37-year-old South American Male Case Study Mini-Series

The purpose of this mini-series is to get in the mind of a treating physician when a patient such as this presents to the clinic or the ED in this case. The first part of this series is the introduction of the case with case history and initial lab testing. Please don’t hesitate to leave comments on what you think the diagnosis is and what other confirmatory tests need to be done if any as well as what treatment should consist of. This is mean’t to stimulate a discussion and there are no wrong answers. I am in no way a physician or at that level or have that education. I am a student with a passion for molecular diagnostics and creating these cases is a good way for me to practice real life scenarios through careful and diligent research as well as help others who think the same way. This case is no way real and all lab values are made up to the best of my knowledge. If anything is incorrect please do not hesitate to email me or  leave a comment.

CASE:

A 37-year-old South American male presented to his annual physical with his primary care physician with general fatigue, decreased appetite and weight loss over the past three weeks. The patient mentioned to his physician that he has had multiple nosebleeds throughout the last few weeks, an occurrence of multiple a week. The patients past medical history is unremarkable. No family history of bleeding tendencies. He is not taking any prescription medication and denies use of recreational drugs and only social use of alcohol. His physician ordered a CBC and a prothrombin time/activated partial thromboplastin time (PT/aPTT). Results are in table 1.

Two days later the patient presented to the emergency room with fever and heavy fatigue, he explained to the attending that it has been hard to do anything the last few days, and has been bed-ridden. Physical exam revealed bilateral bruising on the upper arms and forearms with purpura and petechiae. The attending physician ordered a full coagulation panel, platelet function tests (Ristocetin cofactor assay), bleeding time test for vWD, and full CBC with peripheral blood smear analysis. Results are summarized in table 2.

Later that evening the patient developed a high fever, and back/flank pain and was moved to the ICU. Blood cultures, CRP and a procalcitonin was ordered, results are in table 3.

Positive cultures for Staphylococcus aureus were found after 48-96 hours and the patient was started on a course of vancomycin and monitored closely.

Patient results indicated he was pancytopenic with a hemoglobin of 9.7 g/dL (Ref. 13.5-18.0 g/dL) and RBC count of 3.7×10^3/uL (Ref. 4.20-6.00×10^6 uL) with severe thrombocytopenia at 37×10^3/uL (Ref. 150-450×10^3/uL).

Initial coagulation results revealed significantly elevated PT and aPTT. The bleeding time test along with the results from the RCO indicate platelet dysfunction or acquired inhibition of platelets by accelerated destruction. Platelet aggregation studies were normal. RCO studies indicate factor VIII inhibition or consumption.

The peripheral blood smear confirmed leukopenia and thrombocytopenia and revealed abnormal promyelocytes with abundant azurophilic granulation and multiple auer rods in bundles. RBC morphology showed schistocytes and fragmented cells.

The attending followed up by ordering a complete fibrinogen, D-dimer and a plasminogen panel. Results are in table 4.

The significantly elevated D-dimer, elevation in t-PA and u-PA in combination with the significant decrease in fibrinogen, and plasminogen levels indicates primary hyperfibrinolysis.

The attending sent a blood sample to the Blood Bank laboratory and asked for units of packed red cells, platelets, and fresh frozen plasma (FFP) to be transfused. With the additional blood components, the patient was able to regain control over the thrombocytopenia, hemoglobin, fibrinogen and coagulation factor levels.

A bone marrow aspirate was ordered including cytology, cytochemistry, immunophenotyping, FISH (Fluorescence in situ hybridization), cytogenetics (chromosomal analysis and FISH) and RT-PCR for PML/RARA quantification of transcripts. The attending started the patient on all-trans retinoic acid (ATRA) as induction therapy.

FISH revealed the PML-RARA fusion gene present which was later quantified and confirmed by RT-PCR. PCR sequencing revealed a bcr-3 PML-breakpoint. Chromosomal analysis of the bone marrow identified a t(15;17) classic translocation. Cytochemistry revealed intensely positive reacting cells to myeloperoxidase and Sudan black B. Immunophenotyping results are in table 5.

Table 1:

RBC: 4.10×10^6/uL             4.20-6.00×10^6/uL          

HGB: 12.9 g/dL                    13.5-18.0 g/dL        

HCT: 38.7%                          40-54%

MCV: 88 fL                            80-100 fL

MCH: 33.2 pg                        26-34 pg

MCHC: 32.3 g/dL                  32-36 g/dL

RDW: 13.5%                         11.5-14.5%

RETIC: 0.8%                          0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 6.3×10^3/uL              3.6-10.6×10^3/uL

NEUT: 3.6×10^3/uL             1.7-7.5×10^3/uL

LYMPH: 1.9×10^3/uL          1.0-3.2×10^3/uL

MONO: 0.7×10^3/uL           0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                  0.0-0.3×10^3/uL

BASO: 0                                 0.0-0.2×10^3/uL

PLT: 111×10^3/uL              150-450×10^3/uL

MPV: 7.3 fL                           7.0-12.0 fL

 

PT: 21 seconds                    11-14 seconds

aPTT: 37 seconds               25-35 seconds

Table 2:

RBC: 3.7×10^3/uL              4.20-6.00×10^3/uL

HGB: 9.7 g/dL                     13.5-18.0 g/dL                    

HCT: 28.9%                          40-54%

MCV: 71 fL                            80-100 fL

MCH: 31.8 pg                       26-34 pg

MCHC: 33.1 g/dL                 32-36 g/dL

RDW: 15.1%                        11.5-14.5%

RETIC: 2.3%                         0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 2.5×10^3/uL             3.6-10.6×10^3/uL

NEUT: 1.3×10^3/uL           1.7-7.5×10^3/uL    

LYMPH: 0.7×10^3/uL         1.0-3.2×10^3/uL

MONO: 0.3×10^3/uL          0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                 0.0-0.2×10^3/uL

BASO: 0.1×10^3/uL            0.0-0.3×10^3/uL

PLT: 37×10^3/uL               150-450×10^3/uL

MPV: 19.3 fL                       7.0-12.0 fL

 

Myeloblasts: 7%                  0%

Promyelocytes: 54%         0%

Myelocytes: 3%                    0%

Metamyelocytes: 5%           0%

Bands: 0%                             0%

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PT: 33 seconds                   11-14 seconds

aPTT: 63 seconds              25-35 seconds

BT: 13 minutes                   1-9 minutes

RCO: 30%                              50-150%

Platelet aggregation studies: Normal

Table 3:

Blood Cultures: POS Staph aureus          NEG

Procalcitonin: 0.25 ng/mL                        <0.15 ng/mL

CRP: 23 mg/L                                                0-10 mg/L

Table 4:

Fibrinogen: 67 mg/dL

Plasminogen: Reduced

a2-Antiplasmin: Reduced

t-PA: Elevated

u-PA: Elevated

D-Dimer: >19,000 ng/mL    

Table 5:

CD2                NEG

CD4                NEG

CD13              POS

CD14              NEG

CD16              NEG

CD19              NEG

CD33              POS

CD34              NEG

CD45              POS

CD56              NEG

CD64              POS

CD117            POS

HLA-DR         NEG

 

-Caleb

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Immune Checkpoint Inhibitors Advance Treatment of Glioblastomas.

A few articles ago immunotherapy was mentioned and introduced via the usage and progression in the research of CAR-T therapy and its use with ALL (B-cell) and lymphomas. Another branch of immunotherapy is quietly emerging, called immune checkpoint inhibitors. Specifically for the treatment of Glioblastoma, which is an aggressive form of brain cancer.

Glioblastomas (GBM) arise from the astrocytes that make up the supportive tissue in the brain. Usually found in the cerebral hemispheres of the brain, but it isn’t unusual to find them elsewhere like the spinal cord. They are highly malignant due to the access of a large network of blood vessels. There are two forms of GBM; de novo and secondary. De novo or primary GBM are very aggressive and the most common form. Secondary GBM is slower growing, but still fairly aggressive. Secondary GBM represents about 10% of all GBM diagnoses. There is no known cause for glioblastomas, but they represent about 15.4% of all primary brain tumors and 60-75% of all astrocytomas. Typically affect men more than women are seen in people of older age rather than youth or adolescents although it isn’t unheard of.

Standard treatment for GBM is surgery to remove as much of the tumor as possible in combination with chemotherapy and radiation. Surgery is difficult because of the extensive network that glioblastomas have that reach to multiple areas of the brain, thus the wide array of symptoms that lead to the diagnosis of GBM. Standard chemotherapy agent is temozolamide, trade name Temodar®. Temozolamide is an alkylating agent meaning that it is most active during the resting phase of the cell. It is also cell-cycle non-dependent.

Prognosis of GBM reported in years of medium survival in the context of an equal number of patients do better and an equal number of patients do worse is about 14 months after diagnosis is made. The two year survival rate is 30%. Diagnosis is made by both identifying and characterizing physical appearance and growth rate along with molecular techniques which leads to greater diagnostic accuracy as well as improved patient management and treatment response. Tumors with methylated O-6-Methylguanine-DNA Methyltransferase (MGMT) have been found to respond better to chemotherapy and radiation and allow longer patient survival. The IDH1 gene mutation is present in 12% of all GBM and is used as a predictor of survival and for the efficacy of treatment. IDH1, also called isocitrate dehydrogenase 1, catalyzes the conversion of isocitrate to alpha-ketoglutarate as part of normal brain physiology and metabolism. From these examples, it is important to consider the fundamental biological base of these tumors and to consider each tumor as a separate entity. With the addition of molecular techniques to expand the diagnosis of GBM has opened up researchers to begin journeys of novel treatments.

In GBM there are several growth factor receptors that are over expressed in the tumor cells. EGFR, VEGF, PDGFR to name a few. This over expression allows unchecked proliferation of tumor cells and increased vascular supply to the tumor. Also called tumor-induced immunosuppressive mechanisms. Because of the recent breakthroughs in the mechanisms of leading to complete T-cell activation and immune recognition there has been a push to uncover effective anti-tumor responses.

One such group of new therapies being developed is called immune checkpoint inhibitors. Checkpoint proteins are proteins that signal the immune system that a cell is healthy. Because of the tumor cells ability to manipulate the cells machinery and biology although there may be molecular signals that signify the cell is cancerous the checkpoint proteins on the cells surface signal the immune system to overlook those cancerous markers from the cell. One of the most common checkpoint proteins is called the PD-L1 (Programmed Death Ligand 1) protein and its receptor PD-1. PD-L1 is used to keep the cytotoxic CD8 T-cells from attacking the hosts own healthy cells. Cancer cells can upregulate the expression of PD-L1 that when bound to its receptor; PD-1 not the surface of the T-cell, induces apoptosis of the T-Cell. The immune checkpoint inhibitors are drug-antibodies that bind to the checkpoint proteins, inhibiting them so the signal doesn’t get to the T-cell that the cell is healthy causing the host immune system to target the cancerous cells.

CDR774646-750

There have been multiple FDA approved checkpoint inhibitors; Optivo®, Keytruda®, and Tecentriq®. All for the treatment of metastatic melanoma, lung cancer, Hodgkins lymphoma, renal cancer, and bladder cancer.

The advancement of understanding the complete host immune system response coupled with the understanding of the molecular biology of each individual tumors gives rise to a new generation of immune therapies. There are multiple clinical trials happening now evaluating the response of tumors, not just GBM to the immune checkpoint inhibitors; those established and those in development. There are researchers working constantly to refine and come up with the most effective way to treat these tumors and different cancers. This is one step closer to a cure.

-Caleb

CAR-T, A Promising Future

For many years the pillars of cancer treatment were surgery, chemotherapy, and radiation therapy. Soon, targeted therapies began to emerge such as Gleevec® and Herceptin®. Targeted therapies are those that target specific molecular elements primarily in cancer cells.

Even more recently, immunotherapy has been getting a lot of attention. Immunotherapy is used to strengthen the patients immune system to allow it to attack tumors. A more rapidly emerging immunotherapy is called adoptive cell transfer (ACT). ACT involves collecting and using the patients’ own immune cells to treat their cancer. This article is going to focus on one of the more successful ACT treatments called CAR-T therapy. CAR-T has gained the spotlight as it is being used in small clinical trials to primarily treat patients with advanced blood cancers and seen excellent results.

The backbone of CAR-T therapy is T-cells. T- cells are responsible for directing the immune response and killing host cells infected by pathogens. The therapy includes taking the patients blood and separating the T-cells out. Using a disarmed virus, most commonly lentivirus (HIV), the T-cells are genetically engineered to produce specific receptors on their surface called chimeric antigen receptors (CARs). The receptors allow the T-cell to recognize a specific protein, or antigen on tumor cells.

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The FDA approved CAR-T therapy for treatment of refractory and previously treated with remission ALL in children and adolescents in August 2017. One of the earlier trials using CAR-T CD19 modified T-cells saw 27 out of the 30 patients have a complete response in that all signs of cancer disappeared with many of the patients continuing to show no signs of remission. CD19, also called B-lymphocyte antigen CD19 is a protein that is encoded by the CD19 gene and found on the surface of B-cells. B-cell ALL is the predominant pediatric ALL.

Because of the strong results seen in using CAR-T to treat ALL, there are ongoing trials exploring the therapy in different lymphomas. On October 18th, 2017 the FDA approved Yescarta™ from Kite Pharma for the treatment of large-B-cell lymphomas based on the results of a large trial that was funded by Kite Pharma.

The rapid advances and growth within CAR-T therapy has given hope and another treatment option to those who have undergone standard regimens of treatment unsuccessfully.

There are efforts to expand the use of CAR-T for the treatment of solid tumor cancers. These efforts have been largely unsuccessful as it is increasingly difficult to identify unique antigens on the surface of solid tumors. There is ongoing research into other forms of ACT that may be better suited to treat solid tumor cancers.

There is an ongoing ethics battle as Novartis the owner of the FDA approved CAR-T therapy tisagenlecleucel (Kymriah®) set the price for a single infusion at $475,000. Novartis claims that the cost-effective price is between $600,000-$750,000, but recognize the importance of the therapy, thus the lower price set. ACT therapy is in its infancy, with CAR therapy exploding as more and more research is done. Its hard to understand pharmaceutical companies position as the high price tag may keep the drug out of the hands that may need it the most. People fail to understand that a stem cell transplant on average costs anywhere from $500,000-$600,000, not to mention the accumulation of previous chemotherapy and radiation regimens.

With the discovery of ACT and CAR-T comes a paradigm shift within cancer treatment that is now only showing its true potential. There is much more to be discovered and much more to understand.

PCR, A Surge Forward

What is it?

PCR or polymerase chain reaction is a technique used in molecular biology and diagnostics to amplify a single or few copies of a sequence of DNA or RNA into thousands to millions. Developed in 1983 by Kary Mullis, who also went on to win the Nobel Prize in 1993.

It changed the way for a lot of different domains. PCR is an indispensable technique used in clinical and research laboratories with a broad span of applications. PCR is used for DNA cloning, gene cloning and manipulation, gene mutagenesis and functional analysis of genes for diagnostic or monitoring purposes.

PCR is dependent on thermal cycling; that is exposing the reactants to cycles of repeated heating and cooling which allows different temperature dependent reactions to take hold.

So, how does it work?

The basic PCR set-up requires several specific components and reagents. There needs to be a DNA template that contains the DNA target sequence that is targeted for amplification. DNA polymerase is needed, more specifically taq polymerase as it is heat-resistant. Taq polymerase is an enzyme that is isolated from the thermophilic bacterium Thermus aquaticus. It can survive the high-temperature DNA denaturation phase. Primers that are complementary to the 3′ ends of each sense and anti-sense strands of the DNA target. Primers are specific and complementary to the target sequence and are often selected beforehand. More than likely the primers are artificially synthesized from a commercial biochemical supplier. Deoxynucleoside triphosphates of dNTPs are the building blocks from which taq polymerase synthesizes a new strand.

There are three steps to PCR; The first step, denaturation is the first step in the natural cycle of events and consists of heating the reaction to 94-98 degrees Celsius for 20-30 seconds. This causes DNA denaturation of the dsDNA template by breaking the hydrogen bonds between the complementary base-pairs. The result is two, ssDNA molecules. Annealing is the second step in PCR and the temperature is lowered to 50-65 degrees Celsius (122-149 F) for 20-40 seconds. This allows annealing of the primers to each of the ssDNA molecules. The temperature in the annealing step is critical because you must select a temperature low enough to allow hybridization of the primer to the strands, but high enough for the hybridization to be specific to the target sequence selected to amplify. The primer should only bind to the complementary part of the strand and no where else. If the temperature is too low the primer may bind improperly and cause issues or may not bind at all. Extension and elongation is the final step in PCR where the DNA taq polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture in a 5′ to 3′ direction. The reaction is raised to about 72 degrees Celsius. The 5′-phosphate group is condensed to the 3′-hydroxyl group at the end of the nascent or elongating new DNA strand.

Polymerase_chain_reaction

At the elongation step in each cycle, the number of DNA copies is doubled. Denaturation, annealing, and elongation constitute a single cycle.

PCR can fail for various reasons. PCR is very sensitive to contamination causing DNA amplification of erroneous DNA products. Primer-design techniques are important to improving PCR product yield and in avoiding the production of wrong DNA products. There are multiple primer rules that should be followed; Primers should be between 22-26 bases in length with optimization at 24. Specificity and Tm (Melting temperature) should be between 58-66 degrees Celsius. Both primers should have a similar Tm (+/- 2 degrees). Keep the G-C base pair content between 40-60% , optimization at 50% if possible. Avoid repetitive sequences (AAAA, TATATA) as they can cause mis-priming. Because annealing of the primer is most critical at its 3′ end, a primer that has a high G-C content at its 3′ end is more likely to cause mis-priming.

Some notable analogs of PCR are RT-PCR (Reverse transcriptase PCR) and qPCR (quantitative PCR). RT-PCR is used for amplifying DNA from RNA. Reverse transcriptase enzyme transcribes RNA template into cDNA, which is then amplified. This is widely used for gene expression profiling or to identify the sequence of an RNA transcript. This determines the expression profile of a gene if known. It can be used to map the exons and introns in the gene. qPCR is used to measure the quantity of the target sequence. It quantitatively measures starting amounts of DNA, cDNA, or RNA and measures the amount of copies in the sample. qPCR is highly specific and precise. Usually qPCR methods use fluorescent dyes, most commonly Sybr green or Taqman which measures the amount of amplified product in real time.

PCR and its PCR derivatives have evolved and changed the way clinical labs and diagnostics are performed. There is a wide range of use in medicine.

Check out my previous posts;

Gram-negative diplococci failed to grow at 48 hours in 35 degree Celsius.

FAB Classification of Leukemias and Cytochemical Stain Observations in each.

3 year old evaluated with abdominal pain and anorexia.