USG Guided Fine Needle Aspiration Cytology along with Immunocytochemistry to Diagnose Primary Malignant Mixed Mullerian Tumors: A Three-Year Study from a Tertiary Care Center.

Aims and
Objectives: To study the diagnostic utility of fine-needle aspiration cytology (FNAC) and immunocytochemistry in diagnosing primary malignant mixed Mullerian tumors (MMMT). Materials and
Methods: A 3-year retrospective study carried out in a tertiary care hospital, which included all the gynecological patients who underwent USG-guided FNAC of their abdominopelvic masses. Observations and
Results: Out of the 324 total cases, 05 (1.5%) were reported as primary malignant mixed Mullerian tumors. Out of these 05 cases, 03 were ovarian, 01 was uterine, and 01 involved both uterus and one-sided adnexa. The FNA smears from the masses revealed cytomorphological features of a biphasic neoplasm with elongated pleomorphic spindle cells and dispersed, focal attempted acinar pattern, thus indicating the possibility of MMMT. Immunocytochemistry was further carried out which showed both vimentin and cytokeratin positivity. The diagnosis was confirmed on subsequent biopsy and immunohistochemistry (without any histopathological-cytological discrepancy).
Conclusion: Though the literature is replete in establishing a histo-pathological diagnosis of MMMT, the diagnosis on USG-guided FNAC has been rarely described. Emphasis should be made on the careful examination of small sarcomatous elements in smears. Utilization of cell block and immunocytochemistry with histopathological correlation should be done to avoid misdiagnosis. Copyright:

INTRODUCTION

Malignant mixed Mullerian tumors (MMMTs) of the female genital tract (FGT) are biphasic tumors composed of both malignant epithelial and mesenchymal component; most commonly identified in the uterus.[1] MMMTs generally affect post-menopausal women with risk factors like obesity, nulliparity, exogenous oestrogen, and long-term tamoxifen use.[2] The epithelial components in carcinosarcoma are mucinous, serous, squamous, endometrioid, clear cell, transitional or mixture of these types and sarcomatous components may be either homologous (composed of malignant stromal elements native to the tissue) or heterologous (composed of nonnative sarcomatous tissue such as rhabdomyosarcoma, chondrosarcoma, or osteosarcoma).[23] They are aggressive and have a poor overall survival rate.[4] Diagnosis of MMMTs is a challenge on cytology alone especially due to nonrepresentation of all the components on needle aspirates. These are only described as eight studies in cytology literature.[56789101112] In the current study, we retrospectively analyzed the cytological findings of patients of FGT carcinosarcoma with clinical and histopathological correlation.

MATERIALS

This was a retrospective study done over a period of 3 years (2015–2018). Patients who presented to the Gynecology Department with complaints of abdominal mass with or without additional complaints of bleeding per vaginum, menorrhagia weight loss etc. were advised to consent to ultrasound-guided FNAC from these lesions. FNAC of the affected area was performed with aseptic precautions under supervision and material was aspirated with a 10 ml disposable syringe. The air-dried smears were Giemsa stained. Cell blocks were prepared and few extra unstained smears were kept for immunocytochemistry (ICC). Clinical and biochemical profiles of the patients and treatment regimens were also noted.

A total of 324 USG-guided FNAC for intra-abdominal masses were performed during the period of 3 years for various indications. Out of these, only 4 cases were diagnosed as MMMT on FNAC. One case was studied on scrape cytology upon receipt of excision specimen.

The patients were managed with cisplatin-based chemoradiotherapy; however, follow-up data is available for only three patients. Two of the patients succumbed to the illness within 6 months of starting treatment, only one was alive and under follow-up a year after surgery.

ICC was performed in these cases using cytokeratin, epithelial membrane antigen (EMA), and vimentin (vim) antibody. The gold standard for diagnosis was based on histopathology and immunohistochemistry.

Ethical committee approval was duly obtained for this retrospective study.

RESULTS

Five cases out of 324 USG-guided gynecological FNACs of abdominopelvic masses, during the 3 year study period were diagnosed as MMMT (carcinosarcoma) on cytology with an overall incidence of 1.5%. These 5 patients ranged from 37–57 years. Two out of five cases were young. All the cases presented with an abdominal mass, three had additional complaints of bleeding per vaginum and menorrhagia. The clinical presentations have been depicted in Table 1. CA 125 was known in 4 out of 5 cases and was raised in all 4 cases, i.e., in 100% of patients.

Table 1

Clinical parameters of five cases

Parameters	Age (in years)	Symptoms	Tumor size (in cm2)	Tumor site	Omentum	Lymph node	CA-125 (U/ml)
Case 1	37	Abdominal pain and mass × 2 months - Weight loss and cachexia+	8 × 8 × 12 cm2	Uterus and one Ovary	Negative	ND	387
Case 2[13]	37	Abdominal mass and discomfort × 6 months - Menorrhagia - weight loss × 2 months	10 × 10 × 8 cm2	ovary	Positive	ND	215
Case 3	65	Abdominal mass × 1 month - Postmenopausal bleeding × 1 month	10 × 9 × 7 cm2	Uterus	Positive	ND	Not known
Case 4	40	-Abdominal mass × 4 months - Pain lower abdomen × 4 months	10×9 × 7 cm2	Ovary	Positive	Positive	1608
Case 5	50	-Abdominal mass × 8 months - Menorrhagia × 8 months	7×6 × 4 cm2	Uterus	Positive	ND	263

The FNA smears from the masses revealed cyto-morphological features of a biphasic neoplasm with variable cellularity (moderate to high) comprising of two cell populations; one with round to ovoid dispersed cells with focal attempted acinar pattern with few cells showing cytoplasmic vacoulations, high NC ratio, hyperchromasia, one to two prominent nucleoli were also noted. [Figure 1a] The second population of cells were elongated pleomorphic spindle cells with high nucleo-cytoplasmic ratio and fragile cytoplasm, some spindle cells showed giant cells with bizzare nuclei as well.[Figure 1b] Atypical mitotic figures were noted in both the cell types [Figure 1c]. These cells were dispersed in a necrotic blood mixed background. ICC for a limited panel of CK [Figure 1b], vimentin [Figure 1e] and EMA, epithelial membrane antigen was performed on unstained smears and/or cell blocks (depending upon the availability and quantum of material obtained) which delineated epithelial and sarcomatous components.

Figure 1

(a) FNA: Highly cellular smear with focal attempted acinar pattern plump plasmacytoid cells representing epithelial component (Geimsa 200×). (b) CK positive in these cells (ICC with DAB as chromogen 400×) (c) Brisk mitosis and pleomorphism seen (Geimsa 600×). (d) Plump spindled cells present in slightly cohesive clusters (Geimsa 600×). (e) Vimentin positive in these cells (ICC with DAB as chromogen 400×)

Upon excision of these masses [Figure 2], gross and histopathological examination was carried out. Grossly, the cut surface of tumors in the ovary was solid-cystic, variegated with areas of hemorrhage and necrosis [Figure 3a]. Tumor size ranged from 7–12 cm with a mean size of 10 cm. On microscopic examination, all cases showed features of biphasic neoplasm with the presence of glandular elements as the predominant epithelial component which was confirmed by CK and/or EMA positivity on IHC [Figure 3b and 3c]. One of the cases with scrape cytology (Case 1) also showed presence of squamous and neuroendocrine elements which was later confirmed on IHC for chromogranin. This case has previously been published from our institute.[13] Case 2 also had a population of cells with clear to vacuolated cytoplasm, which on special stains showed positivity for mucicarmine. Mesenchymal component in all the cases was homologous and comprised of spindled out pleomorphic cells with high mitosis ranging from 6 to 8 per high power field and stained positive for vimentin and SMA. None of the cases had any heterologous component. IHC was performed in four cases for confirmation [Table 2].

Figure 2

(a) A CECT- Abdomino-pelvic mass involving the uterus, one sided ovary. (b) Cell block representing both components (HE 200×)

Figure 3

(a) Gross- 10 × 10 × 10 cm2 mass involving the whole uterine cavity. (b) Histopathology showing spindled cells with marked pleomorphism (HE 400×). (c) Epithelial component in form of glands interspersed in cellular spindled stroma (HE 400×) Spindled cells expressing vimentin and glandular component showing CK (IHC with DAB as chromogen 400×)

Table 2

Cytology and histopathology of five cases

Case	Cytology	Histopathology
Case 1	Highly cellular smear, focal attempted acinar pattern, pleomorphic spindle-shaped cells Diagnosis: Poorly differentiated carcinoma	Glandular element along with Spindled out pleomorphic cells; no heterologous component IHC: CK+ Vim+ Final Diagnosis: MMMT
Case 2	Moderately cellular, dispersed and focal attempted acinar pattern with elongated cells Pleomorphism+necrosis+Diagnosis: MMMT	Glandular, squamous, neuro-endocrine Spindled out pleomorphic cells; no heterologous component IHC: CK+ Vim+ Chromo + EMA+ Final Diagnosis: MMMT
Case 3	Moderately cellular, Clusters and scattered plasmacytoid pleomorphic cells Scanty stromal component Diagnosis: MMMT	Glandular Bland spindle-shaped cells with bipolar cytoplasm; no heterologous component IHC: CK+ Vim+ Final Diagnosis: MMMT
Case 4	Highly cellular, round to oval cells in acinar pattern, plump spindled elongated Diagnosis: MMMT	Glandular Spindled out pleomorphic cells; no heterologous component IHC: CK+ Vim+ Final Diagnosis: MMMT
Case 5	Highly cellular smears with round cells forming glandular pattern and oval to spindle shaped cells Diagnosis: MMMT	Glandular Elongated pleomorphic spindle cells; no heterologous component IHC: CK+ Vim+ Final Diagnosis: MMMT

On cytological-histopathological correlation, 04 out of 05 cases had no discrepancy as these cases were diagnosed as MMMT on FNAC and were later confirmed on histopathology. One of the cases was poorly differentiated carcinoma on cytology, only to be later confirmed on histopathology as MMMT making FNAC to be 90% sensitive. This case was revisited and we found that stromal components in the form of plump, ovoid spindle cells with brisk mitotic activity were overlooked, being few in number. Since both cytological and histological diagnosis was high-grade malignancy and all histologically confirmed cases were screened positive on cytology, thus positive and negative predictive value (PPV and NPV) was 100%.

DISCUSSION

Carcinosarcomas/MMMTs constitute an infrequently encountered group of malignant neoplasms, which comprise 1%–3% of all ovarian malignancies and 2%–5% of uterine malignancies.[1415] These tumors are highly aggressive and are poorly responsive to treatment.[2] The relatively low number of reported cases and the difficulty of preoperative diagnosis make it difficult to assess these tumors. Various hypotheses have been proposed to explain the biphasic appearance of MMMTs, but the exact biological nature is still unclear. It has been postulated that these tumors arise from pluripotent Mullerian stem cells, which undergo divergent differentiation into malignant epithelial and stromal elements (combination theory).[3] Another suggests that the two tumor types, epithelial and sarcoma, evolve independently and then collide, suggesting that carcinoma and sarcoma are two independent tumors (collision theory).[3] Current evidence incriminates metaplastic transformation of epithelial component, which gives rise to sarcomatous component, so that these tumors could be regarded as dedifferentiated carcinomas of the ovary (conversion theory).[3] The diagnosis of primary MMMTs rarely suspected preoperatively, as the clinical presentation and radiology is similar to other epithelial tumors as was in all the cases of our study. Table 3 depicts various studies published in cytology literature in this respect. Tumor markers such as CA-125 may be measured, but they may be raised or may be in the normal range, though in all our cases it was raised. Even cytological analysis of ascitic fluid in positive cases may yield malignant epithelial components only in majority of cases.[16] Contrary to widely reported literature and several clinicopathological reports, we found that apart from affecting older and postmenopausal women, MMMT also affects middle-aged women. Three women in our study were premenopausal at the time of diagnosis. The median age at presentation had been reported to be 57–65 years,[17] and in the current series, it was 45.8 years, with a range of 37–65 years.

Table 3

Review of published cytology literature

Author	Anatomic site	Year
Nguyen et al. (Case report)[5]	Intra-abdominal mass	1982
Donat et al. (Case report)[6]	Right ovary	1994
Mourad et al. (case report)[7]	Pelvic mass	1994
Finley et al. (case report)[8]	Vagina and pelvic mass	1996
Ventura et al. (Case report)[9]	Right Pelvic mass	2000
Banik et al. (Case report)[10]	Uterus	2011
Gupta et al. (Case series of 8 cases)[11]	Cervical cytology	2014
Gupta et al. (Case report)[12]	Ovarian scrape cytology	2017

The gross appearance of the tumors was similar to that of those described in other series[17]; solid, cystic masses with varying degrees of hemorrhage and necrosis. Extraovarian/extrauterine extension of the tumor was noted in four cases (80%). These tumors are typically large, ranging from 15–20 cm in diameter.[2] In the present study, tumor size ranged from 7–12 cm with a mean of 10 cm. The characteristic microscopic feature is an intimate admixture of malignant epithelial and stromal elements.

Diagnosis of MMMT via cervicovaginal, endometrial, and peritoneal fluid cytology has also been previously described.[1819] The detection rate of malignant cells in cervical and endometrial samples in histologically proven MMMT is between 56% and 70% and even lower in peritoneal fluid cytology.[20]

In our study, none of the patients was found to harbor heterologous elements on review of the histopathology. Glandular elements were the predominant epithelial component, which were confirmed by CK and/or EMA positivity on IHC. One of the cases also showed the presence of squamous and neuroendocrine elements, which was later confirmed on IHC for chromogranin. In a study from India, endometrioid carcinoma and heterologous rhabdomyosarcoma were the predominant components seen in a series of 27 cases.[21] ICC for cytokeratin and epithelial membrane antigen show strong staining of the epithelial element, while vimentin exhibits strong staining of the mesenchymal element.

Considering propensity to lymphatic metastasis, it is now thought that most carcinosarcomas represent sarcomatous transdifferentiation/transformation of endometrial carcinoma. Studies have listed various shared molecular aberrations in carcinoma and sarcoma components like X-chromosome inactivation, types of TP53 and KRAS mutations, microsatellite instability (MSI), and loss of heterozygosity (LOH) status.[22]

In our study, we were able to conclude that ICC staining is a very useful and reliable method for highlighting the biphasic nature of carcinosarcomas and for distinguishing spindle cell carcinoma or undifferentiated carcinoma from carcinosarcoma. ICC on smears/cell block was reliable and reproducible on histopathology. Atypical spindle cells/sarcomatous component is not always represented in cytology samples as represented by one of our cases, thus a possibility of MMMT should be kept in mind even on finding malignant glandular/epithelial cells.

CONCLUSION

MMMT of FGT is very rare with an incidence of less than 2%. Though the literature is replete in establishing the histo-pathological diagnosis of MMMT, the diagnosis on USG-guided FNAC has been rarely described. The sensitivity of FNAC as in our study was 90%. This highlights the importance of FNAC as a diagnostic tool for this lesion. Emphasis should be made on the careful examination of small sarcomatous elements in smears. Utilization of cell block and immunocytochemistry with histopathological correlation should be done to avoid misdiagnosis.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.