Pathology Week 6 p1-17

Carcinogenesis: Where does cancer come from and how can we avoid it? Learning Objectives: 1. Direct Acting vs Procarcinogen 2. Initiator vs Promoter 3. Two types of Radiation, Which organs 4. Two types of Virus, Mechanistic differences 5. Examples of Virus related malignancy 6. 3 Avoidable Cancer Causes – – –

Carcinogenesis Outline: • Chemical Carcinogenesis – Procarcinogen – Initiator, Promoter • Radiation Carcinogenesis – UV radiation – Ionizing radiation • Viral Carcinogenesis – RNA virus – DNA virus

Mon. 09/20/10 So who cares? • Magnitude of the problem • You're the expert • Future developments

In 2007 1,445,000 new cases and almost 560,000 will die from cancer. You’ll see cancer patients in every specialty. Patients and family ask you for information about disease causes so they can reduce their risk of getting disease. They make conversation with you by probing your knowledge. Mechanisms give us a tool to understand things that are not yet known. Even if the old mechanism is replaced, it contains elements that aid in understanding the new paradigm.

Multistep Carcinogenesis (accumulated mutations over time)

Chemical Carcinogenesis: Chemical carcinogens tend to be bland, unreactive, etc., but somehow they cause cancer. Often, they are procarcinogens – harmless as they are, but the body metabolizes them into something harmful. Promoter – cause proliferation of cells so that mutation is not lost.

Squamous cell carcinoma of the scrotum:  This photograph of a woodcut in the Royal College of Surgeons Museum illustrates how epidemiology of a disease may lead to better understanding of that condition. Sir Percival Pott, a London surgeon, observed that a very rare form of cancer often took the lives of young men in his practice. He also observed in 1775 that this condition occurred only in chimney sweeps and proposed that it may be related to soot. The Danish Chimney Sweeps guild did the appropriate experiment and required all of their members to bathe daily. Scrotal carcinomas almost disappeared on the continent, so British chimney sweeps instituted similar reforms about a hundred years after Pott’s original article. Now we know that the carcinogens in chimney soot are many of the same chemicals found in cigarette smoke.

Chemical Carcinogen: Metabolic Alteration • Electrophiles – electron-seeking chemicals • Direct acting – Weak carcinogens – react w/a number of other chems in the blood (ex: albumin) before they ever get to the DNA – Cytotoxic cancer chemotherapy • Indirect acting – Procarcinogen (most important kind)  Proximate Carcinogen  Ultimate Carcinogen  Metabolites – Procarcinogen – inactive, but converted into reactive intermediate species (proximate carcinogen) that can get into the nucleus or the cell. Ultimate carcinogen – very reactive. If it does not react to something in the cell, it is decomposed. – Strong carcinogens – Mixed function oxidase (P450) activates – CYP1A1 increases lung CA risk 2.4 x – 10% of the population has CYP1A1 – variant of cytochrome p450

Caption from slide: When investigators began painting carcinogenic chemicals on the skin of laboratory animals, or feeding these to the animals, they found that some of the highly reactive chemicals they expected to cause cancer were relatively ineffective and some of the most powerful carcinogens appeared to be stable compounds. The stable compounds had to be absorbed and metabolized by the animal to cause cancer. Metabolic studies of the strong carcinogens revealed that they were absorbed as a stable compound, converted to a les stable intermediate, the proximate carcinogen, often near the target organ and decomposed within the cells to highly reactive free radical ultimate carcinogen that formed covalent bonds with DNA and critical regulatory proteins. We now see that both weak and strong carcinogens attack DNA as cytotoxic electrophiles. Overdose will cause lethal mutation within the cell, and they do so because these electron seeking chemicals react with electron-rich areas of cell components to form covalent bonds by oxidation=reduction reactions. The metabolic activation of procarcinogen to proximate carcinogen often involves the cytochrome P450 mixed-function oxidase enzyme system.

Screening and Testing: • Ames test (10-30%"false negative") • Cell culture – to see if damage is caused by a suspected carcinogen. • Chromosome studies – can karyotype to see if chromosomes have changed • DNA transcription and repair • Animal exposure studies – but there are huge differences between the way animals metabolize drugs and humans metabolize drugs. Not always an accurate predictor. • Epidemiologic (IARC – Intl Assoc of Research on Cancer) – classify chemicals into 3 classes – sufficient, limited, or insufficient evidence that a substance is carcinogenic. Caption from slide: As you might anticipate from the discussion about ultimate carcinogens reacting with DNA, most carcinogens are also mutagens, and many mutation-causing chemicals are also carcinogenic. Suppose you wanted to know if a chemical could cause cancer. Say you were presenting an investigational new drug to the FDA. How would you study its carcinogenicity. The Ames test is a bacterial test for mutagenicity. It turns out that 70 to 90% of carcinogens are positive by the Ames test, and about 80 percent of compounds mutagenic by the Ames test are also carcinogenic.

Initiator-Promotor Experiments:  Figure: Initiator-Promotor Experiments 1&2: Initiator or promoter by itself – no cancer. 3: Promotor before initiator – no cancer. 4: Initiator then spaced-out promotor – no cancer. 5&6: Initiator with appropriate timing of promotor dosages (even if after weeks or months, as in #6)  cancer. Caption from slide: Those investigators who painted purified carcinogens on the ears of rabbits or on the skin of rodents found that these could be empirically divided into two families of chemical. They called these initiator and promoter. Neither one caused cancer by itself, but together they can be powerful.

Initiator Properties: • Chemicals that damage genes, mutagenic (as determined by Ames test) • Single application effective, irreversible • Additive effect of multiple doses • No threshold or maximum • Replication to "fix" initiation • Electrophilic attack, covalent binding

Promoter Properties: • Enhance growth (not mutagenic) • Multiple applications necessary, Reversible effect • Time-dependent, Non-additive • Threshold and Maximum effect seen • Alter expression of genetic message • No covalent binding

Test q: Chemical carcinogenesis investigators have observed that not only must cells be changed to undergo malignant transformation, but the tissue containing the altered cells must also be stimulated to proliferate for the abnormal cells to become grossly evident. The former chemical process is called initiation, and the latter is referred to as promotion. Investigators have also found about promotion that: multiple applications are required. Test q: The concept of initiator and promoter in the process of carcinogenesis was first observed in experimental models of chemical carcinogenesis. Nevertheless the underlying differences between these two processes also apply to many cancer-causing agents or cancer-related clinical patterns. In addition, distinguishing initiator compounds and promoter compounds gave an early indication that carcinogenesis is a multi-step process. An important feature of initiation, which is not characteristic of promotion, would be: causes irreversible, permanent change.

Most malignant cells do not recognize or repair mutations. Accumulate more and more mutations – this is how we get multistep involvement that leads to colonies of cells that can break loose and metastasize to other locations. Progression • Neoplastic evolution toward autonomy • Higher mutation rate (10-100,000x) • Random and spontaneous variant cells • Selection for viability and proliferation • Therapy itself a selective pressure Chemical Carcinogens and ras Gene • Mutations repaired unless p53, Rb or other key regulator genes also damaged • RAS mutations occur in 15-20% of human tumors • Most common oncogene • Each chemical carcinogen causes a set pattern of base substitutions at specific codons – Methylnitrosurea G→A codon 12 – 7,12-Dimethylbenz[a]anthracene A →T codon 61 p53 – tumor suppressor – only need deletion of 1 out of 2 alleles to be oncogenic. RAS – growth-promoting oncogene. No longer subject to normal cellular control. Caption from sldie: Most oncogenic mutations are discovered and eliminated by the cell, unless there is also inactivation of a protective tumor suppressor gene. Many of these tumor suppressor deletions permit the oncogenic mutation to be passed on to a clone of progeny and allow other mutations to introduce polyclonality in the tumor. RAS is one of the most frequently altered oncogenes in tumor cells. These mutations reduce the GTPase activity of the RAS protein. They are found in 15-20% of all human cancers. A useful characteristic of chemical carcinogens is that each one tends to introduce specific mutations in oncogenes such as ras. Methylnitrosourea, for example, causes a transition of guanosine to adenosine at codon 12 of ras, where as dimethylbenzanthracine causes a transversion of adenosine to thymidine at codon 61. Thus, if we sequence the mutant oncogene, we may be able to suggest possible environmental carcinogens that led to the change.

Ras mutation effect:

 RAS receptor has bridging protein that binds to inactive Ras Caption from slide: To review how ras promotes proliferation, recall that an intrinsic kinase type of receptor may activate ras, allowing it to release GDP and bind a new GTP molecule. GTP-bound active ras continuously sends proliferation signals to the nucleus via the map kinase pathway. In normally cycling ras, the bound GTP is quickly hydrolized to GDP, returning ras to the inactive form. Ras cycling requires nucleotide exchange to make the protein active for cell signaling, and GTP hydrolysis to return it to the inactive state. Mutant ras proteins or cofactor mutations cause ras protein to be locked in the GTP-bound active state.

Radiation Carcinogenesis: Radiation comes in two main groups: • Ultraviolet – skin tumors – UVA (320-400 nm) – UVB (280-320 nm) – UVC (200-280 nm)

• Ionizing – therapeutic – occupational – nuclear weapons

Radiation Carcinogenesis Mechanism: • Long latency w/permanent damage • Direct damage (free-radicals) – not only DNA, but also RNA & enzymes • Causes immunodeficiency • Latent oncogenes or virus • Promoter effect of regeneration

Microbial Carcinogenesis • RNA virus (retrovirus) – HTLV-1 – HCV • DNA virus – HPV – EBV – HBV – HSV-8 • Helicobacter pylori (?)

 Caption from slide: Studies of radiation carcinogenesis gave confusing answers to the question, “Is radiation an initiator or a promotor?” Cancers associated with radiation often have a long period between exposure and s clinical appearance of malignancy, like an initiator. Yet epigenetic, promoter-like effects have also been found. Radiation suppresses the immune system, decreasing immune surveillance. In experimental settings it activates latent viral genes in the somatic DNA, potentially causing transformation. And radiation injury causes tissue regeneration, enhancing the growth of transformed as well as normal cells.

 Caption from slide: Three main types of infectious agent have been implicated as environmental agents in carcinogenesis. We’ll talk about RNA and DNA virus here, but you should be aware that there is a clear association between benign gastric ulcer-causing Helicobacter pylorii and both gastric carcinoma and low grade gastric lymphoma. However, in the case of Helicobacter and many other chronic infectious agents, it is difficult to know what part of transformation is due to direct interaction with the organism, and what part is due to the inflammation and repair that always accompanies chronic infection.

Retrovirus • Infectious homologue (v-onc) of Cellular oncogenes (c-onc) • Reverse transcriptase* vRNA *  dsDNA "provirus”  integration  mRNA  viral core proteins (gag) reverse transcriptase(pol) coat glycoproteins(env) • Permissive or NonPermissive host cells - If virus produces daughter particles, eventually it will kill the host and infect other cells (normal expected life cycle of RNA virus). In some conditions, cells may not permit the virus to go through its complete life cycle – non-permissive: provirus remains integrated in the somatic DNA – no daughter particles. • Protein Kinase regulator or paracrine Growth Factor release Retrovirus Examples: • avian leukosis virus – heritable provirus (myc ) • murine leuk/sarc – heritable only (abl ) • other leuk/sarc – feline by saliva only (sis)

• HTLV I = Human T lymphoma Leukemia virus (closely related to HIV) – Japan, Caribbean – tax gene proliferation (stimulates proliferation of T cells) • HCV – blood transfusion – cirrhosis in 17% hepatoma in 50% of those

Test q: An RNA virus now known to participate in some cases of liver cancer is: HCV Test q: A 66y/o female, who has worked all of her life near Tokyo, Japan, has a peripheral white cell count (WBC) of 64,000/µL. Immunophenotyping reveals that 90% of these WBCs are T lymphocytes that are CD4 positive. Which of the following agents is most likely to be involved in this process? Human T-cell lymphotrophic virus (HTLV-I).

Figure: HTLV-1 – Infects T cell – does not become integrated but stimulates tax protein  proliferate. IL-2 = paracrine hormone (induces other cells to proliferate). The huge increase in cells increases the risk of new mutations. DNA Virus: • Animal tumors • Transformation incompatible with viral replication • Tumor-Specific Antigens (TSA) – Membrane bound – Protein kinase

DNA Virus Examples: • Human Papilloma Virus (HPV) – 6,11 vs 16, 18, 31, 33, 35, 51 – E6 binds p53, E7 binds Rb product • Epstein-Barr Virus (EBV) – Herpesvirus 4 – t(8;14) cmyc to chr 2, 14, 22 – Burkitt’s lymphoma, Hodgkin’s, Nasopharyngeal CA • Hepatitis B Virus (HBV) – HBx binds p53 and activates growth promoter genes • Herpesvirus 8 (KSHV) – Kaposi’s sarcoma (endothelial cells)

HPV Carcinogenesis:

EBV and Burkitt’s:

Causes of cancer:

Caption from slide: The last set of slides describes the  prevalence of cancer risk factors, such as tobacco use and physical inactivity, and the prevalence of cancer screening, such as use of mammography. Tobacco use is a major preventable cause of death, particularly from lung cancer. The year 2004 marks the anniversary of the release of the first Surgeon General’s report on Tobacco and Health, which initiated a decline of per capita cigarette smoking in the United States. As a result of the cigarette smoking epidemic, lung cancer death rates showed a steady increase through 1990, then began to decline among men. The lung cancer death rate among US women, who began regular cigarette smoking later than men, continues to increase slightly.

 Obesity has reached epidemic proportions in the United States. The percentage of adults age 20 to 74 who are obese increased from 1960 to 2004 with the largest increases occurring in the 1990s. Similar trends were observed among men and women.

Learning Objectives w/Answers: 1. Direct acting vs Procarcinogen: Direct (weak) alkylating agents Procarcinogen (strong) metabolic activation 2. Initiator vs Promoter: I: irreversible, mutation-like, additive, no threshold, early S phase P: decays, enhance growth, not cumulative, definite threshold, milieu sensitive Both: replication to “fix” initiation 3. Two types of radiation, which organs: UV: skin Ionizing: leukemia, thyroid, lung, breast 4. Two types of virus, mechanistic differences: RNA dsDNA provirus integrates DNA episomal genes integrate 5. Examples of RNA & DNA virus related malignancy: HTLV-1: T-cell leukemia (tax), HCV: HCCA (cirrhosis 17%). HPV (E6/p53, E7/Rb), EBV (EBNA-1 product), HBV (HBx product/p53), HSV-8 (endothelial). 6. 3 avoidable cancer causes: Tobacco, Diet, Occupational

This slide highlights the obesity epidemic as mentioned in the previous slide. In 2005, over 50% of the adults in all states, including District of Columbia, were overweight or obese, compared to just 12 states in 1997.

Cytopathology

Tues. 09/21/10

Objectives: Don’t trust my answers on these. There is probably more info to some of them – did these pretty quick. If anything is wrong, please post a reply on the message board. 1. Know the most important anatomic location to be sampled for an adequate Pap smear: squamous epithelium of exocervix, squamocolumnar junction, endocervical epithelium 2.

Identify koilocytes and high grade dysplastic cells on a Pap smear:

Normal exfoliated superficial squamous epithelial cells. Koilocytes, CIN I

CIN II

CIN III Note the reduction in cytoplasm and the increase in the nucleus-to-cytoplasm ratio as the grade of the lesion increases. This reflects the progressive loss of cellular differentiation on the surface of the cervical lesions from which these cells are exfoliated. Grade of dysplasia: CIN I < CIN II < CIN III. 3.

Know the significance of koilocytes on a Pap smear: sample is potentially pre-cancerous, HPV-related

4. For cervical carcinoma, chart the incidence of CIS, Squamous Carcinoma, and death due to Squamous Cell Carcinoma over the past decade in the U.S. (I’m not sure what he wants on this one…) Cervical carcinoma: th 2000: 8 leading cancer cause of death; 12,800 new cases; 4,600 deaths th 2005: 10 leading cause of cancer death in women; 10,370 new cases; 3,710 deaths 2007: 11,150 new cases; 3,670 deaths 2009: 11,270 new cases; 4,070 deaths

Unspecified year: - 50,000,000 - 9,000,000 - 3,000,000 - 250,000 - 12,000

Pap smears ASCUS LSIL HSIL Cancers

5. When is HPV DNA typing recommended following a Pap smear? From ACOG bulletin: Co-testing using the combination of cytology + HPV DNA testing is an appropriate screening test for women older than 30 years 6. Diagram HPV Thin Prep Pap smear testing (not sure how he would like us to diagram it… See #2 for example of Thin Prep specimen). What tests can be prepared on a Thin Prep specimen? (Pap smear slide, HPV, chlamydia, gonorrhea) Pap test; Chlamydia test; Gonorrhea test combined. 7. What are high-risk HPV DNA types? HPV 16 and 18

Low risk types? HPV 6 and 11 (genital warts)

8. Know the “summary recommendations” from the ACOG practice bulletin (at the end of the paper) Level A (based on good and consistent scientific evidence): – Cervical cancer screening should begin at age 21 years. Screening before age 21 should be avoided because it may lead to unnecessary and harmful evaluation and treatment in women at very low risk of cancer. – Cervical cytology screening is recommended every 2 years for women between the ages of 21-29 years. – Women age 30+ who have had 3 consecutive negative cervical cytology screening test results and who have no history of CIN2 or 3, are not HIV infected, are not immunocompromised, and were not exposed to diethyl stilbestrol in utero may extend the interval between cervical cytology exams to every 3 years. – Both liquid-based and conventional methods of cervical cytology are acceptable for screening. – In women who have had a total hysterectomy for benign indications and have no history of high grade CIN, routine cytology testing should be discontinued. – Co-testing using the combination of cytology + HPV DNA testing is an appropriate screening test for women older than 30 years. Any low-risk women age 30+ who receive negative test results on both cervical cytology screening and HPV DNA testing should be rescreened no sooner than 3 years subsequently. Level B (based on limited and inconsistent scientific evidence): – Sexually active adolescents (ie, females younger than age 21 years) should be counseled and tested for sexually transmitted infections, and should be counseled regarding safe sex and contraception. These measures may be carried out without cervical cytology and, in the asymptomatic patient, without the introduction of a speculum. – Because cervical cancer develops slowly and risk factors decrease with age, it is reasonable to discontinue cervical cancer screening between 65 years and 70 years of age in women who have three or more negative cytology test results in a row and no abnormal test results in the past 10 years. – Women treated in the past for CIN 2, CIN3, or cancer remain at risk for persistent or recurrent disease for at least 20 years after treatment and after initial posttreatment surveillance, and should continue to have annual screening for at least 20 years. – Women who have had a hysterectomy with removal of the cervix and have a history of CIN 2 or CIN3- or in whom a negative history cannot be documented- should continue to be screened even after their period of post-treatment surveillance. Whereas the screening interval may then be extended, there are no good data to support or refute discontinuing screening in this population. Level C (based primarily on consensus and expert opinion): – Regardless of the frequency of cervical cancer, physicians should also inform their patients that annual gynecologic examinations may still be appropriate even if cervical cytology is not performed at each visit. – Women who have been immunized against HPV-16 and HPV-18 should be screened by the same regimen as nonimmunized women.

Cytopathology Lecture: This powerpoint is 273 slides long, so I did not include every single slide (because he did not show every single one in class). Here is ALL of the info he showed in class – text plus photos. Diagnostic Cytology Definition – The art and science of the interpretation of cells from the human body that either exfoliate (desquamate) freely from the epithelial surfaces or are removed from various tissue sources by various clinical procedures. Applications of Diagnostic Cytology: 1. Screening of asymptomatic patients; ex: Pap smear 2. Investigation of symptomatic patients w/lesions Investigation of Symptomatic Patients: - Neoplasm - Non-neoplastic - Infectious If neoplastic, decide… - Benign - Malignant - Type of Neoplasm Cytology Sampling Techniques - EXFOLIATION - Spontaneous - Enhanced (ex: Pap smear) - FNA (putting needle into lump in question) Artificially Enhanced Exfoliation 1. Scraping – cervix, vagina, skin, conjunctiva, oral cavity (Pap smear) 2. Brushing – respiratory tract (bronchi, trachea), GI tract, urinary tract, endocervix, endometrium 3. Lavage (aka washing) – respiratory tract, GI tract, urinary tract Fine Needle Aspiration Cytology - Cysts: neck, thyroid, breast, liver, pancreas, kidney - Solid masses: superficial, deep. Superficial = palpable. The Problem: - 50,000,000 - 9,000,000 - 3,000,000 - 250,000 - 12,000

Pap smears ASCUS LSIL HSIL Cancers

ASCUS = Atypical squamous cell of undetermined significance Note: 50% of patients w/cervical carcinoma have never been screened. Goal is to diagnose PRECURSOR lesions. Cervical Carcinoma: 2000: th - 8 leading cancer cause of death - 12,800 new cases - 4,600 deaths

2005: -

10 leading cause of cancer death in women 10,370 new cases 3,710 deaths

2007: -

11,150 new cases 3,670 deaths

2009: -

11,270 new cases 4,070 deaths

th

Woman’s lifetime risk of diagnosis cervical ca ~0.85%; risk of dying from disease is ~0.30%. Cervical Carcinoma: 220 estimated new cases in Indiana, 2009 Female Genital Tract Cancers – 2009 Statistics Cases Deaths - Breast 194,280 40,170 - Endometrium 42,160 7,780 - Ovary 21,550 14,600 - Cervix 11,270 4,070 - Vulva 3,580 900 Cervical cancer was at the top of the list before the Pap smear. Cervical Carcinoma 5 year survival rate CIS 100% SCC, stage I 80% stage 2/3 30% stage 4 0-15% Test q: A 25y/o female is diagnosed w/squamous cell carcinoma in situ of the cervix. Her 5yr survival rate can be estimated at: 100% Test q: A patient is diagnosed as having squamous cell carcinoma in-situ (CIS) of the cervix. With standard treatment for this disease the cure rate is best estimated as: 100%

The Solution: Universal screening of all women at risk Cervicovaginal Smear Cytology History - Dr. George Papnicolaou - initial publication: 1928 - monograph: 1943 (Diagnosis of Uterine Cancer by Exfoliative Cytology) Cervicovaginal Cytology Objective: Early Detection - precancerous lesions - occult invasive carcinomas - curable disease Cervicovaginal Cytology: Cells of Interest – All Women - squamous epithelium of exocervix - squamocolumnar junction - endocervical epithelium

Obtaining the Pap Smear:

endocervical brush

Cervicovaginal Cytology Lab Processing: Technologist Functions: - smears logged into computer system - identifying numbers assigned - slides are stained

Thin Prep slides – nice round circle.

Cytotechnologist Functions: - screen smears - each smear: 50,000-300,000 cells - minimum screening time, normal smear: 5 min - labor intensive - fatigue - guidelines established by government

Above: the old way to smear – messy.

Above: new, cleaner Thin Prep way. Better ability to diagnose

ThinPrep Imaging System: Image Processor: - PC based, expandable, “networkable” - 300 slides/day throughput - Identifies 22 “fields of view” which contain objects of interest - Automated review scope used by cytotechnologist for screening interpretation and slide marking Optical Cellular Selection (OCS) Algorithms: - Algorithms look for the largest, darkest objects (increased DNA) - Algorithms analyze both single cells and clusters; overlapping nuclei are excluded - A minimum of 2/22 FOV’s are presented representing clusters of cells - Remainder of FOV’s present predominantly single cells if available on the slide Field of View Screening: Cytotechnologists responsible for entire FOV

Field of View Overlap:

-

Shaded area = approximately 50% Programmed to ensure 100% of cell spot is presented to screener

Dotted slides passed to the pathologist:

Above: Final Product. Inspect these.

Bethesda System: - 1988 National Cancer Institute Workshop - Uniform format for cytopathology reports - 1991 Besthesda System: - Statement of Specimen Adequacy - General Categorization - Descriptive Diagnoses - 2001 Bethesda System Specimen Adequacy: - Satisfactory for evaluation - Satisfactory for evalution by limited by… (specify reason) – category deleted 2001 - Unsatisfactory for evaluation… (specify reason) General Categorization: - Negative for squamous intraepithelial lesion or malignancy (NIL) = normal. 85% of cases. - Epithelial cell abnormality: see descriptive diagnoses Benign Cellular Changes (Deleted Category) Infection: - trichomonas vaginalis - fungal organisms: Candida - coccobacilli due to shift in vaginal flora - actinomyces - herpes simplex virus

Normal exocervix.

Flat condyloma.

Epithelial Cell Abnormalities: Squamous Cell - Atypical squamous cells, of undetermined significance (ASCUS) - Atypical squamous cell, cannot exclude HSIL (ASC-H) – not often diagnosed. Worried about high-grade but does not have all the criteria. - Low-grade squamous intraepithelial lesion (LSIL) – one of the main categories diagnosed – encompassing: - HPV - Mild dysplasia/CIN 1 - High-grade squamous intraepithelial lesion (HSIL) – another huge category – encompassing: - moderate and severe dysplasia - CIN2/CIN3/CIS - Squamous cell carcinoma (SCC) – rare. Epithelial Cell Abnormalities: Glandular Cell - endometrial cells in a postmenopausal woman - atypical glandular cells, of undetermined significance (AGC) – imprecise category but warrants further investigation. - endocervical adenocarcinoma - endometrial adenocarcinoma - extrauterine adenocarcinoma (could be metastasis from colon cancer, for example) - adenocarcinoma, NOS

Exophytic condyloma (caused by HPV 6 or 11)

Test q: A 25y/o female present w/an exophytic condyloma on her cervix. What HPV DNA types would you expect? HPV 6, 11 Test q: A 23y/o female has a “thin prep” pap smear which is diagnosed as ASC-US. The preferred next step in her management is: Current thin prep specimen submitted for high risk HPV testing Test q: A routine Pap smear on a 27y/o is reported as a low-grade squamous intraepithelial lesion w/prominent koilocytosis. Which of the following is the most likely etiology for this finding? HPV Type 6

Actual case: LSIL: HB – 29F, G2P2 (has two children), Early coitarche (age of sexual intercourse): 14

Above: cervical biopsy. Shows koilocytes (hallmark of HPV infection and disease).  Can see enlarged nuclei, multinucleated cells, cytoplasm seems a bit cleared out.

These pictures are of koilocytes. Cleared-out cytoplasm, enlarged and hyperchromatic nuclei.

Cytologic Diagnosis for this case: LSIL 07/13; For clinical reasons, underwent hysterectomy 09/17

Histologic Diagnosis: Focal residual moderate dysplasia (CIN 2) w/prominent koilocytotic atypia. Lab Statistics 2009 (N=38,836): 83.8% NIL or M 32541 6.4% ASCUS 2500 5.3% LSIL 2068 – easiest diagnosis to make. 1.2% HSIL 459 1.4% Other 559 1.4% Unsat 553

* STDs in pop culture (insert Dr. Dre and Eminem songs – HIV/HPV) *

st

HSIL – Severe: CD: 27F, New onset sexual activity (relatively late), 1 GYN exam, ThinPrep

High-grade disease. Nuclei enlarged, hyperchromatic, coarse nuclear chromatin. Not a lot of cytoplasm. See chunks of highly dysplastic epithelium. Cytologic Diagnosis: HSIL 06/13; Cervix Biopsy 06/21

No invasive disease. All of it is in situ – basement membrane is intact. Same cells shown as seen in Pap smear. Notice that the basement membrane is intact – especially in bottom left picture. If basement membrane had been penetrated = invasive cancer. Test q: Pap smear from a 29y/o sexually active female shows carcinoma in situ (CIS). Which of the following is consistent w/a diagnosis of CIS? Neoplastic cells fill the entire thickness of the epithelium

Histologic Diagnosis: Severe squamous dysplasia extending into endocervical glands 06/21; Cervix, LEEP 06/28

Above: LEEP – take big chunk of tissue out. Can see the disease going into the endocervical glands. Histologic Diagnosis: Squamous cell carcinoma in situ (CIS) extending into endocervical glands and extending to cauterized edges of several fragments

CP: 31F; Pap test; Chlamydia test; Gonorrhea test COMBINED

Colposcopy – can see abnormal vascular pattern.

Above: CIS, just like last patient. Here, it’s more single cell presentation. Nuclei enlarged, syncitial groups.

Above: CIS. Full-thickness change. Test q: Following an abnormal Pap smear, a 34y/o woman has a cervical biopsy. This demonstrated marked hyperchromatism and increased nuclear/cytoplasmic ratio of the epithelial cells. These changes involved the full thickness of the epithelium but did not penetrate the basement membrane. These changes are best described as: Carcinoma in situ (CIN III)

Above: Endocervical brush. Shows same thing.

Hysterectomy: whole cervix on slides. No residual disease was left. Cervical Cancer Screening Results: Early US Study: Louisville, KY Study Population: low income women (mostly African American); women at high risk - decreased incidence: 75% - decreased mortality: 50% - (15.5/100,000 to 7.4/100,000) Test q: An epidemiologic study analyzes health care Canadian Study: Vancouver, BC benefits of cancer screening techniques applied to a population. Which of the following diagnostic screening - screening program started in 1949 techniques used in health care is most likely to have had - 85% of population at risk is screened the greatest impact on reduction in cancer deaths in - >500,000 cervical smears/year developed nations? Pap smear - 26,000 cases of BIS diagnosed and treated - incidence of invasive SCC decreased: 78% - mortality of invasive SCC decreased: 72% - reductions attributed to screening program

When a screening program is in place, see spike in CIS – we want that. Incidence of invasive disease/mortality is down.

Results of Cervical Cancer Screening Programs Conclusions: - statistically valid drop in cervical cancer incidence and mortality - cervical cytology is the only effective cancer screening test known today - screening has not eradicated carcinoma of the uterine cervix

Management of Abnormal Pap Smears: - Unsatisfactory: - correct the problem - repeat the smear - Benign cellular changes: - treat infection if indicated – inflammation can make reading slides difficult. - follow-up Pap smear as indicated by history - ASCUS: - treat specific infection if present - topical estrogen if atrophic - colposcopy, if: - previous abnormal Pap smear - poor compliance - follow-up Pap smear every 4-6mo for 2yr until 3 consecutive normal results - Reflex HPV Testing

Frequency of Screening: High Risk Population Annual Screening - HPV-infected women - HIV-infected women - smokers - women w/multiple sexual partners

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-

LSIL: HSIL: SCC: -

colposcopy and biopsy repeat smear in 3-6 months colposcopy and biopsy treatment by destruction or excision of the transformation zone colposcopy and biopsy refer patient to gynecologic oncologist

Big, fungating invasive cancers.

Pleomorphic tumor cells – more cytoplasm than in CIS.

Cervical sample.

Cervix.

High power of liver looks like cervix. Treatment Failures: British Columbia Experience: 1985-1988 437 patients w/newly diagnosed invasive cervical cancer - 170 no previous cytology - 45 no previous cytology within 5 years - 39 abnormal cells missed or under called - 32 sampling error

Liver sample.

This was an autopsy case.

Incidence and mortality is greater among African Americans than Caucasians. Cervical Carcinoma: Death Rate Per 100,000 - 1991: 3.49 - 2005: 2.42 - 30.66% decrease = good Causes of Treatment Failure: - initial clinical exam - sampling - lab errors - screening errors - interpretation errors - Physician error: - failure to perform test - failure to understand report - diagnosis of dysplasia not understood - failure to act on cytologic findings - Patient error

Test q: In comparing the 5yr survival rate for women in the US, Black women have a lower survival rate for all cancers (overall rate)

New Screening Alternatives: - Thin-layer preparations - Automated cytology - Cervicography - HPV viral typing

Physicians called the case above negative. Was actually invasive squamous cancer  Malpractice. Quality Assurance Procedures: - 10% random re-screen - Re-screen of high-risk patients - 5-year look-back (index case: HSIL+) - 100% rapid re-screen (new policy)

High Risk Patients - Previous gynecologic malignancy - Previous dysplasia - High-risk HPV-positive - Abnormal bleeding - Lesion identified by physician