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Abstract
Introduction: Detection of malignant cells in cerebrospinal fluid (CSF) samples in suspected cases of malignancy is critical for the management of patients. CSF involvement by nonhaematolymphoid malignancies is less common. We aimed to study the cytomorphologic characteristics of various nonhaematolymphoid malignancies in CSF. Methods: A retrospective cytomorphological analysis of 27 CSF cytology smears reported as positive or suspicious for nonhematolymphoid malignancies from January 2010 to April 2020 over 10 years was carried out. Smears in all cases were prepared by cytospin technique and stained with May-Grunwald-Giemsa (MGG) and papanicolaou (Pap) staining procedures. Cell immunohistochemistry/immunocytochemistry was done wherever cell block/extra slides were available. Results: Twenty-four of 27 cases were interpreted as “positive,” while three were reported as “suspicious” of malignancy. Nineteen of 27 cases were metastatic adenocarcinomas including three suspicious malignancy cases with the primary sites of origin being the breast (10), stomach (2), rectum (1), gall bladder (1), lung (1), and four cases of unknown primary. Of the remaining positive cases, there were five cases of metastatic medulloblastoma, two cases of metastatic pineoblastomas, and one case of metastatic extraskeletal Ewings sarcoma. Each of these metastatic malignancies had at least a single diagnostic cytomorphological clue, similar to those observed in other body cavities and primary malignancy sites. Conclusion: Nonhematolymphoid malignancies are readily diagnosable on CSF cytology, most of them are metastatic. Identification of malignant cells in CSF is critical, as it has therapeutic and prognostic implications.
Keywords: Carcinoma, CSF, cytomorphology, involvement, nonhematolymphoid malignancies
How to cite this article: Introduction 
Neoplastic meningitis is the dissemination and growth of cancer cells within the leptomeningeal space, which is one of the most serious complications that can occur in cancer patients.[1] Its incidence is increasing significantly due to longer survival of cancer patients, owing to advancements in adjuvant therapies. Neoplastic meningitis can be caused by metastasis from solid tumors, which is termed carcinomatous meningitis, or by infiltration by leukemia or lymphoma which is termed as leukemic or lymphomatous meningitis, respectively. The latter is the most common cause of neoplastic meningitis seen in 5%–15% of patients with leukemias, followed by carcinomatous meningitis which is seen in 1%–5% of cases with solid tumors. Meningeal seeding within cerebrospinal fluid (CSF) from a primary brain tumor is seen in 1%–2% of patients.[2]
Common solid tumors causing neoplastic meningitis are carcinoma of the breast, lung, and malignant melanoma.[3] Rare cases of gastric and gall bladder adenocarcinoma presenting with neoplastic meningitis have also been reported. The diagnosis of neoplastic meningitis mainly depends on the detection of malignant cells in CSF by cytological examination, imaging studies, and clinical findings. However, currently, identification of cancer cells in CSF using cytological examination is considered the gold standard test for diagnosis of neoplastic meningitis.[4]
Identification of malignant cells and their categorization is critical, as it has therapeutic and prognostic implications. This retrospective study was conducted to evaluate the cytomorphological characteristics of various nonhematolymphoid malignancies in CSF, that would aid in the accurate diagnosis of neoplastic meningitis.
Methods A retrospective cytomorphological analysis of 27 CSF cytology smears reported as positive or suspicious for nonhematolymphoid malignancies from January 2010 to April 2020 over 10 years was carried out. In all these cases, smears were prepared by cytospin technique and stained with May-Grunwald-Giemsa (MGG) and papanicolaou (Pap) staining procedures. The cell block was prepared in one of the cases and immunocytochemistry (ICC) was carried out on pap-stained smears in three (03) cases, with cytokeratin (02) and Estrogen receptor (ER) (01). In addition, one case had immunohistochemistry (IHC) performed on the cell block preparation with synaptophysin (01). MRI features and details of clinical presentation were not available due to the retrospective nature of the study.
The cytopathologic diagnosis in the CSF cytology smears fell into three categories. (A) Negative for malignant cells when smear was either acellular or showed only a few benign cells (B) Suspicious for malignancy when few atypical cells are seen, in the paucicellular smear or in the background of reactive cells that were not conclusive enough for the diagnosis of malignancy. (C) Positive for malignancy when there were definite malignant cells in a cellular smear. Cases that are suspicious and positive for nonhematolymphoid malignancies are included in this study.
Results A total of 3338 CSF samples were received during 10 years out of which 130 samples were positive for malignancy, which included both hematolymphoid and nonhematolymphoid malignancies. Of these 130 positive smears, 27 were positive for nonhematolymphoid malignancies, including three suspicious cases of malignancy. Of these 27 cases, seven were of pediatric age group (range 4–5 years; mean age, 4.4 years) and 20 were adults (range 29–68 years; mean age, 48.5 years). The male-to-female ratio was 1:1.7. All seven pediatric cases had positive CSF cytology smears for neoplastic meningitis from primary CNS neoplasm, which included five cases of medulloblastoma and two cases of pinealoblastoma. Of the remaining 20 cases, 16 were reported as positive for adenocarcinoma and three as suspicious for adenocarcinoma. The most common primary site of origin being breast (09), followed by stomach (02), unknown primary (02), gall bladder (01), rectum (01), and lung (01). All of these 16 positive cases had either biopsy or FNAC correlation for primary tumors. In addition, there was one case with positive CSF cytology in a known case of extraskeletal Ewing's sarcoma. Of the three suspicious cases for malignancy, one had osteoblastic skeletal metastasis in a bone scan, one was a known case of infiltrating ductal carcinoma, and the other had neither biopsy nor radiological correlation. The distribution of the cases involved by nonhematolymphoid malignancies is tabulated in [Table 1]. The cytomorphological features were evaluated in these cases which may hint us in identifying the site of primary in challenging situations.
Table 1: The distribution of the cases involved by nonhematolymphoid malignanciesCSF involvement by adenocarcinoma (19 cases)
Breast (10 cases):CSF smears in all of these cases were predominantly cellular and arranged either singly or as lose clusters. Individual cells in most of the cases were large and had dense cytoplasm with cytoplasmic blebs and vacuolation. The nuclear membrane showed irregularity with prominent nucleoli. Some of the smears also showed cellular cannibalism (cell in cell appearance with one cancer cell engulfing an adjacent cancer cell) [Figure 1]a and [Figure 1]b.
Figure 1: Metastatic breast carcinoma in cerebrospinal fluid shows highly cellular smears poorly cohesive cells with dense cytoplasm, cytoplasmic blebs, and cellular cannibalism (arrow) (a) cytoplasmic vacuolations (b) (MGG X 400). Inset showing strong ER positivity (DAB X400)CSF smear from one case of lobular carcinoma was sparsely cellular. Though there were definite malignant cells, they exhibited no cytomorphological clue of its primary diagnosis. A suspicious CSF smear in a known case of IDC showed predominantly necrotic debris along with coagulative necrotic cells. There were very occasional scattered atypical cells.
Stomach (02 cases): Smears from both cases were highly cellular with discretely lying cells and an occasional glandular pattern was also noted in one of the smears. The individual cell had an ample amount of cytoplasm with an irregular nuclear membrane and prominent nucleoli. Few of them also had cytoplasmic mucinous vacuoles. However, no definite signet ring cells were observed in the smears examined [Figure 2]a.
Figure 2: Metastatic adenocarcinoma in cerebrospinal fluid from stomach showing discretely lying malignant cells containing moderate cytoplasm, few of them containing mucinous vacuoles (arrow) (a); from rectum showing singly scattered as well as aggregates of tumor cells containing hyperchromatic nuclei in a mild necrotic background (b); from gall bladder showing discretely lying as well as clusters of malignant cells in an inflammatory background (c) (MGG x 400)Rectum (01 case): Smear was cellular with singly scattered as well as tiny aggregates of tumor cells containing round to oval hyperchromatic nuclei, exhibiting minimal pleomorphism in a mild necrotic blood mixed background [Figure 2]b.
Gall bladder (01 case): Smear was highly cellular with discretely lying as well as clusters of tumor cells with moderate cytoplasm and a few contain cytoplasmic vacuoles in an inflammatory background [Figure 2]c.
Lung (01 case): Smear was cellular with numerous singly scattered and occasional clusters of malignant cells which were predominantly degenerated. However, few of the preserved cells show cytoplasmic vacuolation, irregular nuclear membrane, opened chromatin, and occasional prominent nucleoli.
CSF involvement by primary CNS tumors
Medulloblastoma (05 cases): Smears were cellular and arranged predominantly dense closely packed clusters. The individual cells were large-sized with scant cytoplasm, an angulated nucleus with nuclear molding and indentation. The chromatin was mostly coarse and irregular. In one of the smears, vague rosetting was also noted. Cellblock preparation was available in one of the metastatic medulloblastoma case on which IHC showed synaptophysin positivity [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d.
Figure 3: Metastatic medulloblastoma in cerebrospinal fluid smears cluster of densely packed malignant cells on cytospin (a) PAP, x100; Cellblock showing clusters of malignant cells with scant cytoplasm, angulated nuclei, nuclear molding with coarse chromatin (b) H and E 100 X, (c) 400X; Positive Synaptophysin IHC on cell block (d) DAB X 400Pinealoblastoma (02 cases): One of the cases had a sparsely cellular smear, however, showed scattered and occasional clusters of definite primitive appearing malignant cells which had round nuclei with a barely discernible rim of cytoplasm and extremely hyperchromatic nuclei. The other smear was cellular with loose clusters of malignant cells, the morphology of which was poorly preserved.
Extraskeletal Ewings sarcoma (01 case): CSF smear was cellular as well as hemorrhagic and show clusters of medium-sized degenerated malignant cells with scant to moderate amount of cytoplasm containing fine vacuolations. The nuclear chromatin was fine. The two-cell population comprising larger, lighter cells and smaller, darker cells that is characteristic of Ewings sarcoma in cytology material was not evident in the smears examined [Figure 4]a and [Figure 4]b. No special stains or ICC were performed on cytology smears, however, IHC performed on biopsy sections from left hypochondrial swelling showed positivity for FLI-1 and CD99.
Figure 4: Metastatic extraskeletal Ewings sarcoma in cerebrospinal fluid clusters of medium-sized degenerated malignant cells with scant cytoplasm containing fine cytoplasmic vacuolations in a hemorrhagic background (a) MGG, x100, (b) MGG, x400Unknown primary (04 cases): In two cases, which were called positive, smears were cellular and had singly scattered as well as clusters of malignant adenocarcinoma cells. However, no follow-up details could be obtained and the exact primary site remained unknown. In the two suspicious cases, smears were hemorrhagic with scattered atypical cells of poorly preserved morphology. The exact categorization of cells was not possible.
Discussion Malignant cells infiltrate leptomeninges or CSF cavity by direct extension or by hematogenous spread or by lymphatic metastasis.[5] Neoplastic meningitis is a serious complication of cancer that occurs in 1%–5% of patients with solid tumors and is observed most commonly in patients with breast carcinoma, lung carcinoma, and malignant melanoma. The development of neoplastic meningitis portends a significant worsening of prognosis with a shortening of survival ranging from 4 to 16 weeks after its diagnosis.[6] Hence, early diagnosis of the leptomeningeal disease can be crucial because the institution of aggressive treatment such as radiotherapy/intrathecal chemotherapy can increase the patient's chance of survival and relief from symptoms. This is particularly true in the case of small cell carcinoma of the lung which is associated with treatable occult cerebro-meningeal metastasis.[7]
The diagnosis of neoplastic meningitis is mainly depending on the detection of malignant cells in CSF by cytological examination, radiographic manifestations as revealed by neuraxis imaging, and clinical findings. With improved visualization of subarachnoid space and improvement of sensitivity by high field magnetic resonance imaging (MRI), imaging has become the initial even sole diagnostic tool in the MRI era. However, the neuroimaging findings of neoplastic meningitis are diverse, complex, and usually nonspecific. Its lower specificity precludes it from replacing cytology as the gold standard test for diagnosing neoplastic meningitis.[4] In addition to MRI, CSF levels of protein, glucose, and tumor markers can also be used in the detection of CSF dissemination, however, none of these have shown a definite increase in the diagnostic yield compared to the cytological examination.
Accurate diagnosis also requires appropriately processed and well-stained smears. Due to rapid cellular degeneration within the hypo-osmotic CSF, slide preparation should occur ideally within 2 h following aspiration.[8] It is recommended that cell block preparation is to be made in all possible cases which would aid us in performing further IHC as well as help us with better cytomorphology in situ ations of diagnostic dilemmas.
As described with other studies, the commonest nonhematolymphoid malignancy metastasizing to CSF in our study was adenocarcinoma with the most common primary site of origin being breast and the commonest primary CNS neoplasm found in CSF was medulloblastoma. The diagnosis of neoplastic meningitis in a known case of primary is relatively easy when compared to the case of unknown primary which is quite challenging. Hence, we would emphasize the cytomorphological clues in each of these 27 cases, which would help us in challenging situations.
Cytomorphological analysis of smears from the case of medulloblastoma was characterized by tumor cells arranged as dense closely packed clusters with individual cells exhibiting nuclear molding and indentation. Occasional rosetting can also be seen. Smears from pinealoblastoma showed no diagnostic clue except for the formation of occasional clusters. Medulloblastoma and pinealoblastoma are both PNETs, tumor cells of which upon shedding into the CSF will tend to form clusters which are observed in almost all our cases. Cytological features such as nuclear molding, indentation, and rosetting are not pathognomonic for medulloblastoma which can be observed in other PNETs as well and even various studies have reported the difficulty in discerning medulloblastoma from other metastatizing CNS tumors like pinealoblastoma and neuroblastoma.[9]
The other small round blue cell tumor described in our study is Ewings sarcoma, smears of which show medium-sized cell clusters with cytoplasmic fine vacuolations. In cytological material, as in histology, the tumor tends to show a cellular population of small, round malignant cells, usually of two types: larger cells with paler nuclei and dispersed chromatin and a second group of smaller, more hyperchromatic cells with indistinct nuclear chromatin.[10] However, such two cell population was not observed in our smears and no literature has described the cytomorphology, usefulness of special stains like PAS positivity in cytoplasmic vacuoles, and role of ancillary techniques for Ewings sarcoma in CSF smears. The close differential diagnosis of this case pertaining to the age (46 years) and cytomorphology (small round blue cells) would be leukemia/lymphoma infiltration into CSF, however, the presence of clustering precludes the diagnosis of leukemia/lymphoma. It is also important to remember that the most widely used IHC for Ewings sarcoma like CD99 can show positivity in leukemia/lymphoma as well, hence IHC markers for demonstration of malignant cells in cytological material should be chosen with caution, taking into consideration of cytomorphological features and clinical presentation.
Among adenocarcinomas, there was at least a single diagnostic clue of its primary site in the CSF smears studied. Breast adenocarcinoma smears are highly cellular arranged predominantly as loose clusters with individual cells having dense vacuolated cytoplasm. Though cytoplasmic blebs, vacuolations, and cannibalism are not specific to breast adenocarcinoma, these were observed in most of the cases. Similar cytomorphological features of metastatic breast adenocarcinoma in CSF smears are being described in a study conducted by Rao et al., wherein they described that all samples were cellular and showed either singly dispersed cells or clustered malignant cells with individual cells have high nuclear to cytoplasmic ratio, nuclear hyperchromasia, irregular nuclear membrane, prominent nucleoli, and pale vacuolated cytoplasm.[11] Gastric adenocarcinoma cells had a glandular pattern and intracytoplasmic mucinous vacuoles and cells from rectal adenocarcinoma had round to oval hyperchromatic nuclei with a high N:C ratio in a mild necrotic background. The adenocarcinoma cells from the gall bladder were arranged in an inflammatory background. Smear from lung adenocarcinoma was poorly preserved and was not helpful in cytological analysis.
Thus, the cytomorphological features of these malignant cells are similar to those observed in other body cavities and primary sites, however, it is affected by factors like sparse cellularity, artifacts due to cytospin, and the easy tendency of these cells to undergo degeneration in CSF. The centrifugal force used in cytospin preparations can result in cellular distortion including nuclear irregularity, cellular molding, and edge artifact. This study is limited by the lack of clinical presentation and MRI features due to the retrospective nature of the study. Also, ICC and cellblock preparations were not performed which would have otherwise added to the diagnostic yield in cases of unknown primary. Despite newer diagnostic modalities, CSF cytology remains the gold standard method in the diagnosis of neoplastic meningitis. However, further studies have to be conducted on the role of ICCs and cell block preparations in accurately diagnosing the primary site, in cases of unknown primary especially.
Conclusion CSF involvement by nonhematolymphoid malignancies is rare. The cytological examination is the gold standard for the diagnosis of neoplastic meningitis. Cytomorphology helps in establishing the diagnosis from other mimics of meningitis in cancer patients.
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References 
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CheckDOI: 10.4103/joc.joc_66_22
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