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Effects of Therapy on Cytologic Specimens
Introduction
The development of novel therapeutic modalities for treating patients with malignancies and transplantation rejection is an ongoing process, at the time of this writing. The prototype of different genres includes radiation therapy, chemotherapeutic agents, biologic response modifiers, and innovative mechanical/physical/molecular biology techniques. Amidst the diversity of these traditional and novel techniques, common characteristics shared is the ability of the modalities to lead to a wide range of changes that may have uncanny resemblances to neoplastic or physiologic changes in the target cell populations. The radioactive agents may potentially modify the genome, and be propagated in subsequent cell divisions/generations of cells. These changes may be, at times, dauntingly complex with wide variation in cellular architecture, and could be evident in the surviving neoplastic/benign cell populations for many years following the initial therapy. In a variation on the theme of localized effects of the therapeutic modalities, systemic effects may also be seen in patients receiving one of the many types of chemo/hormonal/immunotherapy. The physical/molecular biology techniques cause changes simulating host reparative response and produce systemic effects rather than genomic alterations. In addition, the aforementioned therapies may synergistically interact with radiotherapeutic agents. The following sections entail the background and site-specific examples of each type of therapy with respect to different anatomic sites.
Radiation Biology
Two types of ionizing radiation have been described: particle and electromagnetic. Particle radiation consists of alpha, beta, proton, neutron, meson, or deuteron particles. Electromagnetic radiation consists of X-rays and gamma rays. Both particle and electromagnetic radiation ejects electrons to release energy and exert their effects in <10 −8 s.
The likelihood of any type of radiation having an effect on the target tissue is proportional to the linear energy transfer (LET) or the rate of energy loss with distance. X-rays have low energy transfer and deep penetration, whereas neutrons have a high energy transfer and low penetration.
Radiation is generally measured in terms of rads (rad) and grays (Gy). For most tumors, doses in the range of 2 Gy/treatment/day for 4–6 weeks are usually given in a fractionated manner, allowing a recovery period for the tissues between consecutive doses. The cell recovery is proportional to 1/LET. The higher the LET, the slower is the cell recovery.
The biologic effect(s) associated with a standard dose of radiation is critically dependent on the target tissue and the type of radiation. Bone marrow and gastrointestinal epithelium tissue with a rapid turnover rate are predominantly sensitive, whereas those with a low turnover rate, e.g., brain, are relatively insensitive.
Radiation interrupts cell replication mainly by damaging the DNA. These include double- or single-stranded breaks, loss or alteration of bases, rearrangements and cross-linking of DNA molecules. The most lethal changes occur with double-stranded breaks as these directly affect the integrity of the template for DNA repair and replication. In addition, the radiation changes also affect the cellular proteins, and are transferred to cellular water.
Radiation and chemicals can damage the DNA through various mechanisms. While the ionizing radiation breaks the DNA backbone, chemicals can lead to alteration in the bases, and ultraviolet rays can damage the pyrimidine dimers. The failure of the proofreading mechanisms of the DNA repair enzymes or their infidelity itself leads to genetic instability and triggers carcinogenesis. In this context, the tumor suppressor gene P53 is most susceptible to environmental and radiation insults. Alteration in the P53 gene, either due to therapy or as a part of carcinogenesis, may lead to the inability to interrupt the cell cycle in the event of damage to the DNA as well as to initiate apoptosis of the irreversibly DNA damaged cells. The tumorigenic tissue affected by altered P53 is degraded slowly, if any, and may be detected by immunocytochemical studies.
The radiation changes to cellular water can potentially produce damaging reactive radical species such as active peroxides, hydroxyl radicals (OH•), electrons, hydrogen atoms, and hydrogen peroxide (H 2 O 2 ) molecules. These may subsequently interact with other molecules in the cell to cause cross-linking ( Table 32-1 ). Of note, the interaction of the OH• radicals with the proteins can cause alterations in structural proteins, enzymes, information molecules, and cytoskeletal proteins and even denaturation. These changes can damage the cell membranes and cause lysis of cells. In addition, cohesive property of cells between themselves and with the extracellular matrix may be altered, contributing to the phenotypic changes of dyscohesion and altered relation with the stroma, as seen in malignancy.
TABLE 32-1
Effects of Radiation on Molecules
| Molecules | Effect(s) |
|---|---|
| Nucleic acids | Single- and double-stranded breaks |
| Proteins | Denaturation |
| Water | Radiolysis, generation of free radicals |
| Lipids | Peroxidation |
Intermolecular interactions are constant in nature. A damaged molecule interacting with other counterparts may potentially propagate the altered effects contained therein to the molecules in other systems through multiple mechanisms. Even though the initial changes may not exhibit overt damage, the accumulative damage to the DNA, protein, carbohydrates, and lipids results in a set of morphologic changes over a period of time ( Box 32-1 ). These radiation-induced changes consist of cellular swelling, cytoplasmic vacuolation, altered chromatin pattern, and nuclear vacuoles. Some of these changes such as vacuolation and alterations in the cytoplasm, and mild nuclear chromatin clumping are reversible. Other changes, such as pyknosis and karyorrhexis, however, are considered irreversible. These alterations are mostly associated with double-stranded breaks, which can be measured by fluorescence in situ hybridization (FISH) techniques, in vitro assays, and histone phosphorylation. Radiation-induced cell death can take the cytologic forms of either necrosis or apoptosis.
Box 32-1
Morphologic Changes Due to Radiation
Stroma
The aforementioned changes cause changes in the stroma. In particular, the endothelial cells of the capillaries, sinusoids, and arterioles are the most sensitive to radiation. They initiate and reflect the subsequent vascular changes associated with radiation damage. In the acute phase, endothelial necrosis and thrombosis of small vessels is evident that progresses to vascular dilation and fibrosis in the chronic phase. Progressive fibrosis leads to endothelial proliferation and sclerosis of small arterioles ( Box 32-2 ).
Box 32-2
Radiation Changes in Tissue
-
Vascular dilatation
-
Endothelial cell sensitivity
-
Acute: necrosis and thrombosis
-
Chronic: dilation and fibrosis
-
Progressive fibrosis, endothelial proliferation
-
Sclerosis of small arterioles
Epithelium
Squamous cells are more sensitive to radiation changes than glandular cells. In general, the epithelial cells with squamous differentiation show greater sensitivity to radiation than basal/parabasal cells, intermediate cells, or superficial cells. Those cells with a high mitotic rate are more sensitive than those with a low mitotic rate; this is confirmed by experimental studies that show cells in G 2 and M phase to be more sensitive than those cells in G 1 and G 0 phase. Cells are more resistant in the late S phase than other phases of the cell cycle. The cells with high oxygen content are more likely to generate active oxygen radicals and thereby are more sensitive to radiation changes than those with low oxygen content. Anaplastic tumor cells are more sensitive than well-differentiated tumors ( Table 32-2 ). Many of these changes were initially described as a result of radiation therapy to the uterine cervix, and will be described separately in the section on the female genital tract.
TABLE 32-2
Sensitivity to Radiation
| Sensitivity | |
|---|---|
| Squamous cells | Basal > parabasal > intermediate > superficial |
| Differentiation | Squamous > endocervical |
| Mitotic rate | High > low |
| Oxygen content | High > low |
| Tumor differentiation | Anaplastic > well differentiated |
General Cellular Changes
Radiation cell changes are divided into two phases, acute and chronic. The acute changes occur within 6 months of irradiation and consist of cellular and nuclear enlargement, vacuolation of the cytoplasm, wrinkling and chromatin condensation of the nuclei, abnormal mitoses, multinucleated and bizarre-shaped cells. Even though these changes are rare after the first 6 months of radiation therapy, in most instances, however, these may persist for many years. The malignant cells also show radiation changes similar to those seen in benign cells. However, the nuclear enlargement may be more pronounced than cytoplasmic swelling that leads to a higher nuclear to cytoplasmic ratio. Moreover, as the malignant cells are more sensitive to radiation than benign cells, they usually disappear within 1 month after termination of radiotherapy. Exceptions may occur, as late cells may be in a dormant phase. Generally, radiation-induced cell death needs a round of cell division to initiate the apoptotic effects.
The chronic changes include cytomegaly, epithelial atrophy and aberrant basal cells. Nuclear and cytoplasmic vacuolation and multinucleated forms are usually less evident. Abnormal, increased keratinization associated with orangeophilia or two-tone cytoplasm, and amphophilia of cells, may be present.
The cytoplasmic changes seen in the radiated cells may be partly due to destruction of cytoplasmic organelles with release of cytoplasmic enzymes. There is an increase in nucleolar and cytoplasmic RNA synthesis followed by a decrease in DNA synthesis. Subsequently, there is a progressive loss of RNA as the cells are irradiated successfully. Large glycogen deposits, increased intracellular potassium concentration, and disappearance of alkaline phosphatase are observed.
The ultrastructural changes in radiated cells consist of changes of both the epithelial and stromal cells. The epithelial cells show alterations in nuclear and cytoplasmic organelles. The nuclear changes usually precede the cytoplasmic alterations and include abnormal clearing of chromatin with a peripheral rim of chromatin-like material underneath the nuclear envelope. The cytoplasmic changes include a greater number of lysosomes, accumulation of lipid droplets, dilatation of endoplasmic reticulum, disrupted cristae in mitochondria, mitochondrial swelling, increased fibrils, and disruption of cytoplasmic membranes. The stromal changes comprise reactive fibroblasts with abundant endoplasmic reticulum, increased dilated Golgi apparatus, and cytoplasmic vacuolization.
Key Features of Radiation Therapy
-
Cell enlargement
-
Vacuolization of cytoplasm
-
Vacuolization of nuclei
-
Altered chromatin pattern
-
Multinucleation.
Female Genital Tract
Historically, the post-irradiation changes were described involving lesions of the female genital tract. In one of its kind, serial histologic changes comprising enlargement, vacuolization, and hyalinization were reported in carcinoma of cervix during radium and X-ray treatment. On a similar note, Donaldson and Canti noted distinct early and late cytologic changes in serial biopsies of cervical carcinoma treated with radium. The early changes were an increase of normal/abnormal mitotic figures, cytomegaly and necrosis, while the late changes (after day 7) comprised karyorrhexis and fibrosis. Mitosis was conspicuously absent during the late changes. It was suggested that radiation-induced changes on cervical cancer could be better appreciated on vaginal smears in comparison to biopsies, due to ease of performance and evaluation of greater surface area of the cervix and vagina.
Acute Radiation Changes
In benign cervical and vaginal cells, the frequently seen radiation-induced changes consist of remarkable increase in nucleus and cell size, along with bizarre cell shapes, e.g., tadpole-shaped cell, cytoplasmic vacuolization, and nuclear changes. These changes could be found in any or all layers of the ectocervix, endocervix, and vagina. The increase in cellular size is in proportion to the nuclear increase, thereby usually maintaining a nucleocytoplasmic (N : C) ratio. This ratio is, however, decreased in the superficial cells, as the condensed nuclei do not enlarge with respect to the increase in the cell size. Cytoplasmic vacuolation ranges from the presence of multiple small vacuoles to a single large vacuole displacing the nucleus to one side. The vacuolation is first appreciated in the parabasal cells, but also involves the intermediate and superficial cells later. In addition to the vacuoles, other cytoplasmic changes comprise increased keratinization, amphophilia, and engulfed neutrophils. The nuclear changes consist of hyperchromasia, an increase in size, wrinkling, multinucleation, pyknosis, and karyorrhexis. The chromatin is finely granular and homogeneously distributed. The background is heavily infiltrated by polymorphonuclear leukocytes, histocytes, and other inflammatory cells ( Fig. 32-1 ).
Similar cytologic changes are identified in the malignant cells as benign epithelial cells, albeit few exceptions. The nuclear enlargement may be greater than the cytoplasm, increasing the N : C ratio. The malignant cells may not be easily distinguished from benign cells in the acute phase post-irradiation. The nuclear chromatin of viable malignant cells shows a distinct pattern of contrasting heterochromatin with euchromatin that may be confused with degenerative nuclear changes. An intense stromal reaction comprising reactive fibroblasts, granulation tissue, cellular debris, and endothelial hyperplasia can be identified. Of note, the endothelial hyperplasia with accompanying atypia may be potentially confounded with malignancy. These changes warrant an increase in vigilance among the cytopathologists to exclude an underlying malignancy and, likewise, refrain from overcalling a post-irradiation change as malignant.
Chronic Radiation Changes
The post-radiation changes may persist for many years. The smears are atrophic, predominantly composed of basal and parabasal cells, and some with aberrant changes. Nuclear and cytoplasmic enlargement may be evident in the epithelial cells. The nuclei are hyperchromatic, but with homogeneous distribution. The nuclear changes mentioned in the basal cells are propagated to other epithelial cells during maturation. A characteristic late radiation effect of net-like background comprising amorphous acellular pink proteinaceous material has been shown by Kaufman and colleagues. Multinucleated epithelial cells may be seen. The background is usually clean; however, an occasional foreign-body giant cell with multinucleated histiocytes and fibroblasts may be identified ( Fig. 32-2 ).
Prediction of Prognosis and Recurrence
A periodic post-radiotherapy cytologic examination is indispensable for the assessment of treatment effectiveness as well as detecting persistent/recurrent tumor. The prognostic features include radiation response, host inflammatory reaction, high karyopyknotic index, post-irradiation dysplasia, and the presence of malignant cells.
The radiation response, assessed as the percentage of benign squamous cells showing radiation effect in a vaginal smear, was described by Graham and Graham in 1955. The cytologic features of radiation effect comprise cellular enlargement, vacuolated cytoplasm, multinucleation, and degenerative nuclear changes. A good response to radiation is considered when more than 75% of benign cells show these changes; a poor response is indicated if the changes are 60% or less. This demarcation is not a reliable or reproducible predictor of prognosis, and is currently a mere historic curiosity.
The inflammatory response predominantly comprises a heavy neutrophilic infiltrate that usually appears during or immediately after radiation. A few histiocytes, lymphocytes with phagocytized red blood cells or debris can also be seen. Giant histiocytes can be seen 25 days after radiation. A persistence of degenerating leukocytes and hemolyzed red blood cells is considered as a poor host response, and ultimately an ulcer, fistulae, sinuses, or deep abscesses may ensue.
Post-irradiation dysplasia is the appearance of abnormal cellular changes in benign epithelial cells after radiotherapy. These changes comprise cytoplasmic and nuclear enlargement with an altered N : C ratio, oval to irregular hyperchromatic nuclei with fine to coarse granular chromatin, and eosinophilic or amphophilic staining of the cytoplasm.
The prognostic significance of post-irradiation dysplasia remains a matter of debate. In a study by Wentz and Reagan, 84 patients were reported with post-irradiation dysplasia from 9 months to 12 years after the initial diagnosis of carcinoma. Of all patients with recurrent carcinoma, 80.8% developed post-irradiation dysplasia within 3 years after an initial diagnosis of carcinoma ( Fig. 32-3 ). The 5-year survival rate for the patients with a less than 3-year latent period of post-irradiation dysplasia was 33.8%, as compared with 100% 5-year survival rate for the patients with a greater than 3-year latent period. Post-irradiation dysplasia within 3 years after treatment for cervical carcinoma indicated a poor prognosis. Patten later claimed that post-irradiation dysplasia is a malignancy-associated change. However, these claims have not be received well by other experts.
Altered keratinization may be reflected in the karyopyknotic index (cornification index), which is the ratio of superficial cells showing karyopyknosis to all squamous cells in the smear, expressed as a percentage. A high karyopyknotic index indicates estrogen effect and may be related to the presence of malignancy in contrast to an atrophic pattern usually seen following irradiation.
The presence of malignant cells in a post-irradiation smear, albeit controversial, is considered as a poor response to therapy. In one study by Graham and Graham, there was no significant correlation between the presence of malignant cells in the smear at the end of treatment and the survival of the patients. While the presence of malignant cells at any time after completion of therapy merely indicated a poor response or recurrence, without any prognostic importance, the presence of malignant cells first after 4 months indicated a relatively poor prognosis. There is little or no radiation effect in the malignant cells in cases of persistent cancer. In the wake of identifying unaffected malignant cells in a post-irradiation smear, a thorough colposcopy and biopsies should be performed to rule out any persisting or recurrent lesions. Any cytologic transformation of the benign cells during the immediate post-radiotherapy period should be evaluated for features of malignancy.
DNA ploidy may be of prognostic value. Only a few studies have dealt with the relation of DNA ploidy to radioresponsiveness of invasive cervical carcinoma. These reports suggest that aneuploid tumors are more radiosensitive than diploid tumors. However, it is not certain whether the radioresponsiveness of tumors is related to survival of the patients. The correlation between DNA index and radiosensitivity of tumors other than carcinoma of the uterine cervix is controversial.
Oral Mucosa
Post-radiative changes in the oral mucosa are usually encountered as a result of therapy in head and neck malignancies. The changes are similar to those seen in the uterine cervix and comprise nuclear and cytoplasmic enlargement, cytoplasmic vacuolation, multinucleation, and increased cytoplasmic eosinophilia. In addition to these changes, the presence of cytoplasmic granules (representing RNA accumulation) and perinuclear halo formation have also been described. Previous studies have shown no correlation between the radiation response of oral squamous cells and the local recurrence rate.
It has been shown that serial cytologic assay of nuclear changes including induction of micronucleation, nuclear budding and multinucleation (polynucleation) during radiotherapy is a potentially useful test to predict radiosensitivity. Multinucleation, in particular, was shown to have the greatest correlation with radiosensitivity, suggesting that injury to the cytokinetic apparatus is important in determining tumor radiosensitivity. Previously, a case has been reported in which smears for DNA cytophotometry analysis indicated recurrence of oral cancer on two occasions, prior to the lesion becoming clinically visible. Moreover, in a recent study, it has been shown that tumor DNA content, in addition to p53 and Ki-67 appeared to be a significant prognostic marker for the evaluation of patients with oral cavity and pharyngeal squamous cell carcinomas receiving radiation therapy.
The presence of malignant cells usually indicates persistent or recurrent lesions. A negative cytology does not necessarily rule out an underlying submucosal malignant recurrence or persistence; the surface may be re-epithelialized with benign mucosa, thereby shielding the cytologic changes. Clusters of benign, streaming epithelial cells with prominent nucleoli showing features of repair may be seen. There may be epithelial atrophy and ductal metaplasia in the adjacent salivary gland tissue.
Lower Respiratory Tract
The lower respiratory tract may show radiation-induced cytologic changes as a part of therapeutic irradiation of the chest for various malignancies including those of the chest wall, lung, or mediastinum. The endothelial cells and type II pneumocytes of the lungs are most sensitive to radiation showing necrosis, type II pneumocyte proliferation, and hemorrhage. The bronchial epithelium, in the early stages, shows marked nuclear and cytoplasmic enlargement, formation of bizarre giant cells and atypical clustering. The nuclei are granular to coarse chromatin with prominent nucleoli. The finding of ciliated bronchial cells with/without clusters (Creola bodies) is a sign of benignancy. The changes of acute radiation pneumonitis, including necrosis of bronchial epithelial cells and infiltration of leukocytes, may superimpose an underlying inflammatory or malignant process.
Chronic radiation changes consist of metaplastic changes, chronic inflammation, and interstitial fibrosis. There can be significant metaplasia of the squamous cells, columnar cells, and bronchial submucosal seromucous glands. Of note, the squamous metaplastic changes may have uncanny cytologic resemblances of malignancy including a high N:C ratio, hyperchromasia, irregular clumping of chromatin, and prominent nucleoli ( Fig. 32-4 ), creating a diagnostic pitfall. A thorough clinical history, including current/past irradiation, gains importance in this scenario. On a similar note, radiation changes of pleura and mesothelium are likely to be confused with carcinoma.
The chronic changes associated with radiation pneumonitis include alveolar exudates, migratory organizing pneumonia, and bilateral interstitial pneumonia. These changes are thought to be secondary to the elaboration of cytokines. Changes similar to those seen in chronic radiation are also seen in response to various chemotherapeutic agents including bleomycin, busulfan, and other toxic pulmonary antineoplastic drugs. In cases where chemotherapy follows radiation, a pattern of radiation recall pneumonitis (RRP), a rare reaction in previously irradiated area of pulmonary tissue after application of triggering agents, may be seen.
Urinary Tract
The urinary bladder may show radiation-induced cytologic changes as a part of therapeutic irradiation for bladder and pelvic malignancies. The post-radiotherapy-induced urothelial changes can be appreciated as early as the second day of treatment. These changes comprise marked cellular enlargement accompanied by nuclear swelling and cytoplasmic vacuolation. Other nuclear changes include hyperchromasia, irregular shapes, pyknosis, karyorrhexis, and multinucleation. The nuclear hyperchromasia of malignant cells is usually marked compared with reactive urothelial cells in the same specimen. These features are most easily visualized in superficial urothelial cells ( Fig. 32-5 ). Of note, the cytoplasmic vacuolation and multinucleation are not specific for radiation effect; they have also been found in non-irradiated urothelium secondary to chronic cystitis and/or calculi. In patients with carcinoma of the cervix treated with radiation, the post-irradiated urothelial cells may cause a diagnostic dilemma by mimicking squamous cell carcinoma. The distinction between a locally invasive cervical carcinoma and a high-grade urothelial cell carcinoma with squamous differentiation may be difficult and may need a detailed clinical history to resolve.
Urine cytology is a useful method for the follow-up of patients with irradiated bladder cancer. Esposti and associates reported 89% accuracy in detecting persistent or recurrent carcinoma. Only one patient of 25 with radioresistant tumor had negative cytologic findings. Of 54 patients with recurrences, urine cytology revealed frankly malignant cells in 46 cases. Of all patients with recurrence, 17 had positive urine cytology findings, about 3–24 months preceding the clinical recognition of recurrence.
As a complication of radiotherapy, malignant tumors can develop in the urinary bladder. The lead time for secondary malignancies following radiation is from 2 to 10 years. In this context, urothelial cell carcinoma, squamous cell carcinoma, and sarcoma have been reported, mostly after treatment for carcinoma of cervix.
Breast
Radiation-induced cytologic change in the breast is a relatively new problem attributed to a greater tendency to treat small localized breast cancer conservatively with lumpectomy or quadrantectomy combined with radiation.
Schnitt and coworkers studied the effect of radiation on breast tissue in 30 patients with breast cancer. Epithelial atypia in the terminal duct lobular unit associated with lobular sclerosis and atrophy was the characteristic finding. Epithelial atypia in large ducts, stromal changes, and vascular changes were also seen, albeit less frequently. The atypical epithelial cells maintained polarity and cohesion without evidence of cellular pleomorphism or proliferation. These changes could easily be distinguished from malignancy. Of note, in a study of 120 post-radiotherapy biopsy or mastectomy patients by Moore and coworkers, radiation-induced changes including the stromal vascular and fibroblastic changes, epithelial cell changes of the terminal duct lobular unit and extralobular ducts, and terminal duct lobular unit fibrosis/atrophy did not regress over time, mandating the pathologist to be alert to the possibility of radiation therapy and systemic chemotherapy even without the therapeutic history. Fat necrosis may be present as a palpable breast lesion following breast-conserving therapy. Surgery can be avoided by using fine-needle aspiration (FNA) to evaluate such lesions.
Cytology.
The post-irradiation changes in an FNA biopsy from a recurrent breast mass may be difficult to distinguish from malignancy. The radiated benign ductal and lobular epithelia may be dyscohesive, showing severe atypia with large, pleomorphic, hyperchromatic nuclei and rare bipolar naked nuclei. These cytologic features were in contrast to the histologic epithelial changes described by Schnitt and colleagues. A clue that these changes may represent radiation effects is the hypocellularity of the smears due to fibrosis. The identification of myoepithelial cells, bipolar naked nuclei or a transition between normal and reactive cells within an aggregate of atypical cells may indicate benignancy. Of course, a thorough clinical history of prior radiation would be indispensable. Moreover, radiation-induced changes may also be evident in the pleura and chest wall. The pleural mesothelial cells may show cytologic changes independent of the presence or absence of an underlying malignancy.
Prostate
In comparison to the organ systems, a FNA follow-up of a radiation insult to the prostate after therapeutic irradiation in cases of prostatic carcinoma has been infrequently dealt with.
The post-radiotherapeutic histologic changes of non-neoplastic and neoplastic prostatic tissue have been studied. In non-neoplastic prostate, these changes include atrophy and squamous metaplasia (with or without atypia), stromal fibrosis, and arterial sclerosis. The neoplastic glands, however, were unaffected without architectural distortion or dedifferentiation. Interpretation of post-irradiation prostate needle biopsies is a major diagnostic challenge due to substantial radiation-induced changes in benign and malignant prostatic tissue. In a study of 29 patients by Cheng and coworkers, histologic features that were helpful in the diagnosis of cancer after radiation therapy included infiltrative growth, perineural invasion, intraluminal crystalloids, blue mucin secretions, the absence of corpora amylacea, and the presence of coexistent high-grade prostatic intraepithelial neoplasia. The findings indicated that, while Gleason grade in post-irradiation needle biopsy specimens appeared to provide useful predictive information, the quantification of radiation effect in needle biopsy specimens was inaccurate and potentially misleading. The cytologic changes in the neoplastic glands did not differ from their non-neoplastic counterparts, and were not a useful feature to differentiate between the two.
Cytology.
An FNA biopsy of an irradiated prostate usually yields a hypocellular specimen as a result of ensuing fibrosis within days of radiotherapy. As aforementioned, cytologic changes are not useful in differentiating radiation-induced changes in non-neoplastic from neoplastic glandular epithelium, and consists of cellular and nuclear enlargement, cytoplasmic vacuolation, and bizarre giant nuclear forms ( Fig. 32-6 ). Squamous metaplasia may develop. In the neoplastic epithelium, it is common to find degenerative nuclear changes, such as karyorrhexis, karyolysis, or pyknosis. While the presence of well-preserved nuclei with crisp chromatin, eosinophilic nucleoli, and cellular dyshesion suggests a viable neoplasm, viability of neoplastic cells in an irradiated prostate may be difficult to identify in as much as the nucleus and nucleoli may be inconspicuous due to shrinkage.
Serial rectal examination and biopsy, Tru-Cut, or FNA still remain the accepted methods of monitoring a patient with irradiated prostate carcinoma. A positive biopsy finding 12–18 months after therapy may indicate a residual viable neoplasm. Of note, the biopsy findings can convert from malignant to benign as late as 1–2 years after radiation. On average, after 2.5 years, approximately 20% of the prostate biopsy samples contain viable malignant cells. However, the prognostic significance of persistent malignant cells and the value of a post-irradiated biopsy to assess the tumor response remain controversial, in part because of challenges in evaluating the viability and metastatic potential of the residual malignant cells. Also, the difficulty of post-radiation prostate needle biopsy interpretation includes the distinction of treatment effect in normal prostatic tissue from recurrent or residual tumour. Histologic changes after thermal ablation mainly include lesions observed in prostatic infarcts due to periurethral coagulative-type necrosis of variable volume. In addition, there is a poor correlation between the post-therapy biopsy results and the patient's survival.
Thyroid
The administration of radioactive iodine-131, a form of internal radiation for the treatment of Graves' disease and a few cases of functioning thyroid carcinomas, is common practice. Iodine-131 emits mostly beta rays that centrifugally spread approximately 2 mm, thereby sparing the structures adjacent to the thyroid. The effects of radioactive iodine on the thyroid tissue depend on the dose (radioactivity per gram of thyroid tissue), the duration, and the sensitivity of the thyroid cells to radiation.
Histologically, the post-radioactive treatment effects on the thyroid can be divided into acute and chronic changes. The acute changes include stroma edema, cytoplasmic oxyphilia, and nuclear pyknosis. By the third week, the stromal edema can be replaced by interstitial fibrosis with a decrease in follicle size. Acute inflammation or necrosis is rarely found in therapeutic iodine-131 doses. The chronic changes, after 6 weeks, comprise nuclear atypia with hyperchromasia, interstitial fibrosis, cytoplasmic swelling, oxyphilia (resembling Hürthle cells), bizarre giant forms, small follicles, and vascular alterations. The nuclear changes are most prominent in the oxyphilic cells and may be indistinguishable from malignancy. A picture of oxyphilic cells with lymphocytic infiltrate and multinucleated histiocytes may mimic Hashimoto's thyroiditis. Some authors have reported a 39% incidence of Hashimoto's thyroiditis in the irradiated glands. In the long term, regenerative or adenomatous nodules may be evident. The irradiation therapy may be a risk factor for thyroiditis with an increase in the incidence of papillary thyroid carcinoma as seen at Chernobyl, Three Mile Island, during irradiation for acne, and many more.
Cytology.
The post-irradiated acute cytologic features of thyroid consist of nuclear enlargement with bizarre nuclear forms, clumping of chromatin, cytoplasmic swelling or eosinophilic granularity, and naked nuclei. Intranuclear inclusions and blue-black cytoplasmic pigment granules in the follicular cells have also been described. These features may be indistinguishable from malignancy, and a thorough clinical history including current/past iodine-131 therapy remains indispensable. In the chronic stage, aspiration biopsy may yield scant cellularity owing to fibrosis and follicular atrophy.
Antineoplastic Chemotherapy
Chemotherapy follows first-order kinetics in destroying the tumor cells, targeting a certain percentage after each dose. This phenomenon is related to dose as well as the phase of the tumor cell cycle. Interestingly, the chemotherapeutic cytologic changes share many of the features with those seen in post-radiation changes.
The chemotherapeutic agents can be categorized broadly into alkylating agents, antimetabolites, natural products, biologic response modifiers, hormonal agents, and miscellaneous agents.
The tumor cells comprise an asynchronous and heterogeneous population. The chemotherapeutic agents, including the alkylating agents, antimetabolites, and natural products are cell cycle-specific. Each may target a distinctly different phase of the cell cycle or at one or more point. A combination(s) of the therapeutic agents often acts on different targets/phases of the cell cycle ( Table 32-3 ). Table 32-4 identifies these chemotherapeutic agents and their targets at specific stages of the cell cycle.
TABLE 32-3
Chemotherapeutic Agents
| Agent(s) | Effect |
|---|---|
| Dactinomycin | Inhibits DNA, RNA, protein |
| Doxorubicin (Adriamycin), BCNU | Intercalate DNA |
| Bleomycin, nitrogen mustard | Single- and double-stranded breaks |
| Cisplatin | Intra- and interstrand cross-links |
| Vincristine, vinblastine | Bind tubulin in mitotic spindle |
| 5-Fluorouracil | Inhibits thymidylate synthetase |
| Hydroxyurea | Inhibits ribonucleotide diphosphate reductase |
| Methotrexate | Inhibits dihydrofolate reductase |
| Etoposide | Topoisomerase II inhibitor |
| Paclitaxel (Taxol) | Tubulin binding |
TABLE 32-4
Cell Cycle-specific Therapeutic Effects
| Phase | Therapeutic Agents |
|---|---|
| G1 | Radiation, alkylating agents, cisplatin |
| S | Docetaxel, methotrexate, Ara-C, 6-thioguanine, hydroxyurea, vinblastine, vincristine, doxorubicin |
| G2 | Paclitaxel, bleomycin |
| M | Radiation and alkylating agents |
Chemotherapeutic agents tend to target a smaller number of cells than radiation changes. It is not surprising that, correspondingly, the cytologic changes are also evident in far fewer cells than in the irradiated specimens. Since chemotherapeutic agents are administered systemically, changes at remote sites are evident more commonly than radiation treatment. The chemotherapeutic agents are mutagenic in nature; the risk of other malignancies is increased by many fold, 2–4 years after chemotherapy. This is most evident with alkylating agents. The overall risk of acquiring a secondary malignancy may be enhanced in combination with radiotherapy. These include leukemias, lymphomas, and squamous cell carcinoma. In contrast, the secondary malignancies developing post-radiotherapy are mostly carcinomas and sarcomas.
Historically, approximately 35 chemotherapeutic agents were in use in 1992. The current number of these agents is close to 100. The assessment of the chemotherapeutic effects is dependent on either non-clonogenic assays or clonogenic assays on tumor stem cell populations. The non-clonogenic assays are applied against an unselected tumor cell population ( Box 32-3 ), and most closely resemble tumor cells in vivo. The detection of microscopic evidence of cell degeneration is one of the examples of a non-clonogenic assay. The secondary effects of chemotherapy may be found anywhere in the body site but are most frequently noted in the cervix, lung, and bladder. Of course, these sites are also among the most frequently sampled sites for cytology.
Box 32-3
Clonogenic and Non-clonogenic Assays
Key Features of Chemotherapy
-
Cell enlargement
-
Hyperchromasia
-
Smudged chromatin
-
Nuclear and cytoplasmic vacuolization
-
Dyscohesion.
Bleomycin
Bleomycin is an antineoplastic and antimicrobial polypeptide antibiotic, isolated from the fungus Streptomyces verticillus . Briefly, it acts by binding to the DNA guanine bases with the oxidation of Fe 2+ (ferrous iron) to Fe 3+ (ferric iron). The liberated electron is accepted by oxygen to form toxic superoxides (O 2 − ) and hydroxyl (OH•) radicals. These active oxygen intermediates attack the DNA bases, leading to single- and double-stranded DNA breaks and deletions. The primary toxicity of bleomycin is subacute/chronic pneumonitis with progression to interstitial pneumonitis.
Chest radiography shows bibasilar pulmonary infiltrates that need to be differentiated from metastatic tumor, infectious processes such as Pneumocystis jirovecii and cytomegalovirus, and radiation injury. The diagnosis is often made by open lung biopsy. Other less aggressive diagnostic methods include evaluation of sputum, bronchial lavage, brushings, and FNA cytology.
Bleomycin toxicity is fatal in about 1–2% of patients; an additional 2–3% of patients experience non-lethal pulmonary fibrosis. This toxicity usually does not manifest below a cumulative dose of 400 units. However, a case occurring after a dose of only 60 units has been reported, questioning the existence of a true threshold whatsoever. Other factors synergistically increasing the incidence of bleomycin-induced pulmonary toxicity include age (>70 years), prior radiation therapy, combination chemotherapy in non-Hodgkin's lymphomas, and high partial pressures of oxygen during anesthesia.
The histologic and ultrastructural changes in bleomycin-induced pulmonary toxicity have been well documented in animal models. The pulmonary endothelial cells and pneumocytes are amongst the most sensitive, a similarity to the radiation changes. The earliest changes evident are endothelial and perivascular edema of pulmonary vessels progressing to interstitial edema. The ultrastructural studies show subendothelial blebbing with intracytoplasmic edema. There is necrosis of type I pneumocytes along with a proliferation and desquamation of type II pneumocytes. The cytologic features of type II pneumocytes comprise bizarre elongated cytoplasmic shapes, nuclear pleomorphism, hyperchromasia, occasional mitotic figures, and metaplastic changes. There may be intra-alveolar fibrin deposition due to vascular leakage. The interstitium develops septal fibrosis with collagen/elastin deposition along with smooth muscle proliferation. An end-stage bibasilar fibrosis develops, eventually leading to a sequela of “honeycomb” lung ( Fig. 32-7 ).
Cytology.
A cytologic evaluation of different samples including sputum, bronchial, and FNA biopsy may yield distinctly different findings in bleomycin-induced pulmonary toxicity.
In one study, an increase in ciliocytophoria (30–56.2%) and low-grade dysplasia (10–37.5%) in the sputum of bleomycin-treated patients has been reported as compared with matched controls. However, there was no significant difference in inflammation, or any evidence of atypical columnar cells. In addition, 40% of the treated patients showed low-grade dysplastic squamous cells in the oral cavity. These cells varied in size and shape with amphophilic cytoplasm and irregular hyperchromatic nuclei. These changes were evident as early as 12 weeks after the start of therapy.
An evaluation of bronchial cytology shows a predominance of benign bronchial cells. However, occasional scattered large abnormal columnar cells with bizarre large nuclei, clumped chromatin, and prominent large eosinophilic nucleoli may be found. In spite of these alarming features, the bronchial cells maintain their delicate translucent, basophilic cytoplasm. The presence of well-defined cilia, of course, is a sign of benignancy.
The FNA aspirates may be of low to moderate cellularity, depending on the degree of fibrosis, mainly showing clusters of atypical cells. The cellular changes observed in bleomycin-induced pulmonary toxicity are similar to radiation-induced changes. The atypical epithelial cells form prominent cohesive 2-dimensional sheets with a few scattered single cells. These atypical cells are large, polygonal, pleomorphic, and have well-defined cell membranes with minimal overlap, resembling a reparative epithelial arrangement. Their nuclei are central or eccentric, with a smooth membrane, coarsely granular chromatin, and single or multiple macronucleoli. The cytoplasm is dense (squamoid) and may be two-toned with slight orangeophilia or amphophilia. Perinuclear microvacuoles may be found, with a few scattered lymphocytes and plasma cells in the background. The cytologic appearance may mimic a malignant change ( Fig. 32-8 ), and a detailed clinical history is imperative.
There is no effective therapy available for bleomycin-induced pulmonary fibrosis. The toxic pulmonary changes and subsequent end-stage fibrosis are not unique to bleomycin and may be found in other cases involving antineoplastic drugs, nitrofurantoin, etc. ( Box 32-4 and Fig. 32-9 ).
Box 32-4
Multidrug-resistant Drugs
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Doxorubicin
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Daunorubicin
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Mitoxantrone
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Vincristine
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Vinblastine
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Colchicine
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Paclitaxel (Taxol)
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Etoposide
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Teniposide
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Actinomycin D
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