cancer
• Breast cancer
• Head and neck squamous cell carcinoma
• Bone and soft-tissue sarcoma
• Thyroid cancer
• Esophageal cancer
• Colorectal adenocarcinoma
• Primary astrocytic tumors
• Pancreatic ductal adenocarcinoma
 Targets for CAR-T Constructs (Non-Viral Vectors)
• WT1 – Acute Myelogenous Leukemia, Acute Lymphocytic
Leukemia, Non Hodgkins Lymphoma, Multiple Myeloma,
WT1 + Solid Tumors
• CD19 - B cell (B-ALL), Non Hodgkins Lymphoma, Multiple Myeloma
• WT1 + CD19 – target CD19 downregulated malignancies
• CA 125 (Mucin 16, non shed domain) – Ovarian Cancer, other
CA 125 +Solid Tumors (Endometrial,Fallopian Tube, Lung,
Breast, GI Tumors)
• CD 20 – B Cell Lymphomas
• PSMA – Prostate cancer
• EGFR – Epithelial cancers
• CD 30 – Hodgkins Lymphoma
Cellular retention assay model to determine cell-to-cell mediated recognition of CD19 CAR engineered cells
with CD19+ leukemic AMLK cells
 Determination of sample transgene copy number in HEK293 cells.

A. Showing optimized PCR amplification of the intracellular signaling domain of CAR construct. Lane 1: molecular weight
marker; lane 2: CAR-transfected HEK293 cells. B. Plasmid copy number quantification by qPCR using vector-specific primer
confirming successful transfection with high efficiency.
Representative polyacrylamide gel
electrophoresis profile of CAR
lipofected HEK293 cells.

Presence of 65 kDa band size indicates

successful transgene expression in HEK
293 cells lipofected with CAR construct;
the band thickness increases with
increasing sample concentration.

Negative control set-up (HEK293 cells

lipofected with empty vector) did not
show this 65 kDa size band.
Retention profile of AMLK cells on substrate-anchored CAR-engineered HEK293 cells. A. Without AMLK treatment.
B. AMLK only. C. CD19 IB AMLK. D. Isotype IB AMLK. Blue arrows represent AMLK cells on the surface of HEK293
cells, indicative of retention due to binding of CD19 scFv on HEK293 with CD19 antigen on AMLK cells
Schematic of treatments and mechanism of retention assay (A) and reverse retention assay (B).
 Cellular Therapy for Chronic Diseases
 Cardiac cell regeneration –
post myocardial infarction,
cardiomyopathies
 Diabetes, metabolic
syndromes
 Neuro-Degenerative
Diseases
 Kidney – renal failure
 Liver regeneration –
cirrhosis, hepatitis
 Bone and connective tissue
regeneration – post trauma,
fractures, osteoporosis,
osteoarthritis
 Auto-immune diseases
Vasculitis, Lupus, Rheumatoid
Arthritis
 Lung – lung fibrosis, COPD
 Eye – cornea, retina
 Skin – burns, aesthetics
Adult Stem Cells for Organ Regeneration

151
 36

A.E., 33 years, M, Non-Ischemic Heart Failure

• Viral myocarditis at 29 years of age, developed severe

exercise limitation
• Coronary angiography: no block of arteries but heart
muscle not functional well, only at 23% even with
maximum drug therapy. Severe symptoms with
multiple admissions for SOB, hemoptysis, syncope
• Treatment at The Medical City: Bone marrow
mesenchymal cells induced to blood vessels and
muscle-like cells, infused intravenously. Infusions did
not cause significant side effects.

152
 Pt AE – Non-Ischemic Cardiomyopathy
50

45 normal activity;
sports; no SOB

40
Normal activity;
no SOB

35
Ejection Fraction

severe excercise tiredness;

30 limitation; SOB; mild SOB
hemoptysis

25

20
Stem cell Stem cell infusions
infusions

15
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
153
Months
 40

J. A., 78 years, M, End Stage Kidney Failure

• Diagnosed with hypertension, chronic obstructive pulmonary disease

• Developed end stage kidney failure and started hemodialysis in
November 2006, with a measured GFR of 5 mL/min/1.73 m2.
• Patient symptomatic, in wheel chair, with general malaise, muscle
weakness, joint pain and stiffness
• Treatment at The Medical City: Bone marrow stromal cells with
growth and differentiation factors.
• After 6 months monthly infusions of stem cells and factors:
decreased creatinine, improved blood cell counts, increased
exercise tolerance, physically active, ambulatory without
assistance, increased muscle mass, decreased joint pain and
stiffness.

154
 Summary 1
• Identify genomic markers on risk of different cancers, for prevention, prophylaxis, early
detection and diagnosis, early treatment
• Cancer - heterogeneous disease with heterogeneous population of cells.
• Each patient’s cancer - analyze at the molecular level, understand its biological behavior and
strategy it employs to resist treatment.
• No two patients react or respond to cancer in the same way. No two cancers are the same at
the molecular level, - expression of genes related to aggressive growth, metastatic capacity
and responsiveness or resistance to therapy. One-size-fits-all approach – does not work.
• Subpopulation of cells within a tumor – properties of stem cells - poses problems of drug
resistance. Targeted biotherapies can inhibit activity of drug resistance proteins, promote or
restore drug sensitivity.
• Immune system - critical in controlling cancer: cancers progress by evading immune
surveillance and cancer cells actively suppress immune recognition, including the
stimulation of T-regulatory cells.
• Chemotherapy can decrease T-regulatory cells and allow immune recognition of cancer cells.
• Immune cells - Dendritic Cells, Cytokine Induced Killer Cells, CAR-T cells – can be used
to target the specific antigenic profile of each patient, – and can be engineered to recognize
specific molecules in a patients cancer. The dendritic cells can direct both the production of
specific antibodies and cytotoxic cells directed against the cancer.
 Summary 2
• Personalized combined molecular-targeted therapies for cancer start with 1)
molecular profile to identify specific targets of each patient’s cancer; 2) analysis
of molecular mechanism of evasion of immune surveillance (simply fusing the
patient’s tumor with immune cells is too simplistic because immune evasion is a
fundamental problem in cancer progression; 3) identify the specific molecular
mechanisms of drug resistance in the patient’s tumor – the basic reason for
failure of cancer therapy and develop strategies to overcome drug resistance; 4)
monitor the molecular status of immune reactivity and drug resistance at each
point of therapy and realign therapy accordingly.
• Best as early modality, avoid “chemo, rad burnout” not a “treatment of last
resort,” otherwise the tumor is already resistant to multiple modalities.
• Balance safety, tolerability of treatment relative to potential efficacy. Immune
stem cell therapies may modulate toxic effects of chemotherapy and radiation.
• To overcome drug resistance in systemic treatment, it may be necessary to use
combination of chemotherapy, radiation, targeted molecular therapies and
immune stem cell approaches.
• Immune Cell Therapies can also be used with immune checkpoint inhibitors
such as Anti-Pd1, Anti-Pdl1 antibodies.
 Summary 3
• Identify unique molecular targets, neoantigens, using
genomics, proteomics, metabolomics, reaction of a specific patient to particular
treatments – pharmacogenomics - creating a personalized profile of optimum
targets in the cancer and molecular reaction of the patient during treatment.
• Engineering of human immune cells, products of living
cells, exosomes, microvesicles – can be directed to unique molecular profile of a
patient’s cancer, deliver anti-cancer substances with greater targeting of the
tumor, less to the normal cells.
• Monitor specific immune recognition of specific cancer molecules by engineered
immune cells. Monitor circulating tumor cells, circulating DNA, heterogeneity of
the tumor cells, and monitor the changes in the molecular genetics of the
patients, during treatment, to identify emergence of treatment resistant mutants
and to adjust treatment early, before radiologic evidence of recurrence.
• Adjust treatment strategies based upon tumor response, anticipate and detect
early signs of toxicity treatments on the patients, before the effects create obvious
symptoms and signs.
• Continuous interactions between doctor – scientist and doctor – patient all
throughout the treatment process and post treatment follow- up.
• Innovate mechanisms in financing new hi-tech treatments for greater access by
patients